Vol. 24 (2019)

DOI: 10.1186/s11658-018-0126-9 Volume 24 (2019)
Authors Lin Xie1, Yajuan Chen2, Jingbo Chen3, Hongbin Zhang3, Yedan Liao1, Yonghong Zhou1, Ling Zhou1 and Chen Qing2*
Abstract Objective: To investigate the anti-tumor effects and the mechanism of the compound 13-chlorine-3, 15-dioxy-gibberellic acid methyl ester (GA-13315) in lung adenocarcinoma in vitro and in vivo.
Methods: The antiproliferative effect of GA-13313 on the A549 cell line was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay. A xenograft model of A549 was established to evaluate the anti-tumor effect and histopathological examination was performed to assess the toxicity of GA-13315. Apoptosis was detected by TUNEL staining in tissues and flow cytometry in cells; activation of caspase-3, caspase-8 and caspase-9 was evaluated by immunohistochemical analysis; protein levels of Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), caspase-4, activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage-inducible gene 153 (GADD153) were determined by western blotting. Mitochondrial membrane potential (MMP) was measured by the JC-1 fluorescence probe.
Results: Our results showed that GA-13315 exhibited potent, dose- and time-dependent anti-proliferative activity, and the IC50 values were 37.43 ± 2.73, 28.08 ± 7.76 and 19.29 ± 7.61 μM at 24, 48, and 72 h, respectively. The xenograft experiment revealed that tumor weight and volume were significantly decreased after GA-13315 3 mg/kg and 9 mg/kg (P < 0.05) treatment, and GA-13315 had low toxicity in bone marrow, kidney and colon tissues. GA-13315 triggered remarkable apoptosis in A549 cells at the concentration of 25.6 μM and 32 μM (P < 0.05) and activated caspase-3, − 8 and − 9. Moreover, GA-13315 induced apoptosis through the mitochondrial apoptosis pathway by elevating the Bax/Bcl-2 ratio, releasing cytochrome c and activating caspase-9 in A549 cells. In the endoplasmic reticulum apoptosis pathway, the levels of caspase-4, ATF4, GRP78 and GADD153 were markedly upregulated.
Conclusions: This study suggests that GA-13315 can be considered as a promising chemotherapeutic agent with anticancer activity in treatment of lung cancer in future.
Keywords Gibberellin derivatives, Lung adenocarcinoma, Antitumor, Toxicity
Address and Contact Information 1 Department of Medical Oncology, Third Affiliated Hospital of Kunming Medical University/ Cancer Hospital of Yunnan Province, Kunming, China.
2 School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products, Cancer Hospital of Yunnan Province, Kunming Medical University, NO.1168, West Chunrong Road, Chenggong Developing Area, Kunming 650031, China.
3 Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education School of Chemical Science and Technology, Yunnan University, Kunming, China.
* Corresponding Author: qingchenhhh@qq.com
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DOI: 10.1186/s11658-018-0124-y Volume 24 (2019)
Authors Chen Xu and Junhua Zheng*
Abstract Objective: The tumor susceptibility gene 101 (TSG101) is closely associated with various tumor types, but its role in the pathogenesis of renal cell carcinoma (RCC) is still unknown. This study used RNA interference to silence the expression of TSG101 in RCC cell lines and explore the role of TSG101 in RCC.
Methods: Immunohistochemistry and western blot were performed to detect the expression of TSG101 in 15 paired renal tumor samples. A small interfering RNA (siRNA) targeting TSG101 was transfected into A498 and 786-O cell lines. The Cell Counting Kit-8 (CCK-8) assay and colony formation assay were used to observe the changes in cell proliferation after transfection. Flow cytometry was used to detect the effect on the cell cycle. Western blot was conducted to study the changes of related functional proteins.
Results: The expression of TSG101 was higher in RCC tissues than in adjacent normal tissues. The CCK-8 assay showed that the proliferation and colony formation of the A498 and 786-O cell lines were attenuated after suppression of TSG101. Flow cytometry showed that silencing of TSG101 induced G0/G1 arrest. The western blot results revealed that the levels of cell cycle-related proteins (c-myc, cyclin E1 and cyclin-dependent kinase 2 (CDK2)) were markedly decreased in the siRNA groups.
Conclusions: TSG101 promotes proliferation of RCC cells. This positive effect on tumor growth involves activation of c-myc and cyclin E1/CDK2 and their effect on cell cycle distribution.
Keywords Renal cell carcinoma, TSG101, Cell proliferation, Cell cycle
Address and Contact Information Department of Urology, Tenth People’s Hospital of Tongji University, Yanchang Road 301, Shanghai 200072, China
* Corresponding Author: renalzjh@163.com
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DOI: 10.1186/s11658-018-0129-6 Volume 24 (2019)
Authors Qiang Ma
Abstract Background: We investigated the potential regulatory role of miR-219-5p in esophageal squamous cell carcinoma (ESCC) and looked at the underlying mechanisms in ESCC.
Methods: Real-time PCR was used to determine the levels of miR-219-5p in ESCC tissues and cell lines. The effects of miR-219-5p and cyclin A2 (CCNA2) on cell proliferation and cell cycle progression were evaluated using MTT, colony formation and flow cytometry assays with ESCC cell lines EC9706 and TE-9. Bioinformatics techniques and the luciferase reporter assay were applied to validate CCNA2 as the miR-219-5p target in ESCC cells. The mRNA and protein levels of CCNA2 were measured using real-time PCR and western blotting.
Results: MiR-219-5p expression was significantly lower in ESCC tissues and cells than in healthy tissues. Upregulation of miR-219-5p repressed cell proliferation and induced cell cycle arrest at the G2/M phase. CCNA2 was identified and confirmed as a direct downstream target of miR-219-5p and its expression negatively correlated with miR-219-5p profiles in ESCC tissues. Knockdown of CCNA2 potentiated the effects of miR-219-5p on cell proliferation and cell cycle distribution.
Conclusions: Our results demonstrate that miR-219-5p might function as a tumor suppressor by directly targeting CCNA2 expression. It could serve as a new therapeutic target for ESCC.
Keywords Esophageal squamous cell carcinoma, miR-219-5p, CCNA2, Cell proliferation, G2/M phase arrest
Address and Contact Information Department of Oncology, People’s Hospital of Xintai City, No. 1329 Xinfu Road, Xintai 271200, Shandong Province, China
Corresponding Author: li_master111@163.com
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DOI: 10.1186/s11658-019-0137-1 Volume 24 (2019)
Authors Zhitao Zhang2, Yanzhen Han1* , Guangxin Sun1, Xiaohong Liu1, Xiaoyan Jia1 and Xiangjun Yu1
Abstract Background: Hepatitis B virus (HBV) infection is acknowledged as the main cause of hepatocellular carcinoma (HCC). Moreover, previous studies have revealed that microRNAs (miRNAs) widely participate in regulation of various cellular processes, such as viral replication. Hence, the purpose of this study was to investigate the roles of aquaporin 5 (AQP5) and miR-325-3p in the proliferation and apoptosis of HBV-related HCC cells.
Methods: AQP5 and miR-325-3p expression in both normal and HBV-HCC tissues or cells (both Huh7–1.3 and HepG2.2.15) was detected using qRT-PCR. AQP5 expression was knocked down in HBV-related Huh7–1.3 and HepG2.2.15 cells using small interfering RNA (siRNA) technology. Down-regulation was confirmed using real-time PCR and Western blot analysis. Effects of AQP5 down-regulation on the proliferation and apoptosis were assessed. Dual luciferase reporter gene assay, Western blot and qRT-PCR were employed to evaluate the effect of miR-325-3p on the luciferase activity and expression of AQP5. Moreover, miR-325-3p mimic-induced changes in cellular proliferation and apoptosis were detected through CCK-8 assay, BrdU assay, flow cytometry analysis and ELISA.
Results: In this study, the expression of AQP5 was up-regulated in human HBV-HCC tissue, Huh7–1.3 and HepG2.2.15 cells. Knockdown of AQP5 significantly inhibited the proliferation and promoted apoptosis of HBV-HCC cells. Next, miR-325-3p was obviously down-regulated in HBV-HCC. In concordance with this, MiR-325-3p directly targeted AQP5, and reduced both mRNA and protein levels of AQP5, which promoted cell proliferation and suppressed cell apoptosis in HCC cells. Overexpression of miR-325-3p dramatically inhibited cell proliferation and induced cell apoptosis.
Conclusions: Our findings clearly demonstrated that introduction of miR-325-3p inhibited proliferation and induced apoptosis of Huh7–1.3 and HepG2.2.15 cells by directly decreasing AQP5 expression, and that silencing AQP5 expression was essential for the pro-apoptotic effect of miR-325-3p overexpression on Huh7–1.3 and HepG2.2.15 cells. It is beneficial to gain insight into the mechanism of HBV infection and pathophysiology of HBV-related HCC.
Keywords Hepatocellular carcinoma, miR-325-3p, Hepatitis B virus, AQP5, Proliferation, Apoptosis
Address and Contact Information 1 General Surgery V Ward, Affiliated Hospital of Hebei Engineering University, Handan 056002, Hebei Province, People’s Republic of China
2 Clinical Laboratory, Handan Infectious Disease Hospital, Handan 056002, Hebei Province, People’s Republic of China.
* Corresponding Author: hanyanzhenhebei@163.com
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DOI: 10.1186/s11658-019-0140-6 Volume 24 (2019)
Authors Claudia Krautgasser1†, Markus Mandl1†, Florian M. Hatzmann1, Petra Waldegger1, Monika Mattesich2 and Werner Zwerschke1*
Abstract Background: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1+/CD34+/CD90+/CD105+/CD45/CD31.
Methods: We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software.
Results: The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes.
Conclusions: Applying these findings for future gene expression analyses will help elucidate ASC biology.
Keywords Adipose stromal cells, Adipose stem cells, Adipogenesis, Proliferation, Differentiation, Reference gene
Address and Contact Information 1 Division of Cell Metabolism and Differentiation Research, Institute for Biomedical Aging Research, University of Innsbruck, Rennweg 10, A-6020 Innsbruck, Austria
2 Department of Plastic and Reconstructive Surgery, Innsbruck Medical University, Anichstraße 35, A-6020 Innsbruck, Austria.
* Corresponding Author: werner.zwerschke@uibk.ac.at
Claudia Krautgasser and Markus Mandl have equal contribution
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DOI: 10.1186/s11658-018-0125-x Volume 24 (2019)
Authors Yong Zou1 and Min Kong2*
Abstract Objective: Tetrahydroxy stilbene glucoside (TSG) has been reported to exert a cytoprotective effect against various toxicants. However, the function and mechanism of TSG in palmitic acid (PA)-induced inflammation and apoptosis in cardiomyocytes are still unknown. The present study was designed to investigate the post-transcriptional mechanism in TSG-treated cardiomyocytes’ inflammation and apoptosis induced by PA.
Methods: The mRNA and protein levels were assayed by reverse transcription- quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. The targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Cell proliferation was analyzed by CCK-8 assay. Annexin V-fluorescein isothiocyanate/polyimide (annexin V-FITC/PI) staining was used to evaluate apoptosis using flow cytometry.
Results: TSG restricted the detrimental effects, including the activated inflammatory response and apoptosis, of PA in cardiomyocytes, as well as the up-regulation of miR-129-3p and down-regulation of p-Smad3 expression. In addition, bioinformatics and experimental analysis suggested that Smad3 was a direct target of miR-129-3p, which could inhibit or enhance the expression of p-Smad by transfection with miR-129-3p mimics or inhibitors, respectively. Furthermore, our results demonstrated that overexpression of Smad3 reversed the inhibition of inflammation and apoptosis by overexpression of miR-129-3p in PA-stimulated cardiomyocytes.
Conclusion: TSG targeted to miR-129-3p/Smad3 signaling inhibited PA-induced inflammation and apoptosis in cardiomyocytes.
Keywords Tetrahydroxy stilbene glucoside, miR-129-3p, Smad3, Cardiomyocytes, Inflammation, Apoptosis
Address and Contact Information 1 Department of Cardiovascular Medicine, Wuhan No. 6 Hospital, Hospital Affiliated to Jianghan University, No. 168, Xianggan Road, Wuhan 430016, People’s Republic of China.
2 Department of Pharmacy, Wuhan No. 6 Hospital, Hospital Affiliated to Jianghan University, No. 168, Xianggan Road, Wuhan 430016, People’s Republic of China
* Corresponding Author: kong_minfh@sina.com
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DOI: 10.1186/s11658-019-0142-4 Volume 24 (2019)
Authors Tao Zhang1*† and Lei Xiang2†
Abstract Background: Honokiol is a low-molecular-weight natural product and has been reported to exhibit anti-inflammatory activity.
Objectives: Our study aimed to investigate the influence of honokiol on sepsis-induced acute kidney injury (AKI) in a mouse model.
Material and methods: A cecal ligation and puncture (CLP) surgical operation was performed to establish a sepsis-induced acute kidney injury model in mice. Renal histomorphological analysis was performed with periodic acid-Schiff (PAS) staining. The levels of inflammatory markers in serum were measured by ELISA assay. The mRNA and protein levels were assayed by RT-qPCR and western blotting, respectively. Annexin V-FITC/PI staining was used to evaluate glomerular mesangial cell (GMC) apoptosis.
Results: The results revealed that honokiol significantly increased the survival rate in mice undergoing a CLP operation. Inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, were significantly inhibited in honokiol-treated septic mice compared with the CLP group. In addition, honokiol showed the ability to reverse CLP-induced AKI in septic mice. Furthermore, heme oxygenase-1 (HO-1) expression levels were significantly up-regulated and miR-218-5p was markedly down-regulated in honokiol-treated septic mice as compared to CLP-operated mice. Bioinformatics and experimental measurements showed that HO-1 was a direct target of miR-218-5p. In vitro experiments showed that both honokiol and miR-218-5p inhibitors blocked lipopolysaccharide (LPS)-induced cell growth inhibition and GMC apoptosis by increasing the expression of HO-1.
Conclusions: Honokiol ameliorated AKI in septic mice and LPS-induced GMC dysfunction, and the underlying mechanism was mediated, at least partially, through the regulation of miR-218-5p/HO-1 signaling.
Keywords Honokiol, Sepsis, Acute kidney injury, Heme oxygenase-1
Address and Contact Information 1 Department of of Intensive Care Unit, Tianjin Huanhu Hospital, No. 6 Jizhao Road, Tianjin 300060, People’s Republic of China
2 Department of Neurology, Tianjin Huanhu Hospital, Tianjin 300060, People’s Republic of China.
* Corresponding Author: zhang_taohh@163.com
† Tao Zhang and Lei Xiang contributed equally.
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DOI: 10.1186/s11658-019-0144-2 Volume 24 (2019)
Authors Liyang Chen, Gejun Liu, Wenjun Li and Xing Wu*
Abstract Background: Indian hedgehog (IHH) and Sonic hedgehog (SHH) are important regulators of chondrogenesis. However, activation of IHH and SHH also promotes chondrocyte hypertrophy and ossification during chondrogenesis. The aims of this study were to investigate the effect of microgravity on IHH- and SHH-induced chondrogenic differentiation and to elucidate the role of microgravity in this process.
Methods: Adenovirus plasmids encoding the rabbit IHH gene and SHH genes were constructed in vitro and transfected into rabbit bone marrow-derived mesenchymal stem cells (BMSCs). A rotary cell culture system (RCCS), in which a dynamic three-dimensional culture system combines the mechanical environment with a three-dimensional culture surface, was used for cell culture and differentiation. During the induction of differentiation, expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Toluidine blue and collagen II immunohistochemical staining and annexin V-Cy3 staining were used to indicate investigate cartilage matrix synthesis and hypertrophic hypertrophy, respectively, on day 21 after induction of differentiation.
Results: In this study, IHH and SHH were shown to be equipotent inducers of chondrogenesis in rabbit BMSCs, as evidenced by strong staining for proteoglycans and collagen II, and increased expression of mRNAs and proteins associated with chondrogenesis in an RCCS environment. More importantly, chondrogenic hypertrophy and aging were effectively inhibited in the RCCS environment. In addition, levels of cartilage-related markers in the IHH and SHH transfection groups were initially increased and later decreased in the traditional two-dimensional environment, while cartilage hypertrophy-related factors revealed higher mRNA expression levels during induction.
Conclusions: In summary, microgravity significantly promoted chondrogenic differentiation of BMSCs induced by IHH and SHH and attenuated chondrogenic hypertrophy and aging during chondrogenesis. Furthermore, exogenous IHH and SHH had the same effect on chondrogenic differentiation of BMSCs in the RCCS environment. This study provides further evidence of chondrogenic induction of BMSCs in vitro via IHH and SHH gene delivery.
Keywords Indian hedgehog, Sonic hedgehog, RCCS, Chondrogenic differentiation, Chondrocyte hypertrophy
Address and Contact Information Department of Orthopedics, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai 200072, People’s Republic of China
* Corresponding author: wxing123@yeah.net
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DOI: 10.1186/s11658-019-0141-5 Volume 24 (2019)
Authors Qing Yang1†, Dan Dan Huang2†, Da Guang Li1, Bo Chen1, Ling Min Zhang1, Cui Ling Yuan1 and Hong Hong Huang3*
Abstract Objective: We investigated the protective effect of tetramethylpyrazine (TMP) on injury related to acute myocardial ischemia (AMI) induced by isoproterenol (ISO).
Materials and methods: Rats were randomly assigned to five groups: control, ISO, ISO + propranolol (10 mg/kg), ISO + TMP (10 mg/kg) and ISO + TMP (20 mg/kg). The rats in the three ISO + groups were pretreated with propranolol or TMP, while the rats in the control and ISO groups were pretreated with an equal volume of saline. Afterwards, the rats in the four administration groups were subcutaneously injected with ISO for two consecutive days. The levels of creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in the serum were measured using ELISA. The expressions of B-cell lymphoma-associated X-2 (Bax-2), B-cell lymphoma-2 (Bcl-2), phosphoinositide-3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase 3β (GSK-3β), MDA5 and SOD1 were determined using western blotting assay. The phosphorylation of PI3K, Akt and GSK-3β were also determined using western blotting assay. The left ventricles of the rats were extracted and stained using hematoxylin and eosin (H&E). The ST segment was recorded using electrocardiograms (ECGs).
Results: Administration of TMP (10, 20 mg/kg) reduced the levels of MDA and CK and the activities of SOD and LDH in the serum. Pretreatment with TMP significantly reduced the levels of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α. Treatment with TMP also improved the histopathological alteration and decreased the ST elevation. Furthermore, TMP ameliorated the expressions of Cu, SOD1, MDA5, Bax-2, Bcl-2, p-PI3K, p-Akt and p-GSK-3β in ISO-induced rats.
Conclusions: Tetramethylpyrazine protected against injury due to AMI by regulating the PI3K/Akt /GSK-3β signaling pathway.
Keywords Tetramethylpyrazine, Myocardial ischemia, PI3K, Akt, GSK-3β
Address and Contact Information 1 Blood Transfusion Department, First Hospital of Jilin University, Changchun, Jilin, China. 2 Preclinical School of North Sichuan Medical College, Nanchong, Sichuan, China. 3 Faculty of Chinese Medical Science, Guangxi University of Chinese Medicine, No. 13 Wuhe Road, Qingxiu District, Nanning 530222, Guangxi, China
* Corresponding author: huanghongh1@foxmail.com
Qing Yang and Dan Dan Huang contributed equally to this work.
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DOI: 10.1186/s11658-018-0127-8 Volume 24 (2019)
Authors Bingxia Shi1* , Chao Ma2, Guolin Liu1 and Yanjun Guo1
Abstract Background: LIM kinase 1 (LIMK1) expression levels are closely associated with microRNA (miRNA) processing. Higher levels of LIMK1 are reported during the progression of many cancers. Our study explored the interaction between LIMK1 and miR-106a in oral squamous cell carcinoma (OSCC). Methods: Quantitative RT-PCR was performed to detect the levels of LIMK1 and miR-106a in OSCC tissues and cell lines. The rates of cell proliferation and epithelial– mesenchymal transition (EMT) were assessed to determine the biological functions of miR-106a and LIMK1 in OSCC cells. The mRNA and protein levels of LIMK1 were measured using quantitative RT-PCR and western blotting. Luciferase assays were performed to validate LIMK1 as an miR-106a target in OSCC cells. Results: We found that the level of miR-106a significantly decreased and the expression of LIMK1 significantly increased in OSCC tissues and cell lines. There was a close association between these changes. Knockdown of LIMK1 significantly inhibited the proliferation and EMT of OSCC cells. The bioinformatics analysis predicted that LIMK1 is a potential target gene of miR-106a and the luciferase reporter assay confirmed that miR-106a could directly target LIMK1. Introduction of miR-106a to OSCC cells had similar effects to LIMK1 silencing. Overexpression of LIMK1 in OSCC cells partially reversed the inhibitory effects of the miR-106a mimic. Conclusion: MiR-106a inhibited the cell proliferation and EMT of OSCC cells by directly decreasing LIMK1 expression.
Keywords Oral squamous carcinoma, MicroRNA-106a, LIM kinase 1, Proliferation, Epithelial–mesenchymal transition
Address and Contact Information 1 Oral and Maxillofacial Surgery, Cangzhou Central Hospital, No. 16 Xinhua West Road, Hebei 061000, People’s Republic of China
2 Department of Medical Plastic Surgery, Cangzhou Central Hospital, Hebei 061000, People’s Republic of China.
* Corresponding author: shibingxiacangzhou@163.com
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DOI: 10.1186/s11658-019-0139-z Volume 24 (2019)
Authors Weiyi Li1, Jing Cao2, Jian Liu1, Wenli Chu1, Congqing Zhang1, Shuiling Chen1 and Zefeng Kang1*
Abstract Background: Cyclin-dependent kinase-like 1 (CDKL1) is a member of the cell division control protein 2-related serine–threonine protein kinase family. It is known to occur in various malignant tumors, but its role in neuroblastoma (NB) remains unclear.
Methods: We constructed a CDKL1-silenced NB cell strain (SH-SY5Y) and used real- time PCR and western blotting to confirm the silencing. Functional analyses were performed using the MTT, colony-formation, FACS, wound-healing and transwell invasion assays.
Results: The expression of CDKL1 was significantly upregulated in NB tissue as compared to the adjacent normal tissue. CDKL1 knockdown significantly suppressed cell viability and colony formation ability. It also induced cell cycle G0/G1 phase arrest and apoptosis, and suppressed the migration and invasion ability of SH-SY5Y cells. CDKL1 knockdown decreased the CDK4, cyclin D1 and vimentin expression levels, and increased the caspase-3, PARP and E-cadherin expression levels in SH-SY5Y cells.
Conclusions: Our findings suggest that CDKL1 plays an important role in NB cell proliferation, migration and invasion. It might serve as a potential target for NB therapy.
Keywords Neuroblastoma, SH-SY5Y, CDKL1, Proliferation, Migration, Invasion
Address and Contact Information 1 Eye Hospital, China Academy of Chinese Medical Sciences, No 33 Lugu Road, Shijingshan district, Beijing 100040, China
2 Yinan Branch of Qilu Hospital of Shandong University, Linyi, Shandong, China.
* Corresponding author: zefeng_K2016@126.com
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DOI:10.1186/s11658-018-0131-z Volume 24 (2019)
Authors Zhen-Hua Gong1†, Feng Zhou2†, Chao Shi3, Tie Xiang1, Chang-Kai Zhou1, Qian-Qian Wang1, Ya-Su Jiang1 and Sheng-Feng Gao1*
Abstract Background: Cutaneous squamous cell carcinoma (CSCC) is a common type of skinmalignancy. MicroRNA-221 (miRNA-221) is a critical non-coding RNA in tumor initiationand progression. However, the molecular mechanisms of miRNA-221 in the developmentof CSCC remain unknown. This study investigated the expression of miRNA-221 in CSCCand its potential tumor biological functions.
Methods: MTT assay, colony assay, PCR, and Western blot were adopted.
Results: In this study, miRNA-221 expression was significantly higher in CSCC tissuesand cell lines than in normal tissues and cells (P < 0.05). Further functional experimentsindicated that miRNA-221 knockdown inhibited the proliferation and cell cycle, whileupregulation of miRNA-221 presented the opposite role. The dual reporter gene assaysindicated that PTEN is a direct target gene of miRNA-221. PTEN protein or mRNA levelswere decreased after the cells were transfected with miR-221 mimics.
Conclusions: Taken together, the obtained results indicated that miR-221 plays anoncogenic function in CSCC by targeting PTEN and further suggest that miR-221 maybe a potential target for CSCC diagnosis and treatment.
Keywords Cutaneous squamous cell carcinoma, miRNA-221, PTEN, Proliferation, Treatment
Address and Contact Information 1 Department of Burn and Plastic Surgery, The First People’s Hospital of Nantong, Nantong 226001, China
2 Department of Clinical Laboratory, The First People’s Hospital of Nantong, Nantong 226001, China.
3 Department of Pathology, The First People’s Hospital of Nantong, Nantong 226001, China.
* Corresponding author: 574482029@qq.com
Zhen-Hua Gong and Feng Zhou contributed equally to this work.
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DOI: 10.1186/s11658-019-0145-1 Volume 24 (2019)
Authors Lin Chen*, Weifeng Hu, Guohao Li, Yonglian Guo, Zhihua Wan and Jiajun Yu
Abstract Background: This study aims to investigate the effects of inhibiting microRNA-9-5p (miR-9-5p) on the expression of StAR-related lipid transfer domain containing 13 (StarD13) and the progress of prostate cancer.
Methods: The mRNA expression levels of miR-9-5p and StarD13 were determined in several prostate cancer cell lines. We chose DU145 and PC-3 cells for further research. The CCK8 assay was used to measure the cell viability. The cell invasion and wound-healing assays were respectively applied to evaluate invasion and migration. The expression of E-cadherin (E-cad), N-cadherin (N-cad) and vimentin were measured via western blot. DU145 and PC-3 cells overexpressing StarD13 were generated to investigate the variation in proliferation, invasion and migration. A luciferase reporter assay was used to identify the target of miR-9-5p.
Results: Our results show that miR-9-5p was highly expressed and StarD13 was suppressed in prostate cancer cells. MiR-9-5p inhibition repressed the cells’ viability, invasion and migration. It also increased the expression of E-cad and decreased that of N-cad and vimentin. StarD13 overexpression gave the same results as silencing of miR-9-5p: suppression of cell proliferation, invasion and migration. The bioinformatics analysis predicted StarD13 as a target gene of miR-9-5p. Quantitative RT-PCR, western blot analysis and the dual-luciferase reporter assay were employed to confirm the prediction.
Conclusion: Our results show that miR-9-5p plays a powerful role in the growth, invasion, migration and epithelial–mesenchymal transition (EMT) of prostate cancer cells by regulating StarD13. A therapeutic agent inhibiting miR-9-5p could act as a tumor suppressor for prostate cancer.
Keywords microRNA-9-5p, Prostate cancer, StarD13, Migration, Invasion
Address and Contact Information Department of Urology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, No. 26 Shengli Street, Jiang’an District, Wuhan 430014, China
* Corresponding author: chenlin8279@yeah.net
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DOI: 10.1186/s11658-018-0134-9 Volume 24 (2019)
Authors Jian-yao Wang1, Hao Cheng2, Hong-yan Zhang2, Yong-qin Ye1, Qi Feng1, Zi-min Chen1, Yue-lan Zheng1, Zhou-guang Wu1, Bin Wang1* and Jun Yao3*
Abstract This study was designed to investigate the potential role of microRNA-29c (miR-29c) in biliary atresia-related fibrosis. The expression of miR-29c was determined in 15 pairs of peripheral blood samples from infants with biliary atresia (BA) and infants with non-BA neonatal cholestasis using quantitative real-time PCR. EMT was established by induction with TGF-β1 in HIBEpiC cells. MiR-29c was inhibited by lipofectamine transfection. The expressions of proteins related to epithelial–mesenchymal transition (EMT), i.e., E-cadherin, N-cadherin and vimentin, were determined using quantitative real-time PCR and western blotting. Direct interaction between miR-29c and DNMT3A and DNMT3B was identified using a luciferase reporter assay. The expressions of DNMT3A and DNMT3B were suppressed by treatment with SGI-1027. Patients with BA showed significantly lower miR-29c levels in peripheral blood samples than the control subjects. In vitro, TGF-β1-induced EMT significantly decreased the expression of miR-29c. Downregulation of miR-29c had a promotional effect on BA-related fibrosis in HIBEpiC cells, as confirmed by the decrease in E-cadherin and increase in N-cadherin and vimentin levels. MiR-29c was found to target the 3’UTR of DNMT3A and DNMT3B and inhibit their expression. Suppression of DNMT3A and DNMT3B reversed the effects of miR-29c downregulation on BA-related fibrosis in HIBEpiC cells. These data suggest that BA-related fibrosis is closely associated with the occurrence of EMT in HIBEpiC cells. MiR-29c might be a candidate for alleviating BA-related fibrosis by targeting DNMT3A and DNMT3B.
Keywords Biliary atresia, Epithelial–mesenchymal transition, MiR-29c, Fibrosis, DNMT3A, DNMT3B
Address and Contact Information 1 Department of General Surgery, Shenzhen Children’s Hospital, Shenzhen 518026, Guangdong Province, China
2 Graduate School of China Medical University, Shenzhen 110122, Liaoning Province, China.
3 Department of Gastroenterology, Jinan University of Medical Sciences, Shenzhen Municipal People’s Hospital, Shenzhen 518020, Guangdong Province, China
* Corresponding author: szwb1967@126.com; yj_1108@126.com
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DOI: 10.1186/s11658-019-0147-z Volume 24 (2019)
Authors Yuan Gao1*†, Xiaochun Wu2†, Shuqin Zhao1, Yujun Zhang1, Hailong Ma1, Zhen Yang1, Wanghao Yang1, Chen Zhao1, Li Wang1 and Quanwei Zhang1,2
Abstract Melatonin receptors MT1 and MT2 (genes officially named MTNR1A and MTNR1B, respectively) play crucial roles in melatonin-mediated regulation of circadian rhythms, the immune system, and control of reproduction in seasonally breeding animals. In this study, immunolocalization assay showed that MT1 and MT2 are highly expressed in Leydig cell membrane. To understand the biological function of melatonin receptors in hCG-induced testosterone synthesis, we generated melatonin receptor knockdown cells using specific siRNA and performed testosterone detection after hCG treatment. We found that knockdown of melatonin receptors, especially MTNR1A, led to an obvious decrease (> 60%) of testosterone level. Our further study revealed that knockdown of melatonin receptors repressed expression, at both the mRNA level and the protein level, of key steroidogenic genes, such as p450scc, p450c17 and StAR, which are essential for testosterone synthesis. hCG triggered endoplasmic reticulum (ER) stress to regulate steroidogenic genes’ expression and apoptosis. To further investigate the potential roles of melatonin receptors in hCG-induced regulation of ER stress and apoptosis, we examined expression of some crucial ER stress markers, including Grp78, Chop, ATF4, Xbp1, and IRE1. We found that inhibition of melatonin receptors increased hCG-induced expression of Grp78, Chop and ATF4, but not Xbp1 and IRE1, suggesting that hCG may modulate IRE1 signaling pathways in a melatonin receptor-dependent manner. In addition, our further data showed that knockdown of MTNR1A and MTNR1B promoted hCG-induced expression of apoptosis markers, including p53, caspase-3 and Bcl-2. These results suggested that the melatonin receptors MTNR1A and MTNR1B are essential to repress hCG-induced ER stress and cell apoptosis. Our studies demonstrated that the mammalian melatonin receptors MT1 and MT2 are involved in testosterone synthesis via mediating multiple cell pathways.
Keywords Melatonin receptor, Testosterone, ER stress, Apoptosis
Address and Contact Information 1 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China2 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, Gansu, China.
* Corresponding author: yyshpy@gmail.com

Yuan Gao and Xiaochun Wu contributed equally to this work. Yuan Gao and Xiaochun Wu are considered co-first authors.
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DOI: 10.1186/s11658-019-0136-2 Volume 24 (2019)
Authors Qiangcheng Zeng1†, Yang Wang2,3†, Jie Gao1,4, Zhixiong Yan2, Zhenghua Li1, Xianqiong Zou2, Yanan Li2, Jiahui Wang2 and Yong Guo1,2*
Abstract Background: Mechanical loading is an essential factor for bone formation. A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation.
Methods: Mouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro. Using molecular and biochemical methods, IGF-1 (insulin-like growth factor-1), PGE2 (prostaglandin E2), OPG (osteoprotegerin) and NOS (nitric oxide synthase) activities of the cells were assayed. MiR microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. MC3T3-E1 osteoblasts were stimulated by the mechanical tensile strain, and the miR-29b-3p expression was detected with miR microarray and RT-qPCR. Additionally, the effect of miR-29b-3p on IFG-1 secretion of osteocytes and the influence of conditioned medium of osteocytes transfected with miR-29b-3p on osteoblast differentiation were investigated.
Results: The mechanical strain increased secretions of IGF-1 and PGE2, elevated OPG expression and NOS activities, and resulted in altered expression of the ten miRs, and possible target genes for these differentially expressed miRs were revealed through bioinformatics. Among the ten miRs, miR-29b-3p were down-regulated, and miR-29b-3p overexpression decreased the IGF-1 secretion of osteocytes. The mechanical strain did not change expression of osteoblasts’ miR-29b-3p. In addition, the conditioned medium of mechanically strained osteocytes promoted osteoblast differentiation, and the conditioned medium of osteocytes transfected with miR-29b-3p mimic inhibited osteoblast differentiation.
Conclusions: In osteocytes (but not osteoblasts), miR-29b-3p was responsive to the mechanical tensile strain and regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically strained osteocytes.
Keywords Mechanical tensile strain, Osteocyte, Osteoblast differentiation, miRNA microarray
Address and Contact Information 1 key laboratory of Functional Bioresource Utilization in University of Shandong, Shandong Key Laboratory of Biophysics, Dezhou University, Dezhou 253023, China.
2 Department of Biomedical Engineering, College of Biotechnology, Guilin Medical University, No. 1 Zhiyuan Road, Lingui District, Guilin City 541100, Guangxi, China China.
3 Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, China.
4 Medical Department, Secondary Renmin Hospital of Dezhou, Dezhou 253023, Shangdong, China.
* Corresponding author: guoyong74@163.com
Qiangcheng Zeng and Yang Wang contributed equally to this work.
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DOI: 10.1186/s11658-019-0143-3 Volume 24 (2019)
Authors Yong Yang1,2, Yan Bao1,2, Guo-Kai Yang1,2, Jia Wan1,2, Ling-Juan Du1,2 and Zhen-Huan Ma1,2*
Abstract Overcoming chemorestistance to 5-fluorouracil (5-FU) could offer a new treatment option for highly malignant colon cancer. In our study, differential microRNA expression profiling revealed that miR-214 is downregulated in 5-FU-resistant colon cancer cells compared to normal cells. In vitro, miR-214 could sensitize non-resistant colon cancer cells and 5-FU-resistant colon cancer cellsto 5-FU. Functionally, miR-214 inhibited cell clone formation and cell growth and enhanced 5-FU-inducing cell apoptosis and caspase-3 levels. MiR-214 targeted heat shock protein 27 (Hsp27), as confirmed via dual luciferase reporter assays and western blots. Hsp27 also sensitized HT-29 and LoVo to 5-FU by enhancing cell apoptosis. Overexpression of Hsp27 could block miR-214 with an effect on the sensitivity of colon cancer cells to 5-FU. In conclusion, miR- 214 sensitizes colon cancer cells to 5-FU by targeting Hsp27, indicating a significant role for this miRNA in colon cancer chemotherapy.
Keywords miR-214, Hsp27, 3′-UTR, 5-FU, Colon cancer
Address and Contact Information 1 The Third Department of General Surgery, The Second People’s Hospital of Yunnan Province, Kunming, China
2 Department of Vascular Surgery, The Fourth Affiliated Hospital of Kunming MedicalUniversity, 176 Youth Road, Kunming, Yunnan Province 650021, People’s Republic of China
* Corresponding author: yyyunnan@163.com
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DOI: 10.1186/s11658-019-0138-0 Volume 24 (2019)
Authors Leila Saremi1, Shirin Lotfipanah2, Masumeh Mohammadi1, Hassan Hosseinzadeh3, Mina Fathi-Kazerooni4, Behrooz Johari5 and Zohreh Saltanatpour6*
Abstract Background: The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Several studies have demonstrated a significant association between Pro12Ala polymorphism of the PPARγ2 gene and metabolic disorders. Therefore, this study aimed to evaluate the association of Pro12Ala polymorphism with increased risk of NAFLD in Iranian patients with type 2 diabetes mellitus.
Methods: This cross-sectional study was performed on 145 healthy control subjects and 145 NAFLD patients with a history of type 2 diabetes. Pro12Ala polymorphism genotyping was performed using PCR–restriction fragment length polymorphism (RFLP) technique with the Bs1I restriction enzyme.
Results: Our results demonstrated that CC and GG genotypes of Pro12Ala were found in the participants, but there was no statistically significant difference between NAFLD patients and healthy controls (P = 0.64 and χ2 = 0.21).
Conclusion: This study suggests that Pro12Ala polymorphism of the PPARγ2 gene cannot be considered as a risk factor for NAFLD in the Iranian population.
Keywords Pro12Ala polymorphism, PPARγ2 gene, NAFLD, Type 2 diabetes mellitus
Address and Contact Information 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2 Farhangian University, Shahid Mofatteh Teacher Education Paradise, Tehran, Iran.
3 Department of Biology, Faculty of Science, Yazd University, Yazd, Iran.
4 Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
5 Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
6 Medical Genetic Center, Endocrinology and Metabolism Research Institute (EMRI), Tehran University of Medical Sciences (TUMS), Tehran, Iran.

* Corresponding author: z-saltanatpour@razi.tums.ac.ir; zohre_saltanatpour@yahoo.com
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DOI: 10.1186/s11658-018-0132-y Volume 24 (2019)
Authors Rui Wang1, Boping Wen2 and Dong Sun1*
Abstract Background: MicroRNA (miRNA) plays a vital role in the pathogenesis of intervertebral disc degeneration (IDD). The expression and potential mechanism of miR-573 in human nucleus pulposus (NP) remains to be elucidated. In this study, we aimed to investigate the role of miR-573 in IDD.
Methods: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was applied to examine the expression of miR-573 and Bax in idiopathic scoliosis tissues and IDD tissues. Human NP cells were employed for analysis. Moreover, the proliferation and apoptosis of NP cells were detected using MTT and flow cytometry assay respectively. The expression levels of Bcl-2, cleaved caspase-3, cleaved caspase-9, caspase-3 and caspase-9 in degenerative NP cells were measured by Western blotting assay. Furthermore, a luciferase reporter assay was used to verify the relationship between miR-573 and Bax.
Results: The results revealed that the mRNA expression level of miR-573 was down- regulated whereas Bax was up-regulated notably in degenerative NP cells. In addition, overexpression of miR-573 increased cell viability remarkably, coupled with inhibition of cell apoptosis. The expression level of Bcl-2 was increased while cleaved caspase-3 and cleaved caspase-9 expression levels were decreased in miR-573 overexpression NP cells. Additionally, the bioinformatics analysis underscored that Bax was a direct target gene of miR-573.
Conclusion: These results suggest that overexpression of miR-573 inhibited NP cell apoptosis by down-regulating Bax, which proved to be a novel effective strategy for IDD therapies.
Keywords miR-573, Bax, Nucleus pulposus cells, Intervertebral disc degeneration
Address and Contact Information 1 Department of Massage and Physiotherapy, Guang Xing Hospital, Zhejiang University of Traditional Chinese Medicine, No. 453, Tiyuchang Road, Xihu District, Hangzhou, Zhejiang 310007, People’s Republic of China
2 Department of Rehabilitation, Western Theater General Hospital, Chengdu, Sichuan 610011, People’s Republic of China.
* Corresponding author: sundonghz@126.com
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DOI: 10.1186/s11658-019-0135-3 Volume 24 (2019)
Authors Monique Meyenberg Cunha-de Padua1,2,3, Guilhermina Rodrigues Noleto1*, Carmen Lucia de Oliveira Petkowicz1, Silvia Maria Suter Correia Cadena1, Frédéric Bost3, Jacques Pouysségur2,4 and Nathalie M. Mazure2,3*
Abstract Background: Polysaccharides from various sources have been used in traditional medicine for centuries. The beneficial pharmacological effects of plant-derived polysaccharides include anti-tumor activity.
Methods: Here, we evaluated the anti-cancer effect of the MSAGM:VO complex under hypoxic conditions (1% oxygen). MSAGM:VO is a complex of the hydrolysate of galactomannan (MSAGM) from Schizolobium amazonicum with oxovanadium (IV/V). The hepatocellular carcinoma (HCC) cell line HepG2 was selected as HCC are one of the most hypoxic solid tumors.
Results: Our results showed that the strong apoptotic activity of MSAGM:VO observed in HepG2 cells under normoxic conditions was completely lost under hypoxic conditions. We found a dynamic balance between the pro- and anti-apoptotic members of the Bcl-2 protein family. The expressions of anti-apoptotic Mcl-1 and Bcl-XL increased in hypoxia, whereas the expression of pro-apoptotic Bax decreased. MSAGM:VO strongly induced autophagy, which was previously characterized as a pro-survival mechanism in hypoxia. These results demonstrate total elimination of the anti-cancer activity of MSAGM:VO with activation of autophagy under conditions of hypoxia.
Conclusion: Although this study is a proof-of-concept of the impact of hypoxia on the potential of polysaccharides, further study is encouraged. The anti-tumor activity of polysaccharides could be achieved in normoxia or through raising the activity of the immune system. In addition, combination strategies for therapy with anti-autophagic drugs could be proposed.
Keywords Hepatocellular carcinoma, Hypoxia, MSAGM:VO, Polysaccharides
Address and Contact Information 1 Department of Biochemistry and Molecular Biology, Federal University of Parana, Curitiba, Brazil
2 Institute for Research on Cancer and Aging of Nice, CNRS-UMR 7284-Inserm U1081, University of Nice Sophia-Antipolis, Centre Antoine Lacassagne, 33 Ave. de Valombrose, 06189 Nice, France
3 Present Address: INSERM U1065, C3M, 151 Route de St Antoine de Ginestière, BP2 3194, 06204 Nice Cedex 03, France.
4 Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco, Monaco.
* Corresponding author: guinoleto@yahoo.com.br; mazure@unice.fr
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DOI: 10.1186/s11658-019-0146-0 Volume 24 (2019)
Authors Liang Sun, Jun Xiang Lian and Shu Meng*
Abstract Aim: To investigate the dysregulation of microRNAs (miRNAs) during the differentiation of osteoclasts and the precise roles of miR-125a-5p in the differentiation of osteoclasts.
Methods: The cell model of RAW 264.7 osteoclast precursor cell differentiation induced by RANKL plus M-CSF stimulation was established. During the early stage of osteoclast differentiation, miRNA expression profiles were detected using the biochip technique and analyzed by cluster analysis. TargetScan, miRTarBase and miRDB database analysis was applied to find the key target genes of miR-125a-5p. A dual luciferase experiment was conducted to identify the direct target of miR-125a-5p. MiR-125a-5p mimic transfection and anti-miR-125-5p treatment were conducted to verify the role of miR-125q-5p in osteoclast differentiation. The levels of triiodothyronine receptor auxiliary protein (TRAP), matrix metallopeptidase 2 (MMP-2), MMP-9 and cathepsin K were analyzed by qRT-PCR and western blot assay. The expression levels of MMP-2 and MMP-9 were determined using western blotting and immunofluorescence assay. The migration and invasion of RAW 264.7 cells were assessed by wound healing and Transwell invasion assays. The proliferation of RAW 264.7 osteoclast precursor cells was detected using MTT assay.
Results: There were 44 microRNAs differently expressed during the differentiation of RAW 264.7 osteoclast precursor cells into osteoclasts, 35 of which were up-regulated and 9 were down-regulated. By luciferase reporter assay, it was confirmed that the TNF receptor superfamily member 1B gene (TNFRSF1B) was the target gene of miR-125a-5p. Up-regulation of miR-125a-5p inhibited TNFRSF1B protein expression and promoted osteoclast differentiation whereas down-regulation of miR-125a-5p caused completely opposite results.
Conclusions: In conclusion, overexpression of miR-125a-5p suppresses the expression of TNFRSF1B and promotes osteoclast differentiation. These results reveal the crucial role of miR-125a-5p in the differentiation of osteoclasts.
Keywords miR-125a-5p, Osteoclast, Osteoporosis, TNFRSF1B
Address and Contact Information State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3 of RenMinNanlu, Chengdu 610041, Sichuan, China
* Corresponding author: mengshumens@foxmail.com
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DOI: 10.1186/s11658-019-0149-x Volume 24 (2019)
Authors Hui Su1,2, Defei Zou1,2, Yikun Sun3 and Yiwu Dai1,2*
Abstract Background: As a newfound type of non-coding RNA, circular RNAs (circRNAs) are involved in various physiological and pathological processes via regulation of gene expression. Increasing evidence shows that aberrantly expressed circRNAs play a crucial role in the initiation and progression of many tumors. However, the functions of different circRNAs in gliomas remain elusive.
Methods: The levels of circRNAs, miRNAs, and mRNAs were quantified by qPCR. The interaction between circDENND2A and miR-625-5p was determined by luciferase reporter and pull-down assays. The migratory and invasive capabilities of glioma cells were examined by wound healing and Transwell assays. Immunohistochemistry was performed to analyze the HIF1α level in glioma tissues.
Results: We predicted circDENND2A (has_circ_0002142) to be a hypoxia-responsive circRNA in glioma via a bioinformatic analysis. We found that hypoxia induced the expression of circDENND2A, which promoted migration and invasion of glioma cells. To understand the behaviors of circDENND2A in glioma, we studied the putative miRNAs targeted by circDENND2A and identified circDENND2A as an efficient sponge of miR-625-5p in glioma cells. Phenotype experiments verified that circDENND2A was required for the hypoxia-induced migration and invasion of glioma cells and that this occurred by sponging of miR-625-5p. Notably, glioma tissues overexpressing HIF1α exhibited a high expression of circDENND2A as well as a low expression of miR-625-5p. circDENND2A was negatively correlated with miR-625-5p.
Conclusion: circDENND2A is required for the hypoxia-induced malignancy of glioma cells and functions by sponging miR-625-5p.
Keywords circDENND2A, miR-625-5p, Glioma, Hypoxia, Migration, Invasion
Address and Contact Information 1 Department of Neurosurgery, Chinese PLA General Hospital, Medical School of Chinese PLA, Beijing, People’s Republic of China
2 Department of Neurosurgery, Bayi Brain Hospital, PLA Army General Hospital, Beijing, People’s Republic of China
3 Department of Neurosurgery, The 306th Hospital of PLA, Beijing, People’s Republic of China.
* Correspondng author: dddyyywww@163.com
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DOI: 10.1186/s11658-019-0150-4 Volume 24 (2019)
Authors Shujun Li1†, Zhongyi Sun2†, Tao Chen3, Jingjing Pan2, Yanqing Shen4, Xiaoqing Chen2, Xiaoyu Zhou4, Rui Cheng4 and Yang Yang4*
Abstract Background: Pulmonary surfactant is the complex mixture of lipid and protein that covers the alveolar surface. Pulmonary surfactant deficiency is one of the main causes of neonatal respiratory distress. Recent studies showed that miRNA plays an important role in lung development, but research into miR-431 regulation of pulmonary surfactant are sparse. In this study, we explored the regulatory role of miR-431-5p in the expression of pulmonary surfactant and identified its potential target gene, Smad4.
Methods: The bioinformatics tool TargetScan was used to predict the targets of miR-431. The expression of miR-431-5p was achieved via transfection of miR-431-5p mimics, an miR-431-5p inhibitor and corresponding negative control. The level of miR-431-5p was determined using quantitative real-time PCR. The CCK8 assay was conducted to confirm cell growth 12 h after transfection with miR-431-5p mimics, inhibitor or NC. Smad4 and surfactant-associated proteins in A549 were analyzed using western blot and quantitative real-time PCR.
Results: Smad4 was validated as a target of miR-431 in A549 cells. Overexpression of miR-431 accelerated A549 proliferation and inhibited A549 apoptosis. The mRNA and protein levels for the surfactant proteins (SP-A, SP-B, SP-C and SP-D) were found to be differentially expressed in A549 cells over- or under-expressing miR-431-5p.
Conclusion: Our results show that miR-431-5p is critical for pulmonary surfactant expression and that its regulation is closely related to the TGF-β/Smad4 pathway. These results will help us to study the pathophysiological mechanism of lung developmental diseases.
Keywords miR-431-5p, Pulmonary surfactant, TGF-β/Smads pathway, Lung development
Address and Contact Information 1 Department of Pediatrics, Children’s Hospital of Anhui Medical University, Hefei, China.
2 Department of Pediatrics, The First Affliated Hospital of Nanjing Medical University, Nanjing, China.
3 Department of Cardiothoracic Surgery, The First Affliated Hospital of Anhui Medical University, Hefei, China.
4 Department of Neonates, Children’s Hospital of Nanjing Medical University, No 72, Guangzhou Road, Nanjing 210008, China
* Corresponding author: yy860507@126.com; 15952071803@163.com
Shujun Li and Zhongyi Sun contributed equally to this work.
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DOI: 10.1186/s11658-018-0133-x Volume 24 (2019)
Authors Chao-Ye Duan, Hua-Tao Xie, Xin-Yue Zhao, Wen-Han Xu and Ming-Chang Zhang*
Abstract Background: Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization.
Methods: Rat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs).
Results: COMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63α (p = 0.9177), ABCG2 (p = 0.526), Ki67 (p = 0.0987), and CK3 (p = 0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) (p = 0.0038 for RT-qPCR, p = 0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) (p = 0.0172 for RT-qPCR, p = 0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) (p < 0.0001 for RT-qPCR, p = 0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability (p = 0.0002) and tube formation (p = 0.0002) of HUVECs.
Conclusions: LNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.
Keywords Limbal niche cells, 3T3 cells, Limbal stem cell deficiency, Oral mucosal epithelial transplantation, Corneal neovascularization
Address and Contact Information Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
* Corresponding author: mingchangzhang@hotmail.com
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DOI: 10.1186/s11658-019-0148-y Volume 24 (2019)
Authors Qinglei Kong1†, Caiqian Liang1†, Yi Jin2*†, Yuhang Pan2, Dayue Tong3, Qingcong Kong4* and Jing Zhou2*
Abstract Background: Given the high mortality rate and unclear pathogenesis for liver cancer, investigation of its molecular mechanisms is essential. We focused on the long non-coding RNA (lncRNA) MIR4435-2HG, which was recently reported to be oncogenic in lung cancer and the microRNA miRNA-487a, which has been reported to be oncogenic in hepatocellular carcinoma (HCC). Our aim was to determine if the former has a role in HCC, and to further validate the role of the latter.
Methods: Samples from 64 patients with HCC were taken at The Third Affiliated Hospital of Sun Yat-Sen University. Cell transfection and PCR were applied.
Results: We found that MIR4435-2HG and miRNA-487a were upregulated in tumor tissues compared to adjacent healthy tissues from HCC patients. The expression of MIR4435-2HG was significantly affected by tumor size but not by tumor metastasis. Correlation analysis showed that MIR4435-2HG and miRNA-487a were positively correlated in both the tumor tissues and adjacent healthy tissues from HCC patients. Overexpression of MIR4435-2HG led to upregulation of miRNA-487a in the cells of HCC cell lines, while overexpression of miRNA-487a did not significantly affect MIR4435-2HG. Overexpression of MIR4435-2HG and miRNA-487a promoted the proliferation of cells of HCC cell lines, and miRNA-487a knockdown partially attenuated the enhancing effects of MIR4435-2HG overexpression on cancer cell proliferation.
Conclusion: MIR4435-2HG is upregulated in HCC and promotes cancer cell proliferation possibly by upregulating miRNA-487a.
Keywords Hepatocellular carcinoma, lncRNA MIR4435-2HG, miRNA-487a, Proliferation
Address and Contact Information 1 Department of Emergency Medicine, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou City, Guangdong Province 510630, People’s Republic of China.
2 Department of Pathology, Guangdong Provincial Key Laboratory of Liver Disease Research, The Third Affiliated Hospital, Sun Yat-Sen University, No. 600 Tianhe Road, Guangzhou City, Guangdong Province 510630, People’s Republic of China
3 Department of Forensic Medicine, ZhongShan Medical School, Sun Yat-Sen University, Guangzhou City, Guangdong Province 510080, People’s Republic of China.
4 Department of Radiology, The Third Affiliated Hospital, Sun Yat-Sen University, No. 600 Tianhe Road, Guangzhou City, Guangdong Province 510630, People’s Republic of China
* Corresponding author: zk56932@163.com; zk56932@163.com; zk56932@163.com
Qinglei Kong, Caiqian Liang and Yi Jin contributed equally to this work.
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DOI: 10.1186/s11658-019-0151-3 Volume 24 (2019)
Authors Yuhua Cai1 and Yunpeng Li2*
Abstract Background: MicroRNAs (miRNAs) are pivotal regulators in regulating hypoxia-induced cardiomyocyte injury. This study was designed to evaluate the effects of miR-29b-3p on hypoxic cardiomyocytes.
Methods: Human AC16 cells were cultured under normoxic or hypoxic conditions. Hypoxic injury was confirmed based on alterations in cell viability using CCK-8 assay and apoptosis using flow cytometry and Hoechst staining. Bioinformatics analyses and the dual-luciferase reporter assay were performed to predict and validate the target gene of miR-29b-3p.
Results: We found that hypoxia suppressed cell viability and promoted apoptosis. TNF receptor-associated factor 5 (TRAF5) was a potential target gene of miR-29b-3p. Our in vitro experiments revealed that miR-29b-3p overexpression or TRAF6 knockdown significantly protected cardiomyocytes against hypoxia-induced injury. Moreover, knockdown of TRAF5 knockdown potentiated the protective effects of miR-29b-3p against hypoxia-induced cell injury.
Conclusion: These findings suggest that upregulation of miR-29b-3p could protect cardiomyocytes against hypoxia-induced injury through downregulation of TRAF5. Targeting TRAF5 with miR-29b-3p might be a potential therapeutic method for AMI.
Keywords Cardiomyocyte, Hypoxia, miR-29b-3p, TRAF5
Address and Contact Information 1 Department of Cardiovasology, Jingzhou First Municipal Hospital, Jingzhou, Hubei Province, China.
2 Department of Cardiovasology, Dongfeng Hospital, Hubei University of Medicine, No. 16 Daling Road, Shiyan 442008, Hubei Province, China
* Corresponding author: yunP_litea@126.com
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DOI: 10.1186/s11658-018-0130-0 Volume 24 (2019)
Authors Liang Liu1* , Yanhua Zuo1, Yan Xu2, Zongfang Zhang1, Ying Li1 and Jie Pang1
Abstract Background: This study aimed to investigate the effects of miR-613 on the proliferation, invasion and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs).
Methods: Synovial tissue samples were collected from 20 rheumatoid arthritis (RA) patients and 10 patients with joint trauma undergoing joint replacement surgery. The RASFs were isolated and cultured. MiR-613 and DKK1 expression in both synovial tissues and cells was detected using quantitative real-time PCR (qRT-PCR). Dual luciferase reporter gene assay was employed to evaluate the effect of miR-613 on the luciferase activity of DKK1. Then RASFs were transfected with miR-613 mimics, si-DKK1 and pcDNA-DKK1. Changes in cellular proliferation, invasion and apoptosis were detected through BrdU assay, Transwell invasion assay and flow cytometry analysis, respectively.
Results: MiR-613 was significantly down-regulated in RA tissues and RASFs compared to normal tissues and cells, whereas DKK1 was up-regulated in RA tissues and RASFs. Dual luciferase reporter gene assay showed that miR-613 could specifically bind to the 3′UTR of DKK1 and significantly inhibit the luciferase activity. Moreover, miR-613 significantly reduced the expression of DKK1. Overexpression of miR-613 or knockdown of DKK1 suppressed proliferation and invasion of RASFs, and induced RASF apoptosis. The reverse results were observed when DKK1 was up-regulated in miR-613-overexpressing RASFs.
Conclusions: MiR-613 can inhibit proliferation and invasion and induce apoptosis of RASFs by directly targeting DKK1 expression.
Keywords Rheumatoid arthritis, MicroRNA-613, Rheumatoid arthritis synovial fibroblasts, DKK1, Proliferation, Apoptosis
Address and Contact Information 1 Department of Rheumatology and Immunology, Cangzhou Central Hospital, Cangzhou 061000, People’s Republic of China
2 The Second Nephrology Department, Cangzhou Central Hospital, Cangzhou 061000, People’s Republic of China
* Corresponding author: liuliangcangzhou@163.com
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DOI: 10.1186/s11658-019-0152-2 Volume 24 (2019)
Authors Yuree Byun, Young-Chul Choi, Yunhui Jeong, Gangtae Lee, Sena Yoon, Yongsu Jeong, Jaeseung Yoon and Kwanghee Baek*
Abstract Background: Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor with a pivotal role in physiological and pathological responses to hypoxia. While HIF-1α is known to be involved in hypoxia-induced upregulation of microRNA (miRNA) expression, HIF-1α is also targeted by miRNAs. In this study, miRNAs targeting HIF-1α were identified and their effects on its expression and downstream target genes under hypoxic conditions were investigated. Cell migration under the same conditions was also assessed.
Methods: microRNAs that target HIF-1α were screened using 3′-untranslated region luciferase (3′-UTR-luciferase) reporter assays. The expression levels of HIF-1α and its downstream target genes after transfection with miRNA were assessed using quantitative RT-PCR and western blot analyses. The effect of the miRNAs on the transcriptional activity of HIF-1α was determined using hypoxia-responsive element luciferase (HRE-luciferase) assays. Cell migration under hypoxia was examined using the wound-healing assay.
Results: Several of the 19 screened miRNAs considerably decreased the luciferase activity. Transfection with miR-200c had substantial impact on the expression level and transcription activity of HIF-1α. The mRNA level of HIF-1α downstream genes decreased in response to miR-200c overexpression. MiR-200c inhibited cell migration in normoxia and, to a greater extent, in hypoxia. These effects were partly reversed by HIF-1α expression under hypoxic conditions.
Conclusion: miR-200c negatively affects hypoxia-induced responses by downregulating HIF-1α, a key regulator of hypoxia. Therefore, overexpression of miR-200c might have therapeutic potential as an anticancer agent that inhibits tumor hypoxia.
Keywords microRNA, miR-200c, HIF-1α, Hypoxia
Address and Contact Information Graduate School of Biotechnology, Kyung Hee University, Yongin, Republic of Korea
* Corresponding author: khbaek@khu.ac.kr
Yuree Byun and Young-Chul Choi contributed equally to this work.
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DOI: 10.1186/s11658-019-0156-y Volume 24 (2019)
Authors Hyun-Duk Jang1,2,3, Hye Zin Hwang4, Hyo-Soo Kim1,2,3,5 and Soo Young Lee6*
Abstract Background: In its RING domain, tumor necrosis factor receptor-associated factor 6 (TRAF6) has ubiquitin E3 ligase activity that facilitates the formation of lysine 63-linked polyubiquitin chains. This activity is required to activate nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and plays an important role in the IκB kinase (IKK) complex.
Methods: An in vitro ubiquitination assay was used to establish whether c-Cbl could promote TRAF6 ubiquitination. We assessed direct binding and performed fine mapping between c-Cbl and TRAF6 based on the results of an immunoprecipitation assay with cultured 293 T cells. The luciferase reporter assay was applied to establish if c-Cbl-mediated ubiquitination affected NF-κB activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), and interleukin-1β (IL-1β). An in vivo ubiquitination assay was performed using endogenous immunoprecipitation of TRAF6 in bone marrow macrophages (BMMs) and osteoclasts.
Results: Here, we report on a form of TRAF6 ubiquitination that is mediated by c-Cbl, leading to the formation of lysine 48-linked polyubiquitin chains. The NF-κB activity induced by RANKL and IL-1β treatment is inhibited when c-Cbl is overexpressed, while the NF-κB activity induced by TNFα treatment is not. c-Cbl inhibits NF-κB activity mediated by TRAF6, but not by TRAF2. These findings show that c-Cbl ubiquitin ligase activity is essential for TRAF6 ubiquitination and negative regulation of NF-κB activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-γ (IFN-γ) caused c-Cbl to bind to polyubiquitinated TRAF6.
Conclusions: These findings indicate that the interaction of TRAF6 with c-Cbl causes lysine 48-linked polyubiquitination for both negative feedback regulation and signaling cross-talk between RANKL and IFN-γ.
Keywords Tumor necrosis factor receptor-associated factor 6, Ubiquitin, E3 ligase, C-Cbl
Address and Contact Information 1 National Leading Laboratory for Stem Cell Research, Seoul National University College of Medicine, Seoul, South Korea.
2 Korea Research-Driven Hospital, Biomedical Research Institute, Seoul National University Hospital, Seoul, South Korea.
3 Strategic Center of Cell & Bio Therapy, Seoul National University Hospital, Seoul, South Korea.
4 Department of Biotechnology, The Catholic University of Korea, Bucheon, South Korea.
5 Cardiovascular Center & Department of Internal Medicine, Seoul National University Hospital, Seoul, South Korea.
6 Department of Life Science and the Research Center for Cellular Homeostasis, Ewha Woman’s University, Seoul, South Korea
* Corresponding author: leesy@ewha.ac.kr
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DOI: 10.1186/s11658-019-0153-1 Volume 24 (2019)
Authors HB Jin, L Lu, L Xie, JF Yang, XF Zhang and SL Ma*
Abstract Background: Gemcitabine (2′,2′-difluoro-2′-deoxycytidine;dFdC) is a first-line chemotherapy drug for pancreatic cancer. Recently, a synergistic anti-tumor treatment of dFdC and hyperthermia has achieved good clinical results, but there are few reports on the molecular mechanism influenced by hyperthermia. This study is an initial exploration of the effects of hyperthermia on changes in the concentration of dFdC and its metabolites in pancreatic cancer cells. The aim is to provide a theoretical basis for clinical detection and pharmacokinetic research.
Methods: PANC-1 cells at logarithmic growth phase were used as the experimental object. The MTT assay was performed to determine the half maximal inhibitory concentration (IC50) of dFdC. After PANC-1 cells were cultured in DMEM medium containing IC50dFdC and treated with hyperthermia at 41 °C or 43 °C, changes in the concentration of dFdC, 2′,2′-difluorodeoxyuridine (dFdU) and difluorodeoxycytidine triphosphate (dFdCTP) in the cells were tested using an optimized reverse phase high-performance liquid chromatography (RP-HPLC) protocol.
Results: We found that 41°C and 43°C hyperthermia gave rise to a decrease in dFdC and dFdU content. At 41°C, the levels respectively fell to 9.28 and 30.93% of the baseline, and at 43°C, to 24.76 and 57.80%, respectively. The dFdCTP content increased by 21.82% at 41°C and 42.42% at 43°C.
Conclusion: The two heat treatments could alter the mechanism of dFdC metabolism in PANC-1 cells. The effect of 43 °C hyperthermia is more significant. Our observations may be instrumental to explaining the higher anti-tumor efficacy of this combination therapy.
Keywords Pancreatic cancer, Gemcitabine, Metabolite, Hyperthermia, RP-HPLC
Address and Contact Information Department of Gastroenterology, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China
* Corresponding author: shenglinma2018@163.com
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DOI: 10.1186/s11658-019-0155-z Volume 24 (2019)
Authors Yuxiang Fu, Liewen Lin and Ligang Xia*
Abstract Background: While microRNAs (miRNAs) are known to play a critical role in the progression of colorectal cancer, the role of miR-107 remains unknown. We evaluated its role and explored the underlying mechanism.
Materials & methods: MTT, wound-healing, transwell migration and transwell invasion assays were performed to evaluate the role of miR-107 in SW629 cell proliferation, migration and invasion. Real time-PCR and dual-luciferase reporter gene, TFR1 overexpression and western blotting assays were used to explore the underlying mechanism.
Results: MiR-107 is downregulated in colorectal cancer tissues and several human colorectal cancer cell lines. Low miR-107 expression often indicates a poor survival rate for colorectal cancer patients. MiR-107 suppresses the proliferation, migration and invasion of SW620 cells by negatively regulating transferrin receptor 1 (TFR1).
Conclusion: MiR-107 suppresses the metastasis of colorectal cancer and could be a potential therapy target in colorectal cancer patients.
Keywords Colorectal cancer, microRNA107 (miR-107), Cancer progress, Transferrin receptor 1 (TFR1)
Address and Contact Information Department of Gastrointestinal Surgery, Shenzhen People’s Hospital, The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of South University of Science and Technology, Shenzhen 518055, China
* Corresponding author: doctorw2016@163.com
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DOI: 10.1186/s11658-019-0157-x Volume 24 (2019)
Authors Jun-Li Zhao1*, Mei-Zi Guo2, Jun-Jun Zhu1, Ting Zhang1 and Dan-Yan Min1
Abstract Objective: Peritoneal fibrosis remains a serious complication of long-term peritoneal dialysis (PD) leading to peritoneal membrane ultrafiltration failure. Epithelial–mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Curcumin has been previously shown to inhibit EMT of renal tubular epithelial cells and prevent renal fibrosis. There are only limited reports on inhibition of PMCs-EMT by curcumin. This study aimed to investigate the effect of curcumin on the regulation of EMT and related pathway in PMCs treated with glucose-based PD.
Methods: EMT of human peritoneal mesothelial cells (HMrSV5) was induced with glucose-based peritoneal dialysis solutions (PDS). Cells were divided into a control group, PDS group, and PDS group receiving varied concentrations of curcumin. Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability, and a transwell migration assay was used to verify the capacity of curcumin to inhibit EMT in HMrSV5 cells. Real-time quantitative PCR and western blot were used to detect the expression of genes and proteins associated with the EMT.
Results: High glucose PDS decreased cell viability and increased migratory capacity. Curcumin reversed growth inhibition and migration capability of human peritoneal mesothelial cells (HPMCs). In HMrSV5 cells, high glucose PDS also decreased expression of epithelial markers, and increased expression of mesenchymal markers, a characteristic of EMT. Real-time RT-PCR and western blot revealed that, compared to the 4.25% Dianeal treated cells, curcumin treatment resulted in increased expression of E-cadherin (epithelial marker), and decreased expression of α-SMA (mesenchymal markers) (P < 0.05). Furthermore, curcumin reduced mRNA expression of two extracellular matrix protein, collagen I and fibronectin. Curcumin also reduced TGF-β1 mRNA and supernatant TGF-β1 protein content in the PDS-treated HMrSV5 cells (P < 0.05). Furthermore, it significantly reduced protein expression of p-TAK1, p-JNK and p-p38 in PDS-treated HMrSV5 cells.
Conclusions: Our results demonstrate that curcumin showed an obvious protective effect on PDS-induced EMT of HMrSV5 cells and suggest implication of the TAK1, p38 and JNK pathway in mediating the effects of curcumin in EMT of MCs.
Keywords Peritoneal fibrosis (PF), Peritoneal dialysis, Epithelial-mesenchymal transition (EMT), Curcumin, TGF-β-activated kinase 1 (TAK1)
Address and Contact Information 1 Department of Nephrology, Shanghai University of Medicine & Health Sciences affiliated Zhoupu Hospital, Pudong New District, Shanghai 201318, China
2 Department of Geriatrics, Shanghai University of Medicine & Health Sciences affiliated Zhoupu Hospital, Pudong New District, Shanghai 201318, China.
* Corresponding author: zhaojunli1203@126.com
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DOI: 10.1186/s11658-019-0159-8 Volume 24 (2019)
Authors Hsuan-Yeh Pan1, Abdulla H. Alamri2 and Mallika Valapala1*
Abstract Background: Induction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB.
Methods: Expression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes.
Results: Our results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes.
Conclusions: Collectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.
Keywords Nutrient deprivation, Lysosomal stress, Autophagy
Address and Contact Information 1 School of Optometry, Indiana University, Bloomington, IN 47405, USA
2 State University of New York College of Optometry, 33 42nd St., New York, NY 10036, USA.
* Corresponding author: mvalapal@iu.edu
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DOI: 10.1186/s11658-019-0161-1 Volume 24 (2019)
Authors Guojie Lu and Yaosen Zhang*
Abstract Background: MicroRNAs (miRNAs) have been reported to play crucial roles in cancer cell processes, including proliferation, metastasis and cell cycle progression. We aimed to identify miRNAs that could act as suppressors of cell growth and invasion in non-small cell lung cancer (NSCLC).
Methods: Fifteen paired NSCLC tissue samples and pericarcinomatous normal tissues were collected and preserved in liquid nitrogen. The expression levels of miR-340-5p and ZNF503 mRNA were detected using a qPCR assay. The transfection of plasmids was conducted using Lipofectamine 3000 according to the manufacturer’s protocol. Cell proliferation was determined using a CCK-8 assay. The protein levels of endothelial–mesenchymal transition markers were measured using a western blot assay. Cell invasive ability was evaluated using a transwell assay. TargetScan was used to predict targets of miR-340. A dual luciferase reporter assay was performed to confirm a potential direct interaction between miR-340-5p and ZNF503.
Results: The expression level of miR-340-5p was frequently found to be lower in NSCLC tissues than in matched pericarcinomatous normal tissues. Overexpression of miR-340-5p significantly inhibited the proliferation and invasion NCI-H1650 (a NSCLC cell line), while inhibition of miR-340-5p stimulated cell growth. Using TargetScan, we predicted that ZNF503 could be a target of miR-340-5p. Further mechanistic studies demonstrated that the forced expression of ZNF503 could partially abrogate the miR-340-5p-mediated decrease in NCI-H1650 cell viability and invasion, suggesting that miR-340-5p suppressed cell growth and invasion in a ZNF503-dependent manner.
Conclusion: Our findings indicate that miR-340-5p inhibits NCI-H1650 cell proliferation and invasion by directly targeting ZNF503 and that miR-340-5p can serve as a potential therapeutic target for treating NSCLC.
Keywords Cell proliferation, Metastasis, miR-340-5p, NSCLC, ZNF503
Address and Contact Information Department of Thoracic Surgery, Guangzhou Panyu District Central Hospital, No. 8, Fuyu East Road, Qiaonan Street, Panyu District, Guangzhou 511486, People’s Republic of China
* Corresponding author: 13632332051@163.com
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DOI: 10.1186/s11658-019-0154-0 Volume 24 (2019)
Authors Jing He, Di Qi, Xu-mao Tang, Wang Deng, Xin-yu Deng, Yan Zhao* and Dao-xin Wang*
Abstract Background: Pulmonary edema is one of the pathological characteristics of acute respiratory distress syndrome (ARDS). The epithelial sodium channel (ENaC) is thought to be the rate-limiting factor for alveolar fluid clearance (AFC) during pulmonary edema. The peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone was shown to stimulate ENaC-mediated salt absorption in the kidney. However, its role in the lung remains unclear. Here, we investigated the role of the PPARγ agonist in the lung to find out whether it can regulate AFC during acute lung injury (ALI). We also attempted to elucidate the mechanism for this.
Methods: Our ALI model was established through intratracheal instillation of lipopolysaccharide (LPS) in C57BL/6 J mice. The mice were randomly divided into 4 groups of 10. The control group underwent a sham operation and received an equal quantity of saline. The three experimental groups underwent intratracheal instillation of 5 mg/kg LPS, followed by intraperitoneal injection of 4 mg/kg rosiglitazone, 4 mg/kg rosiglitazone plus 1 mg/kg GW9662, or only equal quantity of saline. The histological morphology of the lung, the levels of TNF-α and IL-1β in the bronchoalveolar lavage fluid (BALF), the level of AFC, and the expressions of αENaC and serum and glucocorticoid-induced kinase-1 (SGK1) were determined. Type 2 alveolar (AT II) cells were incubated with rosiglitazone (15 μM) with or without GW9662 (10 μM). The expressions of αENaC and SGK1 were determined 24 h later.
Results: A mouse model of ALI was successfully established. Rosiglitazone significantly ameliorated the lung injury, decreasing the TNF-α and IL-1β levels in the BALF, enhancing AFC, and promoting the expressions of αENaC and SGK1 in ALI mice, which were abolished by the specific PPARγ blocker GW9662. In vitro, rosiglitazone increased the expressions of αENaC and SGK1. This increase was prevented by GW9662.
Conclusions: Rosiglitazone ameliorated the lung injury and promoted ENaC-mediated AFC via a PPARγ/SGK1-dependent signaling pathway, alleviating pulmonary edema in a mouse model of ALI.
Keywords Acute lung injury, Alveolar epithelial sodium channel, Alveolar fluid clearance, Peroxisome proliferator-activated receptor γ, Serum and glucocorticoid induced kinase-1
Address and Contact Information Department of Respiratory Medicine, The Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong District, Chongqing 400010, China
* Corresponding author: 514342948@qq.com; submitmail@126.com
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DOI: 10.1186/s11658-019-0158-9 Volume 24 (2019)
Authors Zhihua Ren1†, Hongyi He1†, Zhicai Zuo1†, Zhiwen Xu1, Zhanyong Wei2* and Junliang Deng1*
Abstract Common environmental pollutants and drugs encountered in everyday life can cause toxic damage to the body through oxidative stress, inflammatory stimulation, induction of apoptosis, and inhibition of energy metabolism. Silent information regulator 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent deacetylase, is a member of the evolutionarily highly conserved Sir2 (silent information regulator 2) superprotein family, which is located in the nucleus and cytoplasm. It can deacetylate protein substrates in various signal transduction pathways to regulate gene expression, cell apoptosis and senescence, participate in the process of neuroprotection, energy metabolism, inflammation and the oxidative stress response in living organisms, and plays an important role in toxic damage caused by toxicants and in the process of SIRT1 activator/inhibitor antagonized toxic damage. This review summarizes the role that SIRT1 plays in toxic damage caused by toxicants via its interactions with protein substrates in certain signaling pathways.
Keywords SIRT1, Toxicant, SIRT1 activator, Signaling pathway
Address and Contact Information 1 Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China
2 The College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, Henan Province, China
* Corresponding author: weizhanyong@henau.edu.cn; dengjl213@126.com
Zhihua Ren, Hongyi He and Zhicai Zuo contributed equally to this work.
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DOI: 10.1186/s11658-019-0160-2 Volume 24 (2019)
Authors Xiao-Li Ge1, Jin-Li Wang1, Xin Liu2, Jia Zhang3, Chang Liu4 and Li Guo1*
Abstract Background: Accumulating evidence has shown that altered microRNA (miR) modulation is implicated in the pathologies of ischemic stroke. However, it is unclear whether and how hsa-miR-19a-3p mediates cerebral ischemic injury. Herein, we investigated the functional role of miR-19a-3p in cerebral ischemic injury and explored its underlying regulatory mechanism.
Methods: In vivo ischemic/reperfusion (I/R) neuronal injury and in vitro oxygen-glucose deprivation (OGD) were established. Expression of miR-19a-3p was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Glucose uptake, lactate production, and apoptosis were determined. ADIPOR2 was predicted as a target of miR-19a-3p in silico and experimentally validated by qRT-PCR, Western blot analysis and luciferase assay assays.
Results: MiR-19a expression was significantly downregulated and upregulated in rat neurons and astrocytes, respectively (P < 0.01). A significantly elevated level of miR-19a-3p was found in I/R and OGD models in comparison to sham/control groups (P < 0.01). Expression of the glycolysis enzyme markers LDHA, PKM2, HK2, Glut1 and PDK1, apoptosis-related factors levels, apoptosis, glucose uptake, and lactate production were significantly repressed by both I/R and OGD (P < 0.01 in each case). Moreover, miR-19a-3p mimic aggravated, while miR-19a-3p inhibitor alleviated, the above observations. Adipor2 was predicted and confirmed to be a direct target of miR-19a. Furthermore, restoration of Adipor2 reversed miR-19a-3p-induced effects.
Conclusions: Collectively, our results indicate that elevated miR-19a-3p mediates cerebral ischemic injury by targeting ADIPOR2. MiR-19a-3p attenuation thus might offer hope of a novel therapeutic target for ischemic stroke injury treatment.
Keywords Ischemic stroke, miR-19a-3p, ADIPOR2, Glucose metabolism, Apoptosis
Address and Contact Information 1 Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, China
2 Department of Neurosurgery, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, China.
3 Department of Obstetrics, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, China.
4 Department of Rehabilitation, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, China.
* Corresponding author: guoli_med@126.com
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DOI: 10.1186/s11658-019-0162-0 Volume 24 (2019)
Authors Yulong Bao1,3†, Li Wang1†, Lin Shi2, Fen Yun2, Xia Liu2, Yongxia Chen3, Chen Chen2, Yanni Ren2 and Yongfeng Jia1,3*
Abstract Background: Exploration of the genes with abnormal expression during the development of breast cancer is essential to provide a deeper understanding of the mechanisms involved. Transcriptome sequencing and bioinformatics analysis of invasive ductal carcinoma and paracancerous tissues from the same patient were performed to identify the key genes and signaling pathways related to breast cancer development.
Methods: Samples of breast tumor tissue and paracancerous breast tissue were obtained from 6 patients. Sequencing used the Illumina HiSeq platform. All. Only perfectly matched clean reads were mapped to the reference genome database, further analyzed and annotated based on the reference genome information. Differentially expressed genes (DEGs) were identified using the DESeq R package (1.10.1) and DEGSeq R package (1.12.0). Using KOBAS software to execute the KEGG bioinformatics analyses, enriched signaling pathways of DEGs involved in the occurrence of breast cancer were determined. Subsequently, quantitative real time PCR was used to verify the accuracy of the expression profile of key DEGs from the RNA-seq result and to explore the expression patterns of novel cancer-related genes on 8 different clinical individuals.
Results: The transcriptomic sequencing results showed 937 DEGs, including 487 upregulated and 450 downregulated genes in the breast cancer specimens. Further quantitative gene expression analysis was performed and captured 252 DEGs (201 downregulated and 51 upregulated) that showed the same differential expression pattern in all libraries. Finally, 6 upregulated DEGs (CST2, DRP2, CLEC5A, SCD, KIAA1211, DTL) and 6 downregulated DEGs (STAC2, BTNL9, CA4, CD300LG, GPIHBP1 and PIGR), were confirmed in a quantitative real time PCR comparison of breast cancer and paracancerous breast tissues from 8 clinical specimens. KEGG analysis revealed various pathway changes, including 20 upregulated and 21 downregulated gene enrichment pathways. The extracellular matrix–receptor (ECM-receptor) interaction pathway was the most enriched pathway: all genes in this pathway were DEGs, including the THBS family, collagen and fibronectin. These DEGs and the ECM-receptor interaction pathway may perform important roles in breast cancer.
Conclusion: Several potential breast cancer-related genes and pathways were captured, including 7 novel upregulated genes and 76 novel downregulated genes that were not found in other studies. These genes are related to cell proliferation, movement and adhesion. They may be important for research into breast cancer mechanisms, particularly CST2 and CA4. A key signaling pathway, the ECM-receptor interaction signal pathway, was also identified as possibly involved in the development of breast cancer.
Keywords Breast cancer, Transcriptome, Differentially expressed genes, ECM-receptor interaction pathway
Address and Contact Information 1 College of Basic Medicine, Inner Mongolia Medical University, Hohhot, Inner Mongolia, China
2 Department of Pathology, Inner Mongolia Medical University, Hohhot, Inner Mongolia, China.
3 Tumor Molecular Diagnostic Laboratory, The Inner Mongolia Cancer Hospital, Hohhot, Inner Mongolia, China
* Corresponding author: jiayf0471@163.com
Yulong Bao and Li Wang contributed equally to this work.
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DOI: 10.1186/s11658-019-0163-z Volume 24 (2019)
Authors Jinghua Ou1,2, Defeng Guan1 and Yongxiu Yang1,3*
Abstract Background: The aim of this study was to investigate the effect of human umbilical vein endothelial cells on epithelial-to-mesenchymal transition of the cervical cancer cell line SiHa by studying the Notch1/lysyl oxidase (LOX)/SNAIL1 pathway.
Methods: Monocultures of SiHa cells, SiHa cells containing a control sequence, and Notch1-silenced SiHa cells, as well as co-cultures of human umbilical vein endothelial cells with SiHa cells and Notch1-silenced SiHa cells, were established. The invasiveness of SiHa cells in each group was evaluated using a Transwell assay. The mRNA levels of E-cadherin and vimentin were detected using quantitative real-time polymerase chain reaction. The expression levels of the matrix metalloproteinases MMP-2 and MMP-9 were determined in SiHa cells using an immunofluorescence assay and the protein activity was detected by gelatin zymography. Changes in LOX, SNAIL1 and NOTCH1 expression in the SiHa cells in each group were detected using western blotting.
Results: Compared with monocultured SiHa cells, co-cultured SiHa cells showed a significant increase in their invasiveness and expression levels of vimentin, as well as of NOTCH 1, LOX, and SNAIL1, whereas their expression of E-cadherin was significantly reduced and protein activities of MMP-2 and MMP-9 were increased. Compared with SiHa, mono- and co-cultured NOTCH 1-silenced SiHa cells showed significant reductions in their invasiveness and expression levels of vimentin, NOTCH 1, LOX, and SNAIL1, whereas their expression of E-cadherin significantly increased and protein activities of MMP-2 and MMP-9 decreased.
Conclusion: Co-culture with human umbilical vein endothelial cells promoted the epithelial-to-mesenchymal transition of SiHa cells by activating the NOTCH1/LOX/SNAIL1 pathway in SiHa cells, which enhanced their invasive and metastatic capacities. The results of this study may provide a new perspective on cervical cancer metastasis and a theoretical basis for clinical treatment.
Keywords Co-culture, Cervical cancer, SiHa cells, HUVECs, NOTCH/LOX/SNAIL pathway, EMT
Address and Contact Information 1 The First Clinical Medical College of Lanzhou University, NO 1 West Donggang Road, Chengguan District, Lanzhou, Gansu, China.
2 Department of obstetrics and gynecology, Gansu provincial hospital, Lanzhou, Gansu, China.
3 Department of Obstetrics and Gynecology, The First Hospital of Lanzhou University, NO 1 West Donggang Road, Chengguan District, Lanzhou, Gansu, China.
* Corresponding author: yyxfhlz@163.com
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DOI: 10.1186/s11658-019-0164-y Volume 24 (2019)
Authors Linyan Zhu and Liqun Chen*
Abstract Paclitaxel is a well-known anticancer agent with a unique mechanism of action. It is considered to be one of the most successful natural anticancer drugs available. This study summarizes the recent advances in our understanding of the sources, the anticancer mechanism, and the biosynthetic pathway of paclitaxel. With the advancement of biotechnology, improvements in endophytic fungal strains, and the use of recombination techniques and microbial fermentation engineering, the yield of extracted paclitaxel has increased significantly. Recently, paclitaxel has been found to play a large role in tumor immunity, and it has a great potential for use in many cancer treatments.
Keywords Paclitaxel, Anticancer mechanism, Endophytic fungus, Biosynthetic pathway
Address and Contact Information College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China
* Corresponding author: lqchen@fzu.edu.cn
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DOI: 10.1186/s11658-019-0165-x Volume 24 (2019)
Authors Haihong Deng1, Wenbo Ouyang1, Li Zhang3, Xiaoshan Xiao4, Zhiyong Huang3* and Wendian Zhu2*
Abstract Background: TGF-β1 contributes to chronic heart failure. It is known that lncRNA GASL1 can inactivate TGF-β1 in cancer biology.
Methods: All the participants were enrolled in the First People’s Hospital of Zhaoqing during the period June 2012 to June 2013. ELISA, RT-qPCR, vectors, transient transfections and western blot were carried out during the research.
Results: We found that plasma levels of TGF-β1 were significantly higher, while levels of GASL1 in plasma were significantly lower in chronic heart failure (CHF) patients compared to the control group. TGF-β1 and GASL1 were inversely correlated in CHF patients. Low pretreatment plasma levels of GASL1 were closely associated with poor survival of CHF patients. GASL1 expression was not significantly affected by TGF-β1 overexpression in cardiomyocytes, while cardiomyocytes with GASL1 overexpression showed downregulated TGF-β1. Overexpression of GASL1 led to a decreased, while TGF-β1 overexpression led to an increased apoptotic rate of cardiomyocytes under H2O2 treatment. In addition, TGF-β1 overexpression attenuated the effect of GASL1 overexpression.
Conclusion: In conclusion, GASL1 was downregulated in CHF. GASL1 overexpression may improve CHF by inhibiting cardiomyocyte apoptosis through the inactivation of TGF-β1.
Keywords Chronic heart failure, lncRNA GASL1, TGF-β1, Apoptosis
Address and Contact Information 1Department of Anesthesiology, The First People’s Hospital of Zhaoqing, Zhaoqing City, Guangdong Province 526000, People’s Republic of China.
2Department of General Surgery, The First People’s Hospital of Zhaoqing, No. 9 Donggang East Road, Duanzhou District, Zhaoqing City, Guangdong Province 526000, People’s Republic of China 3Department of Anesthesiology, Fuwai Hospital Chinese Academy of Medical Sciences, No. 12 Langshan Road, Shenzhen City 518057, People’s Republic of China
* Corresponding author: huzhgy@gmail.com; jl59911@163.com
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DOI: 10.1186/s11658-019-0169-6 Volume 24 (2019)
Authors Sichuang Tan2, Shuai Zhao2,6, Xuefei Xiao5, Lan Xiao4, Jinliang Xie3 and Sipin Tan1*
Abstract Human bronchial epithelium (HBE)-Dp71 anti-sense(AS)cells with stably transfected Dp71 siRNA plasmids were prepared for further exploration of Dp71 biological traits in cells other than PC12. HBE-Dp71AS cells displayed increased DNA damage induced by H2O2. Apoptosis of HBE-Dp71AS cells induced by H2O2 was increased via enhancing caspase 3, caspase 8 and caspase 9. HBE-Dp71AS cells also displayed decreased proliferation and clonogenic formation. RAD51 was proved to be a new binding partner of Dp71 by co-immunoprecipitation (Ip) and immunofluorescence. Reduced RAD51 mRNA and protein levels were observed in HBE-Dp71AS cells. Decreased lamin B1, focal adhesion kinase (FAK), phosphorylated focal adhesion kinase (p-FAK) and phosphorylated protein kinase B (p-AKT) were detected in the HBE-Dp71AS cells, which functioned together with RAD51 as the molecular explanations for the character alterations of HBE-Dp71AS cells.
Keywords Dp71, DNA damage, Apoptosis, RAD51, FAK
Address and Contact Information 1 Key Laboratory of Sepsis Translational Medicine of Hunan, Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan Province 410008, People’s Republic of China
2 Department of Thoracic Surgery, the Second Xiangya Hospital, Central South University, 139 Ren-min Road, Changsha, Hunan Province 410011, People’s Republic of China. 3 Center of Transplant Surgery, Xiangya Hospital, Central South University, Changsha, Hunan Province 410008, People’s Republic of China.
4 Department of Traditional Chinese Medicine, the Third Xiangya Hospital, Central South University, Changsha, Hunan Province, People’s Republic of China.
5 Department of Emergency and Critical Care Medicine, the Third Xiangya Hospital, Central South University, Changsha, Hunan Province, People’s Republic of China.
6 Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, People’s Republic of China.
* Corresponding author: springtan@csu.edu.cn
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DOI: 10.1186/s11658-019-0167-8 Volume 24 (2019)
Authors Ronggui Lin1†, Ming Li2†, Meiqin Luo3, Tianhong Teng1, Yu Pan1 and Heguang Huang1*
Abstract Background: Impairment of the blood–brain barrier (BBB) could result in secondary cerebral edema and life-threatening pancreatic encephalopathy in patients with severe acute pancreatitis (SAP). Mesenchymal stem cells (MSCs) have been widely adopted in clinical research because of their pleiotropic functions. The aim of this study was to investigate the impact of MSCs on BBB permeability in SAP and the potential mechanisms driving these effects.
Methods: Sprague-Dawley rats were randomly assigned to the control, SAP and SAP+MSCs groups. Pancreatic impairment was assessed. The serum levels of amylase, TNF-α and IL-10, expression levels of claudin-5, Bax, Bcl-2 and MMP-9, and the BBB permeability were measured. Endothelial cell apoptosis was evaluated.
Results: SAP rats showed BBB impairment with increased permeability and secondary cerebral edema, which was confirmed using the Evans blue assay and the calculation of the brain dry/wet ratio. Treatment with MSCs decreased the serum levels of amylase and TNF-α, increased the serum levels of IL-10, attenuated the apoptosis of brain microvascular endothelial cells, upregulated claudin-5 expression and downregulated MMP-9 expression. This treatment attenuated the increased BBB permeability in SAP rats.
Conclusions: MSCs attenuated the impairment of the BBB and decreased its permeability, producing protective effects in SAP rats.
Keywords Severe acute pancreatitis, Blood–brain barrier, Mesenchymal stem cell, Inflammatory response, Endothelial cell
Address and Contact Information 1 Department of General surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, Fujian 350001, People’s Republic of China
2 Department of Histology and Embryology, Hunan University of Medicine, Huaihua, Hunan, China.
3 Department of Orthopedics, Fujian Medical University Union Hospital, Fuzhou, Fujian, China.
* Corresponding author: Heguanghuang2@163.com
Ronggui Lin and Ming Li contributed equally to this work.
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DOI: https://cmbl.biomedcentral.com/track/pdf/10.1186/s11658-019-0171-z Volume 24 (2019)
Authors Mingxiao Liu, Xiangyu Han, Hongyun Liu, Danyang Chen, Yue Li and Wei Hu*
Abstract Background: Deer antler is the only mammalian organ that can be completely regenerated every year. Its periodic regeneration is regulated by multiple factors, including transforming growth factor β (TGF-β). This widely distributed multi-functional growth factor can control the proliferation and differentiation of many types of cell, and it may play a crucial regulatory role in antler regeneration. This study explored the role of TGF-β1 during the rapid growth of sika deer antler.
Methods: Three CRISPR-Cas9 knockout vectors targeting the TGF-β1 gene of sika deer were constructed and packaged with a lentiviral system. The expression level of TGF-β1 protein in the knockout cell line was determined using western blot, the proliferation and migration of cartilage cells in vitro were respectively determined using EdU and the cell scratch test, and the expression levels of TGF-β pathway-related genes were determined using a PCR array.
Results: Of the three gRNAs designed, pBOBI-gRNA2 had the best knockout effect. Knockout of TGF-β1 gene inhibits the proliferation of cartilage cells and enhances their migration in vitro. TGF-β signaling pathway-related genes undergo significant changes, so we speculate that when the TGF-β pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role.
Conclusions: TGF-β1 is a newly identified regulatory factor of rapid growth in sika deer antler.
Keywords CRISPR-Cas9, Sika deer, Transforming growth factor-β1, Cartilage cells
Address and Contact Information College of Life Sciences, Jilin Agriculture University, Changchun 130118, Jilin Province, China
* Corresponding author: huweilab@126.com
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DOI: 10.1186/s11658-019-0170-0 Volume 24 (2019)
Authors Xuan Luo1, Yun Xia2, Xu-Dong Li3 and Jun-Yi Wang1*
Abstract Background: The study aimed to investigate the effect of oxidative stress on Prestin expression, and explore the transcription factors (TFs) that are involved in regulating the expression of Prestin in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells upon oxidative stress.
Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression level of Prestin. Reverse chromatin immunoprecipitation (reverse ChIP) assay was performed to identify proteins that could bind to the Prestin gene. Small interfering RNA (siRNA) and chromatin immunoprecipitation (ChIP) experiments were used to further verify the results. HEI-OC1 cells were incubated with four different concentrations of tert-butyl hydroperoxide (t-BHP) for 24 h or 48 h to construct the oxidative stress model.
Results: Oxidative stress induced Prestin increase at the mRNA level but with a concomitant decrease at the protein level. TF activating enhancer binding protein-2δ (AP-2δ) screened by reverse ChIP assay was demonstrated to bind to transcriptional start site 1441 of the Prestin promoter region and negatively regulate the expression of Prestin by siRNA and ChIP experiments. Furthermore, AP-2δ was down-regulated under oxidative stress.
Conclusions: In conclusion, oxidative stress inhibits the expression of Prestin protein, and the transcription mechanism is triggered to compensate for the loss of Prestin protein. AP-2δ is one of the important TFs that suppresses transcription of the Prestin gene, and AP-2δ suppression further boosted Prestin mRNA activation under oxidative stress.
Keywords Prestin, AP-2δ, Oxidative stress, Transcriptional regulation, HEI-OC1 cells
Address and Contact Information 1 Department of Labor Health and Environmental Hygiene, School of Public Health, Guangdong Pharmaceutical University, Guangzhou 510310, China
2 Department of Labor Health and Environmental Hygiene, School of Public Health, Guangdong Pharmaceutical University, Guangzhou 510310, China.
3 Key Laboratory, Occupational Disease Prevention and Control of Hospital of Guangdong Province, Guangzhou 510300, China.
* Corresponding author: wjy925@163.com
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DOI: 10.1186/s11658-019-0166-9 Volume 24 (2019)
Authors Lixun Chai and Gengpu Yang*
Abstract Background: MiR-216a-5p has been reported to be associated with several tumors, including prostate cancer and melanoma. However, its expression level and potential role in esophageal squamous cell carcinoma (ESCC) remain uncertain.
Results: Here, we found that miR-216a-5p expression was significantly down-regulated in clinical ESCC tissues and cells. Functional assays were performed to evaluate the biological effects of miR-216a-5p on cell proliferation and cell apoptosis by CCK-8 assay and flow cytometry in ESCC cell lines, EC9706 and TE-9. The results showed that miR-216a-5p overexpression repressed cell proliferation and induced cell apoptosis. Through bioinformatics prediction and luciferase reporter assay, we revealed that miR-216a-5p could directly target tectonic family member 1 (TCTN1). Moreover, TCTN1 was obviously suppressed by miR-216a-5p overexpression. In addition, TCTN1 expression was significantly increased and inversely correlated with the levels of miR-216a-5p in ESCC tissues. More importantly, down-regulation of TCTN1 imitated, while restoration of TCTN reversed the effects of miR-216a-5p on cell proliferation and apoptosis. At the molecular level, we further found that TCTN1 overexpression reversed the effects of miR-216a-5p transfection on the expression of PCNA, Bcl-2 and Bad.
Conclusions: Our results demonstrate that miR-216a-5p might serve as a tumor suppressor in ESCC cells through negatively regulating TCTN1 expression, indicating the possibility that miR-216a-5p and TCTN1 might be attractive targets for ESCC therapeutic intervention.
Keywords ESCC, miR-216a-5p, TCTN1, Proliferation, Apoptosis
Address and Contact Information Department of Thoracic Surgery, Shanxi Dayi Hospital, No. 99 Dragon City Street, Taiyuan 030012, Shanxi Province, China
* Corresponding author: yang_Gpu@163.com
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DOI: 10.1186/s11658-019-0168-7 Volume 24 (2019)
Authors Mei Liu, Can Gong, Renyuan Xu, Yu Chen and Xiaodong Wang*
Abstract Background: Chemotherapy based on paclitaxel (PTX) is the standard treatment for a range of cancers, including triple-negative breast cancer (TNBC), but the increasing development of resistance has reduced/has negatively impacted its clinical utility. A previous study demonstrated that miR-5195-3p could suppress lung cancer cell growth. This study was designed to investigate whether miR-5195-3p attenuates chemoresistance to PTX by regulating target genes in TNBC cells.
Methods: The study used both PTX-resistant tumor tissues and PTX-resistant TNBC cell lines. The expression of miR-5195-3p was determined using quantitative real-time PCR. Cell viability, cell cycle distribution and apoptosis were analyzed using CCK-8 and flow cytometry assays. The target genes of miR-5195-3p were predicted with bioinformatics analysis and confirmed using the luciferase reporter assay.
Results: MiR-5195-3p expression was lower in PTX-resistant tumor tissues and PTX-resistant TNBC cell lines. Upregulation of miR-5195-3p enhanced the sensitivity of PTX-resistant TNBC cells to PTX treatment. EIF4A2 was confirmed as a potential target of miR-5195-3p. EIF4A2 knockdown imitated the effects of miR-5195-3p on chemosensitivity, while restoration of EIF4A2 rescued them.
Conclusion: These data demonstrate that miR-5195-3p might be a potential therapeutic target to reverse chemoresistance in TNBC through its targeting of EIF4A2.
Keywords Triple-negative breast cancer, miR-5195-3p, EIF4A2, Chemosensitivity
Address and Contact Information Department of Breast Surgery, West China Hospital of Sichuan University, 37 Guoxue Lane, Wuhou District, Chengdu 610041, Sichuan Province, China
* Corresponding author: wang_xdong2018@126.com
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DOI: 10.1186/s11658-019-0173-x Volume 24 (2019)
Authors Yihua Pei1,2†, Qin Yao1,2†, Yingchao Li3, Xin Zhang4 and Bozhen Xie3*
Abstract Background: In recent years, microRNA-211 (miR211) has been considered as a tumor suppressor in multiple malignancies. However, the function of miR211 in human osteosarcoma has not been explored intensively so far. In this study, the relationship between miR211 and EZRIN was analyzed in human osteosarcoma.
Methods: The expression levels of miR211 and EZRIN were measured in both human osteosarcoma cells and tissues. The direct regulatory relationship between miR211 and EZRIN was evaluated using dual-luciferase assay. The effect of miR211 and EZRIN overexpression on cell proliferation, migration/invasion, and apoptosis was detected.
Results: The expression of miR211 was obviously lower in osteosarcoma tissues than paracancerous tissues. EZRIN was identified as the direct target of miR211, and up-regulation of miR211 increased the percentage of cell apoptosis, and suppressed cell proliferation as well as cell migration/invasion via directly regulating EZRIN.
Conclusions: Our study indicated that miR211 has an important role in the development and progress of osteosarcoma, and it might become a novel target in the diagnosis and treatment of human osteosarcoma.
Keywords miR211, EZRIN, Osteosarcoma, Cell viability, Cell apoptosis
Address and Contact Information 1 Central laboratory, ZhongShan Hospital XiaMen University, Xiamen 361004, China.
2 Fujian Provincial Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma (Xiamen University Affiliated ZhongShan Hospital), Xiamen 361004, China.
3 Department of Spine Surgery, ZhongShan Hospital XiaMen University, No. 201 Hubin South Road, Xiamen 361004, China.
4 Department of Rehabilitation, ZhongShan Hospital XiaMen University, Xiamen 361004, China.
* Corresponding author: yaoqin20180101@sina.com
Yihua Pei and Qin Yao contributed equally to this work.
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DOI: 10.1186/s11658-019-0172-y Volume 24 (2019)
Authors Chunnian Zhang*, Liqin Gu, Xiafang Li and Jianzhong Wang
Abstract Background: Synuclein-γ has been demonstrated to be highly expressed in various human cancers including cervical cancer, and has been shown to play a critical role in tumor aggressiveness. We aimed to investigate the role of Synuclein-γ in human cervical cancer in vitro and in vivo.
Method: Reverse transcription-quantitative polymerase chain reaction assay and Western blot assay were used to detect the mRNA and protein expression, respectively. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay were performed to measure the viabilities of cancer cells. Flow cytometry assay was used to detect the cell cycle and apoptosis. Moreover, an animal experiment was performed to evaluate the biological behavior of Synuclein-γ in vivo.
Results: In the current study, we found that Synuclein-γ was obviously over-expressed in cervical cancer tissues compared to the adjacent non-cancer tissues. Cervical cancer cells transfected with Synuclein-γ siRNA demonstrated significant inhibition of cancer proliferation (P < 0.01), cell cycle arrest at G0/G1 phase, and cell apoptosis (P < 0.05). Moreover, down-regulation of Synuclein-γ significantly inhibited cervical cancer growth in vivo. In addition, protein levels of AKT, c-Myc and Cyclin D1 were much lower in the Synuclein-γ siRNA-treated groups than that in the control group.
Conclusions: Synuclein-γ inhibition reduced cervical cancer tumor growth through the AKT pathway. This effect represented a therapeutic opportunity and provided a novel target for cervical cancer treatment.
Keywords Synuclein-γ, Knockdown, Cervical cancer, Growth, AKT
Address and Contact Information Department of Gynaecology, Ganzhou People’s Hospital of Jiangxi Province, No. 18, Meiguan Avenue, Ganzhou city 341000, Jiangxi Province, China
* Corresponding author: Chunnianzhang@163.com
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