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Silencing of c-Ski augments TGF-β1-induced epithelial-mesenchymal transition in cardiomyocyte H9C2 cells.

Silencing of c-Ski augments TGF-β1-induced epithelial-mesenchymal transition in cardiomyocyte H9C2 cells.
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Ling J, Cai Z, Jin W, Zhuang X, Kan L, Wang F, Ye X,


Ling J, Cai Z, Jin W, Zhuang X, Kan L, Wang F, Ye X, (click to view)

Ling J, Cai Z, Jin W, Zhuang X, Kan L, Wang F, Ye X,

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Cardiology journal 2018 03 23() doi 10.5603/CJ.a2018.0009
Abstract
BACKGROUND
The shRNA lentiviral vector was constructed to silence c-Ski expression in cardiac muscle cells, with the aim of exploring the role of c-Ski in transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transitions (EMT) in H9C2 cells.

METHODS
RT-PCR and western blot were used to detect c-Ski expression at protein and messenger ribonucleic acid (mRNA) levels in 5 different cell lines. Then, lentiviral vector was constructed to silence or overexpress c-Ski in H9C2 cells. MTT and/or soft agar assay and transwell assay were used to detect cell proliferation and migration, respectively. The expression levels of c-Ski under different concentrations of TGF-β1 stimulation were detected by RT-qPCR and immunocytochemical analysis. In the presence or absence of TGF-β1 stimulation, the proteins’ expression levels of α-SMA, FN and E-cadherin, which are closely correlated with the process of EMT, were measured by western blot after c-Ski silencing or overexpression. Meanwhile, the effect of c-Ski on Samd3 phosphorylation with TGF-β1 stimulation was investigated.

RESULTS
There is a high expression of c-Ski at protein and mRNA levels in H9C2 cell line, which first demonstrated the presence of c-Ski expression in H9C2 cells. Overexpression of c-Ski significantly increased H9C2 cell proliferation. The ability of c-Ski gene silencing to suppress cell proliferation was gradually enhanced, and inhibition efficiency was the highest after 6 to 7 d of transfection. Moreover, H9C2 cells with c-Ski knockdown gained significantly aggressive invasive potential when compared with the control group. TGF-β1 stimulation could dose-independently reduce c-Ski expression in H9C2 cells and lead to obvious down-regulated expression of E-cadherin. Interestingly, c-Ski could restore E-cadherin expression while suppressing α-SMA and/or FN expression stimulated by TGF-β1. However, shRNA-induced c-Ski knockdown aggravated only the TGF-β1-induced EMT. Moreover, c-Ski-shRNA also promoted the phosphorylation of Samd3 induced by TGF-β1.

CONCLUSION
c-Ski expression in cardiac muscle cells could be down-regulated by TGF-β1. Silencing of c-Ski gene was accompanied by down-regulation of E-cadherin, up-regulation of α-SMA and/or FN and Smad3 phosphorylation induced by TGF-β1, promoting EMT process. Therefore, c-Ski may be closely associated with TGF-β1-induced EMT and play an important role in cardiac fibrosis development and progression.

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