Silencing of c-Ski augments TGF-beta 1-induced epithelial-mesenchymal transition in cardiomyocyte H9C2 cells

Ling, J; Cai, ZR; Jin, W; Zhuang, XH; Kan, LH; Wang, F; Ye, XL

Ye, XL (reprint author), Ningbo Univ, Ningbo Inst Med Sci, Dept Pharmacol, Ningbo 315020, Zhejiang, Peoples R China.

CARDIOLOGY JOURNAL, 2019; 26 (1): 66

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 beta 1 (TGF-beta 1)-induced epithelial-mesenchymal transitions (EMT) in H9C2 cells. Methods: Real-time polymerase chain reaction (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-beta 1 stimulation were detected by RT-qPCR and immunocytochemical analysis. In the presence or absence of TGF-beta 1 stimulation, the proteins' expression levels of alpha-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-b1 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-beta 1 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 alpha-SMA and/or FN expression stimulated by TGF-beta 1. However, shRNA-induced c-Ski knockdown aggravated only the TGF-beta 1-induced EMT. Moreover, c-Ski-shRNA also promoted the phosphorylation of Samd3 induced by TGF- beta 1. Conclusions: c-Ski expression in cardiac muscle cells could be down-regulated by TGF- beta 1. Silencing of c-Ski gene was accompanied by down-regulation of E-cadherin, up-regulation of alpha-SMA and/or FN and Smad3 phosphorylation induced by TGF- beta 1, promoting EMT process. Therefore, c-Ski may be closely associated with TGF-beta 1-induced EMT and play an important role in cardiac fibrosis development and progression.

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