沉默C-SKI对大鼠心肌细胞H9C2细胞增殖与侵袭能力的影响研究

Downregulation of C-SKI enhances invasion of H9C2 cardiac muscle cells

目的通过慢病毒载体沉默心肌细胞细胞系H9C2中C-SKI(c-ski)基因的表达,探讨C-SKI基因沉默后对心肌细胞细胞增殖、凋亡及迁移的调控作用。方法应用RT-PCR及蛋白质印迹方法检测SW620,HT-29,LoVo,H9C2及RKO细胞系中C-SKI的表达状况;设计人C-SKI基因的shRNA慢病毒载体,在293T细胞中包装病毒并感染C-SKI高表达的心肌细胞细胞系,运用蛋白质免疫印迹的方法鉴定其对C-SKI基因的沉默效果;C-SKI基因沉默后通过MTT实验检测其对细胞增殖能力的影响,并应用Transwell迁移实验检测细胞的体外侵袭迁移能力。结果 C-SKI在H9C2中高表达,构建C-SKIshRNA慢病毒载体沉默H9C2细胞中C-SKI的表达,蛋白质免疫印迹结果提示,C-SKI稳定沉默的H9C2细胞构建成功。在稳定沉默C-SKI基因后,MTT法显示C-SKI沉默不会影响细胞的增殖能力。细胞小室实验结果显示,C-SKI沉默后H9C2侵袭和迁移能力均增强(P<0.001)。结论C-SKI基因能够一定程度上抑制心肌细胞的迁移和侵袭,进而参与了心肌细胞纤维化的发生发展。

Objective To explore the role that C-SKI plays in proliferation,apoptosis and migration of CRC cells,by shRNA lentiviral vector was constructed to silence C-SKI expression in cardiac muscle cells（CRC）cells.Methods RT-PCR and western-blot（WB）were used to detect C-SKI expression profile in 5 different CRC cell lines,in order to confirm the target cell line.In vitro,the lentiviral vector was constructed to silence C-SKI expression in H9C2 cells.Then,methyl thiazolyltetrazolium（MTT）and soft agar assay were used to detect cell proliferation.Additionally,cell apoptosis and cell migration after C-SKI knockdown were measured by the method of transwell assay,respectively.Results The expression of C-SKI in protein and mRNA level in different cell lines of colorectal cancer were detected,among which the H9C2 cells showed significant high C-SKI expression than that in the other four cell lines and also protein had significant difference.After stably silencing C-SKI in H9C2,MTT showed that there was no statistical significantly differences in cell proliferation in the C-SKI knockdown group.Moreover,H9C2 cells with CSKI silencing gained significant aggressive potential in migration than other tested cells.Conclusion C-SKI may be closely associated with CRC cell proliferation,apoptosis as well as migration,which indicate that it plays a pivotal role in CRC development and progression.