过表达连接子蛋白40(Cx40)通过引起细胞周期阻滞抑制H9c2大鼠心肌细胞增殖

Overexpression of connexin 40(Cx40)inhibits the proliferation of H9c2 cardiomyocytes in rats by cell cycle arrest

目的 建立过表达连接子蛋白40(Cx40)的H9c2心肌细胞稳定株,初步探讨慢病毒载体介导的Cx40蛋白过表达对H9c2细胞增殖的影响及其相关机制。方法 采用实时定量PCR从H9c2细胞株扩增Cx40基因片段,与慢病毒载体质粒pLVX-IRES-Puro连接,获得重组pLVX-Flag-Cx40表达载体。通过与包装质粒共转染人胚肾HEK293T细胞,获得携带Flag-Cx40的重组慢病毒颗粒。感染的H9c2细胞经嘌呤霉素(puromycin)筛选出稳定表达株(H9c2-Flag-Cx40细胞)。采用Western blot法检测Cx40蛋白表达情况;通过CCK-8法检测Cx40对H9c2细胞增殖的影响;流式细胞术检测细胞周期的变化;实时定量PCR检测细胞周期蛋白D1(cyclin D1)的mRNA表达;Western blot法检测cyclin D1蛋白表达;免疫共沉淀(Co-IP)实验检测H9c2细胞Cx40和Yes相关蛋白(YAP)的相互结合情况;分离胞质、胞核蛋白,利用Western blot法检测Cx40对YAP细胞定位的影响。结果 测序结果显示成功构建重组pLVX-Flag-Cx40表达载体,Flag-Cx40重组慢病毒可在H9c2细胞过表达,H9c2-Flag-Cx40稳定细胞株构建成功。与对照组相比,过表达Cx40明显降低H9c2细胞的增殖速率,细胞周期阻滞在G0/G1期,cyclin D1表达量降低。H9c2-Flag-Cx40稳定转染细胞的胞质中YAP表达量明显增多,而在胞核中的表达量明显减低,Cx40通过在胞质中与YAP结合,阻止其进入细胞核发挥转录共激活作用。结论 慢病毒介导Cx40基因过表达使H9c2细胞的细胞周期阻滞在G0/G1期,并抑制其增殖。

Objective To establish a stable strain of H9c2 cardiomyocytes overexpressing Cx40 and preliminarily investigate the effect of lentiviral vector-mediated Cx40 protein overexpression on the proliferation of H9c2 cells and its related mechanisms.Methods The Cx40 gene fragment was cloned from H9c2 cells by PCR and linked with lentivirus vector pLVX-IRES-Puro to obtain the recombinant plasmid pLVX-Flag-Cx40.Recombinant lentiviral particles carrying Flag-Cx40 were obtained by cotransfection with packaging plasmids into HEK293T cells.A stable expression strain(H9c2-Flag-Cx40 cell)was screened from infected H9c2 cells by purinomycin.The expression of Cx40 protein was detected by Western blot analysis,and the effect of Cx40 on H9c2 cells proliferation was determined by CCK-8 assay;cell cycle changes were measured by flow cytometry;the expression of the cell cycle protein cyclin D1 was detected by qRT-PCR and Western blot analysis.Co-immunoprecipitation(Co-IP)immunoprecipitation and Western blot analysis were used to identify the binding of Cx40 and Yes associated protein(YAP)in H9c2 cells;cytoplasmic and cytosolic proteins were isolated to detect the effect of Cx40 on the localization of YAP using Western blot analysis.Results Sequencing results showed that the recombinant pLVX-Flag-Cx40 expression vector was successfully established.A stable transfected cell line containing recombinant Flag-Cx40 lentivirus(H9c2-Flag-Cx40 cell)was successfully constructed from H9c2 cells.Compared with the control group,overexpression of Cx40 significantly reduced the proliferation of H9c2 cells,arrested the cell cycle at G0/G1 and reduced cyclin D1 expression.A significant increase in YAP expression was observed in the cytoplasm of the H9c2-Flag-Cx40 stable cell line,while the expression in the nucleus was significantly reduced.Cx40 bound to YAP in the cytoplasm and prevented it from entering the nucleus to play the role of transcriptional coactivation.Conclusion Overexpression of Cx40 induces cell-cycle arrest at GO/G1 phase and inhibits the proliferation in H9c2 cells.