柯萨奇病毒B3调控CAPN总体活性促进H9c2细胞凋亡的机制

Study on the mechanism of Coxsackie virus B3 regulating the overall activity of CAPN and promoting H9c2 cell apoptosis

目的探讨柯萨奇病毒B3(CVB3)调控进H9c2细胞凋亡的潜在机制。方法 CVB3感染大鼠心肌细胞H9c2后,通过Annexin-PI染色和流式细胞仪检测H9c2的凋亡水平。抽取感染CVB3和未感染的H9c2细胞的总RNA后进行高通量测序。使用siRNA敲低测序结果中差异较大的基因,并检测H9c2细胞的凋亡水平。结果CVB3感染大鼠心肌细胞H9c2后,细胞的凋亡水平上升(P<0.05)。高通量测序发现感染CVB3和未感染的H9c2细胞表达差异大于5倍的基因有40个。siRNA敲低这些基因后,发现当敲低CAPN1、CAPN2、CAPN3、CAPN10时,H9c2细胞凋亡水平下降(P<0.05)。感染CVB3后,H9c2细胞中CAPN1、CAPN2、CAPN3、CAPN10的蛋白水平上升。感染CVB3后12 h、24 h、48 h H9c2细胞中总体Calpain活性上升(P<0.05)。感染CVB3的同时使用Calpain抑制剂Z-LLY-FMK处理,Calpain总体活性和H9c2细胞凋亡水平无显著变化(P>0.05)。结论 CVB3通过上调CAPN1、CAPN2、CAPN3、CAPN10的表达水平提高了Calpain的总体活性来诱导大鼠心肌细胞H9c2凋亡。

Objective To investigate the potential mechanism of Coxsackie virus B3(CVB3)regulating the apoptosis of H9c2 cells.Methods After CVB3 infects rat cardiomyocytes H9c2,the apoptosis level of H9c2 was detected by An-nexin-PI staining and flow cytometry.High-throughput sequencing was performed after extracting total RNA from infected CVB3 and uninfected H9c2 cells.Use siRNA to knock down genes with large differences in sequencing results and detect the level of apoptosis in H9c2 cells.Results After CVB3 infects rat cardiomyocytes H9c2,the apoptotic level of the cells increased(P<0.05).High-throughput sequencing found that the expression of infected CVB3 and uninfected H9c2 cells differed more than 40 genes.These genes were knocked down by siRNA.After gene,it was found that when CAPN 1,CAPN2,CAPN3,CAPN 10 were knocked down,the apoptosis level of H9c2 cells decreased(P<0.05).After infection with CVB3,the protein levels of CAPN1,CAPN2,CAPN3 and CAPN10 in H9c2 cells increased.After CVB3 infection,the overall Calpain activity in H9c2 cells increased at 12 h,24 h,and 48 h(P<0.05).When CVB3 was infected with the Calpain inhibitor Z-LLY-FMK,there was no significant change in the overall activity of Calpain(P>0.05),and the level of H9c2 cell apoptosis No significant change(P>0.05).Conclusion CVB3 increases the overall activity of Calpain by up-regulating the expression levels of CAPN 1,CAPN2,CAPN3,and CAPN 10 to induce the apoptosis of rat cardiomyocytes H9c2.