丙戊酸钠下调SH-SY5Y细胞中Bcl-2表达的作用及机制分析

Sodium valprovate upregulates miR-34a to inhibit Bcl-2 expression in human neuroblastoma SH-SY5Y cells in vitro

目的探讨丙戊酸钠(sodium valproate,VPA)在神经细胞中下调Bcl-2表达的作用及可能机制。方法用不同浓度的VPA处理SH-SY5Y细胞,定量PCR检测Bcl-2 mRNA与miR-34a水平变化,Western blot检测Bcl-2蛋白水平变化,报告基因系统检测Bcl-2启动子活性变化,联合转录抑制剂处理细胞,分析Bcl-2 mRNA稳定性变化,联合miR-34a模拟物及抑制剂处理细胞,进一步分析miR-34a在VPA下调Bcl-2表达中的作用。Annexin V/PI双染结合流式分析细胞凋亡情况。结果 VPA可浓度依赖性下调SH-SY5Y细胞中Bcl-2的mRNA与蛋白水平(P<0.05)。VPA暴露不改变Bcl-2的启动子活性(P>0.05),但显著降低了其mRNA的稳定性(P<0.05)。同时,VPA可上调SH-SY5Y细胞中miR-34a的表达(P<0.05),联合使用miR-34a的抑制剂可逆转VPA对Bcl-2表达的下调作用,且单独的miR-34a模拟物也可以下调细胞中Bcl-2的表达。VPA暴露还可引起SH-SY5Y细胞的凋亡增加(P<0.05)。结论 VPA可通过上调SH-SY5Y细胞中miR-34a水平而抑制Bcl-2的表达。

AbstractObjectiveTo investigate the inhibitory effect of sodium valproate （VPA） exposure on Bcl-2 expression and explore the possible mechanism in human neuroblastoma SH-SY5Y cells in vitro. Methods SH-0SY5Y cells exposed to different concentrations of VPA were examined for expression levels of bcl-2 mRNA and miR34a using qRT-PCR and for Bcl-2 protein expression using Western blotting. The changes in the promoter activity of bcl-2 gene in VPA-treated cells were analyzed using a reporter gene assay, and the stability of bcl-2 mRNA was evaluated after transcription inhibition. A miR34a mimic and a miR-34a antagonist were used to analyze the role of miR-34a in VPA-induced Bcl-2 downregulation in the cells. The apoptosis of SHSY5Y cells following VPA exposures was assessed using flow cytometry with Annexin V and PI staining.Results exposure dosedependently downregulated the expression of Bcl-2 at both the mRNA and protein levels in SHSY5Y cells （P〈0.05）. VPA treatment significantly decreased the stability of bcl-2 mRNA （P〈0.05） without affecting its transcriptional activity （P〉0.05）, and obviously up-regulated the expression of miR-34a in SH-SY5Y cells （P〈0.05）. Treatment of the cells with the miR-34a mimic alone inhibited the expression of Bcl-2, and application of the miR-34a antagonist obviously reversed the inhibitory effect of VPA on Bcl-2 expression in SH-SY5Y cells. VPA exposure also significantly increased the apoptosis of SH-SY5Y cells （P〈0.05）. Conclusion VPA exposure down-regulates the expression of Bcl-2 possibly by up-regulating miR-34a level in SH-SY5Y cells. 