Objective: To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells.
Methods: Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist.
Results: VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells.
Conclusions: VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.
目的: 探讨丙戊酸钠（VPA）对SH-SY5Y细胞中miR-34c-5p/ATG4B信号通路的激活作用及对自噬的影响。
方法: 人神经母细胞瘤细胞SH-SY5Y用含10%胎牛血清的DMEM培养基常规培养，然后使用不同剂量VPA处理SH-SY5Y细胞24 h，采用定量PCR分析VPA处理对ATG4B mRNA与miR-34c-5p表达的影响，免疫印迹法分析VPA处理对ATG4B蛋白表达的影响，将含ATG4B启动子片段的报告质粒转染SH-SY5Y细胞，然后通过荧光素酶报告基因系统分析VPA处理对ATG4B启动子活性的影响，用VPA单独或联合转录抑制剂放线菌素D分别处理SH-SY5Y细胞不同时间，定量PCR分析检测不同处理对ATG4B mRNA稳定性的影响，用VPA单独或联合miR-34c-5p抑制剂处理细胞24 h，通过检测不同处理对ATG4B表达变化的影响，进而判断miR-34c-5p在VPA下调ATG4B表达中的作用，同时通过Western blot检测不同处理对LC3-Ⅱ表达的影响，分析细胞自噬水平变化。
结果: VPA可剂量依赖性降低SH-SY5Y细胞中ATG4B的mRNA和蛋白水平（P < 0.05）。VPA处理不影响SHSY5Y细胞中ATG4B的启动子活性（P>0.05），但显著下调其mRNA的稳定性（P < 0.05）。同时，VPA处理明显增强SH-SY5Y细胞中miR-34c-5p的表达水平（P < 0.05）。使用miR-34c-5p抑制剂与VPA联合处理SH-SY5Y细胞，可逆转VPA对ATG4B表达的下调作用。VPA处理还下调了SH-SY5Y细胞中LC3-Ⅱ的表达水平（P < 0.05）。
结论: 结论VPA可能通过激活miR-34c-5p/ATG4B信号通路而抑制SH-SY5Y细胞自噬。
Keywords: ATG4B; SH-SY5Y cells; autophagy; miR-34c-5p; sodium valproate.