miR-143-5p对镉致肾细胞凋亡的调控作用及其机制

Regulatory effects and mechanisms of miR-143-5p on cadmium-induced apoptosis in LLC-PK1 cells

目的 :探讨mi R-143-5p对镉诱导LLC-PK1细胞凋亡的调控作用及其机制。方法 :通过基因芯片技术筛选出由镉引起的差异表达mi RNAs,并用实时定量PCR方法验证基因芯片结果的可靠性;应用Lipofectamine 2000瞬时转染mi R-143-5p的模拟物和抑制剂建立mi R-143-5p高表达和低表达的模型,并结合q RT-PCR技术验证转染效果;Hoechst 33258染色和Annexin V-PI双染法检测细胞凋亡;生物信息学分析结合实时定量PCR和Western blot验证mi R-143-5p靶基因的表达;Western blot分析mi R-143-5p对细胞凋亡相关通路的调控作用。结果:镉可增强LLC-PK1细胞的mi R-143-5p表达水平(P<0.01);转染mi R-143-5p模拟物或抑制剂后,与对照组(mi R-NC)比较,mi R-143-5p表达明显上调或下调(P<0.01);mi R-143-5p的过表达可促进LLC-PK1细胞凋亡(P<0.01);mi R-143-5p在AKT3的m RNA和蛋白水平上发挥靶向调控作用;mi R-143-5p过表达能抑制pAkt和p-Bad蛋白表达,促进caspase-9和caspase-3蛋白上调。结论 :mi R-143-5p可能通过作用于靶基因AKT3,并抑制Akt/Bad信号通路,促进镉诱导LLC-PK1细胞的凋亡。

Objective：To study the regulatory effects and mechanisms of miR-143-Sp on cadmium-induced apoptosis in LLC-PK1 cells, nethods：Microarray analysis was performed to detect dysregulated expression of miRNAs caused by cadmium. Reliability of microarray analysis was validated by quantative real-time PCR. Over-expression and low expression of miR-143-Sp were simulated by its mimic and inhibitor transient transfection with Lipofectamine 2000,and the effect was verified by qRT-PCR. Hoechst 33258 staining and AnnexinV-FITC/PI method were used to detect apoptosis. Target gene of miR-143-Sp was validated by bioinformatics analysis,real-time PCR and Western blot. Western blot was performed to analyze the regulatory effect of miR-143-Sp on apoptosis- related pathway. Results：The expression of miR-143-5p was up-regulated by cadmium（P 〈 0.01）. Compared with the negative control （miR-NC）group,the expression of miR-143-Sp was significantly up-regulated after miR-143-Sp mimic and down-regulated by inhibitor transfection（P 〈 0.01）. Over-expression of miR-143-Sp markedly increased LLC-PK1 cells apoptosis （P 〈 0.01）. The mRNA and protein levels of AKT3 were both targeted and regulated by miR-143-5p. Over-expression of miR-143-Sp reduced protein levels of p- Akt and p-Bad and increased expression of caspase-9 and caspase-3. Conclusion：MiR-143-Sp may promote cadmium-induced apoptosis via targeting AKT3 and inhibiting Akt/Bad signal pathway in LLC-PK1 cells.