miR-143-5p对镉致肾细胞凋亡的调控作用及其机制

目的:探讨miR-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miRNAs,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miR-143-5p的模拟物和抑制剂建立miR-143-5p高表达和低表达的模型,并结合qRT-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miR-143-5p靶基因的表达;Western blot分析miR-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miR-143-5p表达水平(P < 0.01);转染miR-143-5p模拟物或抑制剂后,与对照组(miR-NC)比较,miR-143-5p表达明显上调或下调(P < 0.01);miR-143-5p的过表达可促进 LLC-PK1细胞凋亡 (P < 0.01);miR-143-5p在AKT3的mRNA和蛋白水平上发挥靶向调控作用;miR-143-5p过表达能抑制p-Akt和p-Bad蛋白表达,促进caspase-9和caspase-3蛋白上调?结论:miR-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?

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

Objective:To study the regulatory effects and mechanisms of miR-143-5p on cadmium-induced apoptosis in LLC-PK1 cells. Methods: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-143-5p 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-143-5p was validated by bioinformatics analysis,real-time PCR and Western blot. Western blot was performed to analyze the regulatory effect of miR-143-5p 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-143-5p was significantly up-regulated after miR-143-5p mimic and down-regulated by inhibitor transfection (P < 0.01). Over-expression of miR-143-5p 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-143-5p reduced protein levels of p-Akt and p-Bad and increased expression of caspase-9 and caspase-3. Conclusion:MiR-143-5p may promote cadmium-induced apoptosis via targeting AKT3 and inhibiting Akt/Bad signal pathway in LLC-PK1 cells.