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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。
AIM: To investigate the regulatory effect of mi R-143-5p on cadmium-induced LLC-PK1 cell apoptosis and its mechanism. Methods: MicroRNAs differentially expressed by cadmium were screened by gene chip technique and the reliability of gene chip results were verified by real-time quantitative PCR. MiR-143-5p mimics and inhibitors were transiently transfected into mice by Lipofectamine 2000 R-143-5p were detected by real-time PCR and q-RT-PCR was used to confirm the transfection efficiency. Hoechst 33258 staining and Annexin V-PI double staining were used to detect apoptosis. Bioinformatics analysis combined with real- The expression of mi R-143-5p target gene was verified by Western blot, and the effect of mi R-143-5p on the apoptosis-related pathway was analyzed by Western blot. Results: The expression of mi R-143-5p in LLC-PK1 cells was enhanced by cadmium (P <0.01). Compared with the control group (mi R-NC), mi R-143-5p mimics or inhibitors were transfected by cadmium, (P <0.01). The overexpression of mi R-143-5p could promote the apoptosis of LLC-PK1 cells (P <0.01). The expression of mi R-143-5p in AKT3 m RNA and protein levels. Mi R-143-5p overexpression can inhibit the expression of pAkt and p-Bad protein and up-regulate caspase-9 and caspase-3 protein. Conclusion: mi R-143-5p may promote cadmium-induced apoptosis in LLC-PK1 cells by acting on the target gene AKT3 and inhibiting the Akt / Bad signaling pathway.