目的：探讨Notch3在顺铂诱导的肾小管细胞损伤中的表达变化及其潜在相关的上游microRNA调控机制。 方法：体外培养人肾小管上皮细胞（HK-2细胞），分别转染miR-192-5p模拟物（miR-192-5p mimic）、阴性对照模拟物（NC mimic），并分为空白对照组与顺铂组（20μmol/L），处理24h后实时定量PCR法检测miR-192-5p及Notch3的mRNA表达水平；Western印迹分别检测细胞Notch3和凋亡相关蛋白cleaved-caspase3的蛋白表达水平；流式细胞术检测细胞凋亡率；构建含Notch3 3'端非编码区（3'-UTR）的双荧光素酶报告基因载体，采用双荧光素酶报告基因法检测靶基因Notch3的荧光活性。 结果：与空白对照组相比，顺铂组处理24h后HK-2细胞的miR-192-5p表达降低，Notch3 mRNA和蛋白表达升高，凋亡相关蛋白cleaved-caspase3 表达及细胞凋亡率升高（均P<0.05）；转染miR-192-5p mimic并予顺铂处理24h后，与NC mimic+顺铂组相比，miR-192-5p mimic+顺铂组靶基因Notch3 mRNA和蛋白表达水平均显著下调，cleaved-caspase3的蛋白表达水平及细胞凋亡率降低（均 P<0.05），差异均有统计学意义。双荧光素酶报告基因结果显示，与miR-192-5p+pmiR-Notch3 3’-UTR-Mut共转染组比较，miR-192-5p+pmiR-Notch3 3’-UTR-WT组荧光素酶活性显著降低，差异有统计学意义（P<0.05），证实了miR-192-5p与Notch3之间的直接作用关系。 结论：miR-192-5p可通过直接抑制Notch3的表达而减轻顺铂所致的肾小管上皮细胞凋亡。
Objective：To investigate the expression changes of Notch3 in cisplatin-induced renal tubular cell injury and the potential upstream regulation mechanism of its related microRNA. Methods：Human renal tubular epithelial cells (HK-2 cells) were cultured in vitro and transfected with miR-192-5p mimic (miR-192-5p mimic) and negative control mimic (NC mimic), respectively. Then, they were divided into blank control group and cisplatin treatment group (20μmol/L). After 24 hours, The mRNA expression levels of miR-192-5p and Notch3 were quantified by real-time quantitative PCR and the protein expression levels of Notch3 and apoptosis-related protein cleaved-caspase3 were detected by Western blotting. The apoptosis rate of cells was evaluated by flow cytometry. The reporter plasmids containing the 3'non-coding region (3'-UTR) of Notch3 was constructed and the luciferase activity of target gene Notch3 were detected by dual luciferase report assay. Results：After 24 hours of cisplatin treatment, compared with the blank control group, the expression of miR-192-5p was reduced, while the mRNA and protein expression of Notch3 increased, and the expression of apoptosis-related protein clear-caspase3 protein and apoptosis rate increased in HK-2 cells (all p <0.05). Then, compared with NC mimic group treated with cisplatin, overexpression of miR-192-5p markedly decreased the mRNA and protein expression of Notch3, which was predicted to be the target gene of miR-192-5p. Meanwhile, the protein expression of apoptosis-related protein cleaved-caspase3 and apoptosis rate was significantly lower than that of cisplatin group (all p<0.05). The dual luciferase reporter assay showed that miR-192-5p+pmiR-Notch3 3’-UTR-WT co-transfection group had lower Notch3 luciferase activity than miR-192-5p+pmiR-Notch3 3’-UTR-Mut group, demonstrating the direct interaction between miR-192-5p and Notch3 (P<0.05). Conclusions: miR-192-5p can attenuate the apoptosis of renal tubular epithelial cells induced by cisplatin through directly inhibiting the expression of Notch3.