miR-126-5p通过靶向 Peli2影响糖尿病肾病肾小管上皮细胞上皮 -间质转化发生

目的 探讨微小RNA-126-5p(miR-126-5p)通过靶向pellino E3泛素蛋白连接酶家族成员2(Peli2)影响糖尿病肾病肾小管上皮细胞上皮-间质转化(EMT)发生的分子机制.方法 本研究起止时间为2018年12月至2019年12月.体外培养人肾小管上皮细胞HK-2(美国ATCC),分别使用5 mmol/L、30 mmol/L的D-葡萄糖处理细胞48 h,分别记作对照组、模型组.采用实时荧光定量聚合酶链反应(qRT-PCR)与蛋白免疫印迹法(Western blot)分别检测miR-126-5p、Peli2的表达水平.利用脂质体转染法分别将miR-126-5p寡核苷酸模拟物(miR-126-5p mimics)及阴性对照mimic NC序列(miR-NC)、Peli2小分子干扰RNA(si-Peli2)及其阴性对照(si-NC)转染至HK-2细胞,使用30 mmol/L的D-葡萄糖处理细胞48 h.Western blot检测上皮型钙黏蛋白(E-Cadherin)、神经型钙黏蛋白(N-Cadherin)、波形蛋白(Vimentin)、锌指蛋白(Snail)表达量;双荧光素酶报告实验验证miR-126-5p与Peli2的靶向关系.结果 与对照组相比,模型组miR-126-5p[(1.00±0.10)比(0.32±0.03)]、E-Cadherin[(0.86±0.09)比(0.35±0.04)]的表达水平显著降低(P<0.05),Peli2[(0.42±0.04)比(1.05±0.11)]、N-Cadherin[(0.45±0.05)比(0.99±0.10)]、Vi?mentin[(0.32±0.03)比(0.92±0.09)]、Snail[(0.22±0.02)比(0.75±0.08)]的表达水平显著升高(P<0.05);转染miR-126-5p mimics,与转染miR-NC相比,E-Cadherin[(0.34±0.03)比(0.75±0.08)]的表达水平显著升高(P<0.05),N-Cadherin[(1.02±0.11)比(0.53±0.05)]、Vimentin[(0.95±0.10)比(0.48±0.05)]、Snail[(0.72±0.07)比(0.36±0.04)]的表达水平显著降低(P<0.05);转染si-Peli2后,与转染si-NC相比,E-Cadherin[(0.24±0.03)比(0.70±0.07)]的表达水平显著升高(P<0.05),N-Cadherin[(1.05±0.12)比(0.50±0.05)]、Vimentin[(0.96±0.10)比(0.38±0.04)]、Snail[(0.74±0.07)比(0.40±0.04)]的表达水平显著降低(P<0.05);miR-126-5p能够特异性结合Peli2,Peli2是miR-126-5p的靶基因;Peli2过表达后可明显逆转miR-126-5p过表达对高糖诱导的HK-2细胞EMT的影响.结论 miR-126-5p通过靶向Peli2抑制糖尿病肾病肾小管上皮细胞EMT发生.

miR-126-5p affects EMT occurrence in renal tubular epithelial cells of diabetic nephropathy by targeting Peli2

Objective To explore the molecular mechanism of miR-126-5p affecting EMT in renal tubular epithelial cells of diabetic nephropathy by targeting Peli2.Methods The start and end time of this research was from December 2018 to December 2019. Human renal tubular epithelial cells HK-2(ATCC) were cultured in vitro, and the cells were treated with D-glucose at 5 mmol / L and 30 mmol / L for 48 h, respectively. They were recorded as NC group and HG group, respectively. qRT-PCR and Western blot were used to detect the expression levels of miR-126-5p and Peli2, respectively. The liposome transfection method was used to transfect miR-126-5p mimics, miR-NC, si-Peli2, and si-NC to HK-2 cells, and the cells were treated with 30 mmol / L D-glucose for 48 h. Western blot was used to detect the expression of E-Cadherin, N-Cadherin, Vimentin, and Snail. The dual luciferase reporting experiment verified the targeting relationship between miR-126-5p and Peli2.Results Compared with the NC group, the expression levels of miR-126-5p [(1.00±0.10) vs. (0.32±0.03)] and E-Cadherin [(0.86±0.09) vs. (0.35±0.04)] in the Model group were significantly reduced (P<0.05), and the expression levels of Peli2 [(0.42±0.04) vs. (1.05±0.11)], N-Cadherin [(0.45±0.05) vs. (0.99±0.10)], Vimentin [(0.32±0.03) vs. (0.92±0.09)], and Snail [(0.22±0.02) vs. (0.75±0.08)] were significantly increased (P<0.05). After miR-126-5p mimics or si-Peli2 transfection, the expression level of E-Cadherin [(0.34±0.03) vs. (0.75±0.08)] was significantly higher than that of miR-NC or si-NC (P<0.05), the expression levels of N-Cadherin [(1.02±0.11) vs. (0.53±0.05)], Vimentin [(0.95±0.10) vs. (0.48±0.05)], Snail [(0.72±0.07) vs. (0.36±0.04)] were significantly reduced (P<0.05). After si-Peli2, the expression level of E-Cadherin [(0.24±0.03) vs. (0.70±0.07] was significantly higher than that of si-NC (P<0.05), the expression levels of N-Cadherin [(1.05±0.12) vs. (0.50±0.05)], Vimentin [(0.96±0.10) vs. (0.38±0.04)], Snail [(0.74± 0.07) vs. (0.40±0.04)] were significantly reduced (P<0.05). miR-126-5p could specifically bind to Peli2, and Peli2 was a target gene of miR-126-5p. Peli2 overexpression could significantly reverse the effect of miR-126-5p overexpression on EMT in HK-2 cells induced by high glucose.Conclusion miR-126-5p inhibits EMT in renal tubular epithelial cells of diabetic nephropathy by targeting Peli2.