METTL3/miR-126调控TGF-β1/smad2对高糖诱导的肾小管上皮细胞增殖的影响

Effects of METTL3/miR-126 modulation of TGF-β1/smad2 on hyperglycemia-induced renal tubular epithelial cell proliferation

目的:探讨类甲基化转移酶3(METTL3)/miR-126对高糖(HG)诱导的大鼠肾小管上皮细胞(NRK-52E)增殖的影响及其作用机制。方法:将NRK-52E细胞分为对照组(5.5 mmol/L葡萄糖)、HG组(35 mmol/L葡萄糖)、HG+siRNA-NC组和HG+METTL3 siRNA组。采用MTT法检测细胞增殖,流式细胞仪检测细胞周期,酶联免疫吸附试验(ELISA)法检测细胞上清液中转化生长因子(TGF)-β1蛋白含量,RT-qPCR法检测miR-126和METTL3基因表达,western blotting检测METTL3和smad2蛋白表达,RNA免疫共沉淀技术(RIP)检测METTL3与miR-126的结合情况。结果:与对照组比较,HG组细胞增殖率和G1期细胞所占百分比降低,G2期细胞所占百分比及METTL3、TGF-β1、smad2、miR-126表达升高(P<0.05);与HG组比较,HG+METTL3 siRNA组细胞增殖率升高,G1期细胞所占百分比降低,S期细胞所占百分比升高,METTL3、TGF-β1、smad2、miR-126表达降低(均P<0.05)。HG组与HG+siRNA-NC组各指标比较,差异均无统计学意义(P>0.05)。RIP实验结果显示,METTL3siRNA可降低成熟miR-126的含量(P<0.05)。结论:METTL3在HG诱导的肾小管上皮细胞中的表达上调,抑制细胞增殖,其可能与甲基化修饰miR-126并激活TGF-β1/smad2信号通路有关。

Objective:To investigate the effects of methyltransferase-like 3 (METTL3)/miR-126 on hyperglycemia (HG)-induced proliferation of normal rat kidney-52E (NRK-52E) and its mechanism.Methods:NRK-52E cells were divided into control group (5.5 mmol/L glucose),HG group (35 mmol/L glucose),HG + siRNA- NC group and HG+METTL3 siRNA group.Cell proliferation was detected by MTT,cell cycle was detected by flow cytometry,transforming growth factor (TGF)- β1 protein content in cell supernatant was detected by enzymelinked immunosorbent assay (ELISA),gene expression of miR- 126 and METTL3 was detected by RT- qPCR METTL3 and smad2 protein expression was detected by western blotting,and the binding of METTL3 to miR-126 was detected by RNA immunoprecipitation (RIP).Results:Compared with the control group,the cell proliferation rate and the percentage of G1- phase cells were decreased in the HG group,and the percentage of G2-phase cells and the expression of METTL3,TGF-β1,smad2 and miR-126 were increased in the HG+METTL3siRNA group (P<0.05).Compared with the HG group,the cell proliferation rate was increased,the percentage of G1-phase cells was decreased,the percentage of S-phase cells was increased,and the expression of METTL3TGF-β1,smad2,and miR-126 was decreased in the HG + METTL3 siRNA group (P<0.05).There was no statistically significant difference between the HG group and the HG + siRNA-NC group in any index (P>0.05).The results of RIP experiment showed that METTL3siRNA could reduce the content of mature miR-126(P<0.05).Conclusion:METTL3 expression is upregulated in HG-induced renal tubular epithelial cells and inhibit cell proliferation,which may be related to the methylation modification of miR-126 and the activation of TGF-β1/smad2 signaling pathway.