miR-30c-2-3p靶向MPO介导高糖诱导的肾小球系膜细胞凋亡及炎性反应

The miR-30c-2-3p targeting MPO-mediated high glucose-induced apoptosis and inflammatory response in mesangial cells in vitro

目的探讨miR-30c-2-3p对高糖诱导的肾小球系膜细胞凋亡及炎性反应的影响及其机制。方法用终浓度为30 mmol/L的高糖溶液刺激细胞作为高糖组(HG),同时以终浓度为5 mmol/L的糖溶液处理细胞作为对照组(NG);设置miR-NC组、miR-30c-2-3p组、anti-miR-NC组、anti-miR-30c-2-3p组、HG+miR-NC组、HG+miR-30c-2-3p组、HG+miR-30c-2-3p+pcDNA3.1组、HG+miR-30c-2-3p+pcDNA3.1-MPO组,转染均采用脂质体法。qRT-PCR检测miR-30c-2-3p的表达水平;Western Blot检测蛋白表达;流式细胞术检测细胞凋亡;ELISA法检测TNF-α和IL-6的表达;双荧光素酶报告基因检测实验检测荧光活性。结果高糖可以抑制miR-30c-2-3p的表达(P<0.05);过表达miR-30c-2-3p表达可抑制高糖作用的细胞HMC凋亡,抑制IL-6、TNF-α的表达(P<0.05)。miR-30c-2-3p靶向调控MPO的表达,过表达MPO能逆转miR-30c-2-3p过表达对高糖作用的HMC细胞凋亡及IL-6、TNF-α表达的抑制作用(P<0.05)。结论miR-30c-2-3p能抑制高糖作用的HMC细胞凋亡,其机制主要是下调MPO及炎性因子IL-6、TNF-α的表达。可为糖尿病肾病的治疗提供新思路和新靶点。

Objective To investigate the effects of miR-30c-2-3p targeting MPO-mediated high glucose-induced apoptosis and inflammatory response in mesangial cells in vitro,and to explore its action mechanism.Methods The cells were stimulated with high glucose solution at a final concentration of 30mmol/L as high glucose group(HG group),which were treated with a final concentration of 5mmol/L sugar solution as control group(NG group),moreover,there were miR-NC group,miR-30c-2-3p group,anti-miR-NC group,anti-miR-30c-2-3p group,HG+miR-NC group,HG+miR-30c-2-3p group,HG+miR-30c-2-3p+pcDNA3.1 group and the HG+miR-30c-2-3p+pcDNA3.1-MPO group which were transfected by liposome method.The qRT-PCR was used to detect the expression levels of miR-30c-2-3p,and Western Blot was used to detect the protein expression levels,and flow cytometry was used to detect apoptosis,and ELISA was used to detect the expression levels of TNF-αand IL-6.In addition double luciferase reporter gene detection was used to detect the fluorescence activity.Results The high glucose could inhibit the expression of miR-30c-2-3p,and the overexpression of miR-30c-2-3p could inhibit the apoptosis of HMC in high glucose treated cells and could inhibit the expression levels of IL-6 and TNF-α.The miR-30c-2-3p targeted the regulation of MPO expression and the overexpression of MPO could reverse the inhibitory effects of miR-30c-2-3p overexpression on HMC cell apoptosis and IL-6,TNF-αexpression.Conclusion The miR-30c-2-3p can inhibit the apoptosis of HMC cells treated with high glucose,and its action mechanism is mainly to down-regulate the expression levels of MPO and inflammatory factors including IL-6 and TNF-α,which may provide an new ideas and new targets for the treatment of diabetic nephropathy.