miR-141靶向FZD4调节Wnt/β-catenin信号通路促进糖尿病肾病的进展

MiR‐141 promotes the development of diabetic nephropathy through regulating WNT/β-catenin signaling pathway by targeting FZD4

目的探讨miR-141在糖尿病肾病进展中的作用及其潜在机制。方法通过脂质体转染法分别将miR-141、miR-141 mimic、miR-141 sponge转入人肾小球系膜细胞中,高糖培养构建体外模拟糖尿病肾病模型。利用流式分析和ELISA法检测miR-141对细胞凋亡和炎症反应的影响。TargetScan数据库预测miR-141的下游靶基因,通过双荧光素酶报告基因实验验证两者的结合作用。过表达靶基因FZD4,检测FZD4对miR-141在高糖培养的肾小球系膜细胞中发挥的具体作用。利用Western印迹法检测下游Wnt/β-catenin信号通路及下游靶基因的变化。结果miR-141的上调促进了细胞凋亡以及肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的表达(P<0.05);miR-141的下调抑制了细胞凋亡以及TNF-α、IL-6的产生(P<0.05)。miR-141负调控FZD4的表达,FZD4的过表达逆转了miR-141对高糖培养肾小球系膜细胞凋亡和炎症因子分泌的促进作用(P<0.05)。敲低miR-141显著促进了Wnt/β-catenin的表达,抑制FZD4则减弱了Wnt/β-catenin的表达(P<0.05)。siβ-catenin可以消除miR-141的干扰及对照高糖培养肾小球系膜细胞凋亡比例和炎症因子分泌的差异(P<0.05)。结论miR-141通过靶向FZD4调控Wnt/β-catenin信号通路,促进糖尿病肾病的进展。

Objective To explore the effect of microRNA‐141(miR‐141)on the development of diabetic nephropathy(DN)and its potential mechanism.Methods The miR-141,miRNA-141 mimic,and miR-141 sponge were transfected into human renal mesangial cells by liposome transfection,and the cells were cultured with high glucose to simulate a diabetic nephropathy model in vitro.Flow cytometry and ELISA were used to investigate the effect of miR-141 on apoptosis and inflammatory response.The downstream target gene of miRNA-141 was predicted by the TargetScan database and the binding effect was verified by a dual luciferase reporter assay.The target gene FZD4 was subsequently overexpressed to detect the specific effect of FZD4 on miR-141 in high-glucose cultured renal mesangial cells.In addition,Western Blot was used to detect changes in the downstream Wnt/β-catenin signaling pathway and downstream target genes.Results Upregulation of miR‐141 significantly promoted cell apoptosis and the expressions of tumor necrosis factor-α(TNF‐α)and interleukin-6(IL‐6)(P<0.05).However,downregulation of miR‐141 inhibited cell apoptosis and the production of TNF‐αand IL‐6(P<0.05).Moreover,miR‐141 negatively regulated the FZD4 abundance,and the overexpression of FZD4 reversed the inhibitory effect of miR‐141 on the development of high-glucose cultured mesangial cells(P<0.05).Besides,the knockdown of miR‐141 significantly promoted the expressions of Wnt/β-catenin and its downstream target proteins(P<0.05),while the inhibition of FZD4 could weaken the expression of Wnt/β-catenin.Siβ-catenin can eliminate the interference of miR-141 and the reference cells’differences in terms of the proportion of apoptotic cells and inflammatory factor secretion of renal mesangial cells cultured with high glucose(P<0.05).Conclusion MiR‐141 can promote the DN progression through regulating Wnt/β-catenin signaling pathway by targeting FZD4.