miR-494-3p靶向调控RCAN1对高糖诱导的足细胞损伤的影响

RCAN1-targeted effects of miR-494-3p on high glucose-induced podocyte injury

目的 探讨miR-494-3p对高糖(HG)诱导的小鼠肾足细胞(MPC5)损伤的影响及其对钙调磷酸酶调节蛋白1 (RCAN1)的调控作用。方法 HG诱导MPC5细胞建立细胞损伤模型,随机分为NC组、HG组、HG+anti-miR-NC组、HG+anti-miR-494-3p组、HG+pcDNA组、HG+RCAN1组、HG+anti-miR-494-3p+si-NC组、HG+anti-miR-494-3p+si-RCAN1组。采用实时PCR (qRT-PCR)检测miR-494-3p、RCAN1 mRNA表达量;流式细胞术检测细胞凋亡;ELISA法检测白细胞介素(IL)-1β、肿瘤坏死因子-α (TNF-α)水平;双荧光素酶报告实验检测miR-494-3p mimic对Wt-RCAN1荧光素酶活性的影响;Western blotting检测RCAN1蛋白表达量。结果与NC组比较,HG组miR-494-3p表达量升高,凋亡率、IL-1β、TNF-α水平升高,RCAN1 mRNA及蛋白水平降低(P <0.05);与HG+anti-miR-NC组比较,HG+anti-miR-494-3p组凋亡率、IL-1β、TNF-α水平降低(P <0.05);miR-494-3p mimic可降低Wt-RCAN1荧光素酶活性(P <0.05);与HG+pcDNA组比较,HG+RCAN1组凋亡率、IL-1β、TNF-α水平降低(P <0.05);与HG+anti-mi R-NC+si-NC组比较,HG+anti-miR-494-3p+si-RCAN1组凋亡率、IL-1β、TNF-α水平升高(P <0.05)。结论 沉默miR-494-3p可通过促进RCAN1表达抑制MPC5细胞凋亡、炎症反应,从而减轻HG诱导的MPC5细胞损伤。

Objective To investigate the effect of miR-494-3p on high glucose(HG)-induced mouse renal podocyte(MPC5)injury and RCAN1 regulation.Methods A cell damage model was established using HG to induce injury in MPC5 cells,which were subsequently divided into the following groups:NC group,HG group,HG+anti-miR-NC group,HG+anti-miR-494-3p group,HG+pcDNA group,HG+RCAN1 group,HG+anti-miR-494-3p+si-NC group,and HG+anti-miR-494-3p+si-RCAN1 group.qRT-PCR was performed to determine the mRNA expression levels of miR-494-3p and RCAN1,apoptosis was evaluated using flow cytometry,and the levels of IL-1βand TNF-αwere determined by ELISA.Additionally,dual-luciferase reporter assays were conducted to assess the effect of miR-494-3p mimics on Wt-RCAN1 luciferase activity,and Western blotting was performed to detect RCAN1,Bcl-2,and Bax protein expression.Results miR-494-3p expression,apoptosis rate,and Bax protein,IL-1β,and TNF-αlevels were increased,and RCAN1 mRNA and protein levels were decreased in the HG group compared with the NC group(P<0.05).Additionally,the apoptosis rate and IL-1βand TNF-αlevels were decreased in the HG+anti-miR-494-3p group compared with the HG+anti-miR-NC group(P<0.05).miR-494-3p mimics were found to reduce Wt-RCAN1 luciferase activity(P<0.05).The apoptosis rate and IL-1βand TNF-αlevels were decreased in the HG+RCAN1 group compared with the HG+pcDNA group(P<0.05).Moreover,the apoptosis rate and IL-1βand TNF-αlevels were increased in the HG+anti-miR-494-3p+si-RCAN1 group compared with the HG+anti-miR-NC+si-NC group(P<0.05).Conclusion Silencing miR-494-3p inhibited MPC5 cell apoptosis and inflammatory response by promoting RCAN1 expression,thereby reducing HG-induced MPC5 cell damage.