miR-132-5p靶向S100A9介导高糖诱导的肾小球系膜细胞凋亡及炎症反应

miR-132-5p mediates apoptosis and inflammatory of high glucose-induced mesangial cells by targeting S100A9

目的:探讨微小RNA-132-5p(microRNA-132-5p,miR-132-5p)靶向S100钙结合蛋白A9(S100 calcium binding protein A9,S100A9)对高糖诱导的肾小球系膜细胞凋亡及炎症反应的影响。方法:人肾小球系膜细胞用DMEM正常糖培养基培养作为对照组,用DMEM高糖培养基培养作为高糖组;miR-NC、miR-132-5p、anti-miR-NC、anti-miR-132-5p转染至正常培养的肾小球系膜细胞中,记为miR-NC组、miR-132-5p组、anti-miR-NC组、anti-miR-132-5p组;将miR-NC、miR-132-5p、si-NC、si-S100A9转染至肾小球系膜细胞中再用高糖培养基培养,记为高糖+miR-NC组、高糖+miR-132-5p组、高糖+si-NC组、高糖+si-S100A9组;将miR-132-5p分别与pcDNA、pcDNA-S100A9转染至肾小球系膜细胞中再用高糖培养基培养,记为高糖+miR-132-5p+pcDNA组、高糖+miR-132-5p+pcDNA-S100A9组。实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测miR-132-5p和S100A9 mRNA表达水平;Western blot检测S100A9、Bcl-2相关X蛋白(Bcl-2 associated X,Bax)、B细胞淋巴瘤/白血病-2蛋白(B cell lymphoma/leukemia-2,Bcl-2)表达;双荧光素酶报告实验验证miR-132-5p和S100A9的靶向关系;流式细胞术检测细胞凋亡;酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素(interleukin,IL)-8、IL-6水平。结果:高糖诱导的肾小球系膜细胞中miR-132-5p表达水平降低,S100A9表达水平升高(P<0.05)。miR-132-5p靶向调控S100A9,过表达miR-132-5p或抑制S100A9表达,高糖诱导的肾小球系膜细胞中细胞凋亡率降低,Bax表达水平降低,Bcl-2表达水平升高,TNF-α、IL-8、IL-6水平降低(P<0.05)。S100A9过表达逆转miR-132-5p过表达对高糖诱导的肾小球系膜细胞凋亡和炎症反应的抑制作用。结论:过表达miR-132-5p通过靶向下调S100A9抑制高糖诱导的肾小球系膜细胞凋亡和炎症反应。

Objective:To investigate the effect of microRNA-132-5p(miR-132-5p)targeting S100 calcium-binding protein A9(S100A9)on the apoptosis and inflammatory of mesangial cells induced by high glucose.Methods:Human mesangial cells were cultured in DMEM normal glucose medium as control group,and cultured in DMEM high glucose medium as high glucose group.miR-NC,miR-132-5p,anti-miR-NC,anti-miR-132-5p were transfected into normal cultured mesangial cells and recorded as miR-NC group,miR-132-5p group,anti-miR-NC group,anti-miR-132-5p group;miR-NC,miR-132-5p,si-NC,si-S100A9 were transfected into mesangial cells and cultured in a high glucose medium,and recorded as high glucose+miR-NC group,high glucose+miR-132-5p group,high glucose+si-NC group,high glucose+si-S100A9 group;miR-132-5p was transfected with pcDNA and pcDNA-S100A9 into mesangial cells and cultured with high glucose medium,and recorded as high glucose+miR-132-5p pcDNA group,high glucose+miR-132-5p+pcDNA-S100A9 group.Real-time quantitative PCR(RT-qPCR)was used to detect miR-132-5p and S100A9 mRNA expressions.Western blot was used to detect S100A9,Bcl-2 associated X protein(Bax),and B cells lymphoma/leu-kemia-2 protein(Bcl-2)expression;dual luciferase assay was used to verify the targeting relationship between miR-132-5p and S100A9;flow cytometry was used to detect apoptosis;enzyme-linked immu-nosorbent assay(ELISA)detected the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-8 and IL-6.Results:The expression of miR-132-5p in mesangial cells induced by high glucose was decreased,and the expression of S100A9 was increased(P<0.05).miR-132-5p targetedS100A9,overexpressedmiR-132-5porinhibited S100A9 expression.Apoptosis was reduced in high glucose-induced mesangial cells,Bax expression was reduced,Bcl-2 expression was increased,and TNF-α,IL-8,IL-6 levels were decreased(P<0.05).S100A9 overexpression reversed the inhibitory effect of miR-132-5p overexpression on apoptosis and inflammatory of mesangial cells induced by high glucose.Conclusion:Overexpression of miR-132-5p can inhibit apoptosis and inflammatory of high glucose-induced mesangial cells by down-regulating S100A9.