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

摘要:

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。

Abstract:

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.