LncRNA-NEAT1调控miR-182-5p表达对狼疮性肾炎肾系膜细胞损伤的影响  

The effect of LncRNA-NEAT1-mediated miR-182-5p expression regulation on lupus nephritis mesangial cell injury

作　　者：张路路[1] 谢锐[1] 廖志敏[1] 吴刚[1] 万波[1] 孙威 周莲红 ZHANG Lu-lu;XIE Rui;LIAO Zhi-min;WU Gang;WAN Bo;SUN Wei;ZHOU Lian-hong(Department of Nephrology,the Second People′s Hospital of Liangshan Prefecture,Liangshan 615000,Sichuan,China;不详)

机构地区：[1]凉山州第二人民医院肾内科,四川凉山615000 [2]凉山州第二人民医院检验科,四川凉山615000

出　　处：《广东医学》2024年第1期99-105,共7页Guangdong Medical Journal

基　　金：四川省医学科研课题(S19055)。

摘　　要：目的探讨长链非编码RNA(LncRNA)核旁丛组装转录本1(NEAT1)在狼疮性肾炎(lupus nephritis,LN)中通过微小RNA(miR)-182-5p/叉头盒蛋白O1(FoxO1)/β-连环蛋白(β-catenin)轴对肾系膜细胞损伤的影响。方法收集2019年3月至2021年7月凉山州第二人民医院收治的32例LN患者(LN组)外周血和32例体检健康者(健康对照组)外周血,实时荧光定量聚合酶链反应(qRT-PCR)法检测外周血单个核细胞中NEAT1以及miR-182-5p、FoxO1、β-catenin mRNA表达水平,酶联免疫吸附(ELISA)法检测血清炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6和IL-1β含量。采用20%LN患者血清处理肾系膜细胞的方法构建LN肾系膜细胞模型,造模完成后将细胞分为对照组(正常培养,不转染)、模型组(加入5μg/mL脂多糖培养)、si-NC组(转染si-NC后加入5μg/mL脂多糖培养)、si-NEAT1组(转染si-NEAT1后加入5μg/mL脂多糖培养)、si-NEAT1+anti-miR-NC组(共转染si-NEAT1和anti-miR-NC后加入5μg/mL脂多糖培养)、si-NEAT1+anti-miR-182-5p组(共转染si-NEAT1和anti-miR-182-5p后加入5μg/mL脂多糖培养)。qRT-PCR法检测各组细胞NEAT1、miR-182-5p表达水平;ELISA法测定各组细胞炎症因子水平;乳酸脱氢酶(LDH)试剂盒检测各组细胞LDH含量;细胞计数试剂盒8(CCK-8)实验检测各组细胞增殖活力;流式细胞仪分析各组细胞凋亡情况;免疫印迹法检测各组细胞FoxO1、β-catenin蛋白表达水平。结果与健康对照组比较,LN组患者外周血单个核细胞中NEAT1、FoxO1、β-catenin mRNA表达水平以及血清中炎症因子TNF-α、IL-6、IL-1β含量均显著上升,miR-182-5p表达水平显著下降(P<0.05)。与对照组比较,模型组肾系膜细胞增殖活力、FoxO1和β-catenin蛋白水平以及LDH、TNF-α、IL-6和IL-1β水平均明显增加,miR-182-5p表达水平明显降低(P<0.05)。敲低NEAT1后,细胞增殖活力和炎症反应减弱,FoxO1和β-catenin蛋白表达明显下调(P<0.05);抑制miR-182-5p表达可减轻NEAT1敲Objective To investigate the effect of long non-coding RNA(LncRNA)nuclear paraspeckle assembly transcript 1(NEAT1)on the damage of renal mesangial cells in lupus nephritis(LN)through micro RNA(miR)-182-5p/forkhead box protein O1(FoxO1)/β-catenin axis.Methods The peripheral blood of LN patients(LN group)and physical examination of healthy persons(healthy control group)who were admitted to the Second People′s Hospital of Liangshan Prefecture from March 2019 to July 2021 were collected.Real time fluorescence quantitative polymerase chain reaction(qRT-PCR)method was used to assess the expression levels of miR-182-5p,FoxO1,β-catenin mRNA in peripheral blood mononuclear cells.Enzyme linked immunosorbent assay(ELISA)method was used to measured serum inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-1βcontents.The LN renal mesangial cell model was constructed by treating 20%serum of LN patients with kidney mesangial cells.After modeling,the cells were divided into control group(normal culture,no transfection),model group(cultured in 5μg/mL lipopolysaccharide),si-NC group(transfected with si-NC,and cultured in 5μg/mL lipopolysaccharide),si-NEAT1 group(transfected with si-NEAT1,and cultured in 5μg/mL lipopolysaccharide),si-NEAT1+anti-miR-NC group(cotransfected with si-NEAT1 and anti-miR-NC,and cultured in 5μg/mL lipopolysaccharide)and si-NEAT1+anti-miR-182-5p group(cotransfected with si-NEAT1 and anti-miR-182-5p,and cultured in 5μg/mL lipopolysaccharide).The qRT-PCR method was used to detect the expression levels of NEAT1 and miR-182-5p of cells in each group.ELISA method was used to determine the levels of cell inflammatory factors in each group.Lactate dehydrogenase(LDH)kit was used to detect the LDH content of cells in each group.Cell counting kit 8(CCK-8)experiment was used to detect cell proliferation activity in each group.The flow cytometry was used to analyze cell apoptosis in each group.Western blotting was used to assess the expression levels of NEAT1,FoxO1 andβ-catenin pro

关 键 词：核旁从组装转录本1 狼疮性肾炎 miR-182-5p/叉头盒蛋白O1/β-连环蛋白轴 肾系膜细胞 

分 类 号：R593.242[医药卫生—内科学] R329.21[医药卫生—临床医学]