LncRNA NEAT1/miR-129-5p轴通过调控Wnt/β-catenin通路活化参与LPS诱导的肾小管上皮细胞损伤  

LncRNA NEAT1/miR-129-5p axis was involved in LPS-induced renal tubular epithelial cell injury through regulating Wnt/β-catenin pathway activation

作　　者：徐琳[1] 姚东升[1] 周奕菁 黄迪[1] Xu Lin;Yao Dongsheng;Zhou Yijing;Huang Di(Department of Nephrology,Shuguang Hospital,Institute of Chinese Medicine and Nephrology,Key Laboratory of Liver and Kidney Diseases of the Ministry of Education,Shanghai Key Laboratory of Traditional Chinese Medicine Clinics,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Department of Nephrology,Jiaxing Hospital of Traditional Chinese Medicine Affiliated to Zhejiang University of Chinese Medicine,Zhejiang 314000,China)

机构地区：[1]上海中医药大学附属曙光医院肾病科、上海市中医药研究院肾脏病研究所、肝肾疾病病证教育部重点实验室(上海中医药大学)、上海市中医临床重点实验室(20DZ2272200),201203 [2]浙江中医药大学附属嘉兴市中医医院肾病科,嘉兴314000

出　　处：《中华细胞与干细胞杂志(电子版)》2025年第1期41-50,共10页Chinese Journal of Cell and Stem Cell(Electronic Edition)

基　　金：国家自然科学基金(82174289);上海市白玉兰人才计划浦江项目(24PJD112);上海市中医药研究院科技发展项目(24YJS05)。

摘　　要：目的探究LncRNA NEAT1(NEAT1)在脂多糖(LPS)诱导肾小管上皮细胞损伤中的作用及其分子机制。方法选择人肾小管上皮细胞HK-2进行体外培养,不同浓度LPS处理HK2细胞。流式细胞术测定不同浓度LPS处理细胞后的细胞凋亡率。RT-qPCR和Western blot法检测细胞NEAT1、miR-129-5p、磷酸化(p-)β-Catenin和β-Catenin的表达。实验分组-1为mimic NC组,miR-129-5p mimic组;miR-NC组和miR-129-5p inhibitor组。上述4个分组细胞共转染NEAT1-WT或NEAT1-MUT。采用双荧光素酶报告基因分析并验证上述分组细胞中NEAT1和miR-129-5p的直接序列互作和负调控关系。实验分组-2为空白对照组,si-NC组和si-NEAT1组。分别转染si-NC和si-NEAT1,并检测NEAT1和miR-129-5p的相对表达水平。细胞完成转染后均处理以1μg/mL LPS,联合处理24 h,实验分组-3为LPS组(1μg/mL LPS),si-NC+miR-NC组(共转染si-NC+miR-NC),si-NEAT1+miR-NC组(共转染si-NEAT1+miR-NC),si-NC+miR-129-5p inhibitor组(共转染si-NEAT1+miR-NC),si-NEAT1+miR-129-5p inhibitor组(共转染si-NEAT1+miR-129-5p inhibitor)。CCK-8和流式细胞术检测细胞活力、细胞周期和细胞凋亡;Western blot检测细胞Bcl-2、Bax、cleaved caspase-3、β-catenin、p-β-catenin、cyclin D1和c-myc蛋白表达。多组间比较采用单因素方差分析,组间两两比较采用Turkey&apos;s检验。结果与0μg/mL LPS组相比,1μg/mL LPS诱导HK-2后细胞活力[(83.70±1.40)%比(100.00±1.70)%]、miR-129-5p相对表达水平(0.81±0.07比1.00±0.13)、磷酸化(p-)β-catenin水平(0.78±0.05比1.00±0.14)下降,NEAT1相对表达水平(1.33±0.12比1.00±0.08)、细胞凋亡率[(32.80±1.30)%比(5.20±0.20)%]增高;与mimic NC组相比,miR-129-5p mimic组细胞中miR-129-5p表达(2.34±0.22比1.00±0.06)升高,NEAT1-WT荧光素酶活性(0.36±0.03比1.00±0.08)降低(P均<0.05),而NEAT1-MUT荧光素酶活性差异无统计学意义(P>0.05)。与si-NC组相比,si-NEAT1组细胞中miR-129-5p表达(2.18±0.24比1.04±0.13)升高;LPS处理后,与转�Objective To investigate the role and mechanisms of LncRNA NEAT1(NEAT1)on lipopolysaccharide(LPS)-induced renal tubular epithelial cell damage.Methods Human renal tubular epithelial cells HK-2 were cultured in vitro,LPS was used to treat HK-2 cells under different concentrations.Flow cytometry was used to determine the apoptosis rate of cells treated with different concentrations of LPS.The expression of NEAT1,miR-129-5p,phosphorylated(p-)β-Catenin andβ-Catenin were detected by RT-qPCR and Western blot.Experimental groups-1 were mimic NC group,miR-129-5p mimic group;miR-NC group,miR-129-5p inhibitor group.The above four groups were co-transfected with NEAT1-WT or NEAT1-MUT.Dual luciferase reporter gene experiment was used to analyze and verify the direct sequence interactions and negative regulatory relationships between NEAT1 and miR-129-5p in the above groups of cells.Experimental group-2 were the blank control group,si-NC group and si-NEAT1 group.si-NC and si-NEAT1 were transfected,respectively.The relative expression levels of NEAT1 and miR-129-5p were detected in the above three groups of cells.Cells were treated with 1μg/mL LPS after transfection,and co-treated for 24 h.Cells were divided into LPS group(1μg/mL LPS),si-NC+miR-NC group(co-transfected with si-NC+miR-NC),si-NEAT1+miR-NC group(co-transfected with si-NEAT1+miR-NC),si-NC+miR-129-5p inhibitor group(co-transfected with si-NC+miR-129-5p inhibitor),si-NEAT1+miR-129-5p inhibitor group(co-transfected with si-NEAT1+miR-129-5p inhibitor).CCK-8 and flow cytometry experiment were used to detect cell viability,cell cycle and apoptosis rate;the expression of Bcl-2,Bax,Cleaved caspase-3,β-catenin,p-β-catenin,cyclin D1 and c-myc proteins in cells were detected by Western bot.One-way ANOVA analysis of variances was used for multi-group comparisons,and pairwised comparisons between groups were performed by post hoc Turkey&apos;s test.P<0.05 was considered statistically significant.Results Compared with LPS group(0μg/mL),the cell viability[(83.70±1.40)%vs(

关 键 词：肾小管上皮细胞 脂多糖 LncRNA NEAT1 miR-129-5p 

分 类 号：R73[医药卫生—肿瘤]