lncRNA NEAT1调控骨关节炎软骨细胞焦亡的机制  

Mechanism of lncRNA NEAT1 regulating chondrocyte pyroptosis in osteoarthritis

作　　者：唐捷 张丕军 林维 李超艺 TANG Jie;ZHANG Pi-jun;LIN Wei;LI Chao-yi(Department of Joint Surgery,Second Affiliated Hospital of Hainan Medical University,Haikou Hainan 570311,China)

机构地区：[1]海南医科大学第二附属医院关节外科,海南海口570311

出　　处：《局解手术学杂志》2025年第3期199-205,共7页Journal of Regional Anatomy and Operative Surgery

基　　金：海南省卫生健康行业科研项目(22A200068)。

摘　　要：目的探讨长链非编码RNA(lncRNA)核富集丰富转录本1(NEAT1)对骨关节炎(OA)软骨细胞焦亡信号轴的影响和潜在机制。方法收集6例OA患者的膝关节软骨组织和6例正常软骨组织。使用TargetScan 7.2和StarBase预测miR-26a与lncRNA NEAT1的靶向关系,双荧光素酶报告基因实验验证lncRNA NEAT1与miR-26a的靶向调控作用。将培养的软骨细胞系分为对照组(不进行药物干预)、IL-1β组(添加10μg/L IL-1β诱导体外OA)、IL-1β+NEAT1-EV组(添加10μg/L IL-1β,联合转染20μg/L的NEAT1-EV)、IL-1β+NEAT1-OE组(添加10μg/L IL-1β,联合转染20μg/L的NEAT1-OE)、IL-1β+NEAT1-OE+mimic-NC组(添加10μg/L IL-1β,联合转染20μg/L的NEAT1-OE和50μg/L的mimic-NC)、IL-1β+NEAT1-OE+mimic组(添加10μg/L IL-1β,联合转染20μg/L的NEAT1-OE和50μg/L的miR-26a mimic)。qRT-PCR法检测OA软骨组织和细胞中lncRNA NEAT1和miR-26a的表达水平。CCK-8法检测各组细胞的活力,蛋白免疫印迹法检测各组Caspase1和cleaved-Caspase1蛋白表达及细胞焦亡标志物[NOD样受体蛋白3(NLRP3)、白细胞介素18(IL-18)和切割模式的Gasdermin D(cleaved-Gasdermin D)]的表达。结果OA软骨组织中lncRNA NEAT1的表达水平高于正常软骨组织(P<0.05),而miR-26a的表达水平低于正常软骨组织(P<0.05)。双荧光素酶报告基因实验证实,lncRNA NEAT1与miR-26a具有靶向调控作用。与对照组比较,IL-1β组细胞中lncRNA NEAT1、cleaved-Caspase1、NLRP3、IL-18、cleaved-Gasdermin D的表达水平均上调(P<0.05),而miR-26a表达水平下调(P<0.05),细胞活力降低(P<0.05)。与IL-1β+NEAT1-EV组比较,IL-1β+NEAT1-OE组细胞中lncRNA NEAT1、cleaved-Caspase1、NLRP3、IL-18、cleaved-Gasdermin D的表达水平均上调(P<0.05),而miR-26a表达水平下调(P<0.05),细胞活力下降(P<0.05)。与IL-1β+NEAT1-OE+mimic-NC组比较,IL-1β+NEAT1-OE+mimic组细胞中cleaved-Caspase1、NLRP3、IL-18、cleaved-Gasdermin D的表达水平均下调(P<0.05),而miR-26a表达水平上调(P<0.05),细胞活�Objective To investigate the effect of long non-coding RNA(lncRNA)nuclear enriched abundant transcript 1(NEAT1)on the chondrocyte pyroptosis signaling axis in osteoarthritis(OA)and its potential mechanism.Methods Knee joint cartilage tissues were collected from 6 OA patients and 6 healthy individuals.The targeting relationship between miR-26a and lncRNA NEAT1 was predicted by TargetScan 7.2 and StarBase,and the targeting regulatory effect of lncRNA NEAT1 and miR-26a was verified by dual luciferase reporter assays.The cultured chondrocyte lines were divided into the control group(without drug intervention),IL-1βgroup(with 10μg/L of IL-1βfor inducing OA in vitro),IL-1β+NEAT1-EV group(with 10μg/L of IL-1βand transfected with 20μg/L of NEAT1-EV),IL-1β+NEAT1-OE group(with 10μg/L of IL-1βand transfected with 20μg/L of NEAT1-OE),IL-1β+NEAT1-OE+mimic-NC(with 10μg/L of IL-1βand co-transfected with 20μg/L of NEAT1-OE and 50μg/L of mimic-NC),and IL-1β+NEAT1-OE+mimic(with 10μg/L of IL-1βand co-transfected with 20μg/L of NEAT1-OE and 50μg/L of miR-26a mimic).The expression levels of lncRNA NEAT1 and miR-26a in OA cartilage tissues and cells were detected using qRT-PCR.The cell viability was determined using CCK-8 assay.The expression of Caspase1 and cleaved-Caspase1,as well as the pyroptosis markers[NOD-like receptor family pyrin domain containing 3(NLRP3),interleukin-18(IL-18)and cleaved-Gasdermin D]were detected using Western blot.Results In the OA cartilage tissue,the expression level of lncRNA NEAT1 was higher than that in the normal cartilage tissue,while the expression level of miR-26a was lower than that in the normal cartilage group(P<0.05).The dual luciferase reporter assays confirmed the targeted regulatory effect of lncRNA NEAT1 and miR-26a.Compared with the control group,the IL-1βgroup showed upregulation of lncRNA NEAT1,cleaved-Caspase1,NLRP3,IL-18,and cleaved-Gasdermin D expression levels(P<0.05),downregulation of miR-26a expression level(P<0.05),and a decrease of cell viability(P<0.05).Com

关 键 词：lncRNA NEAT1 miR-26a 骨关节炎 软骨细胞 半胱天冬酶1 细胞焦亡信号轴 

分 类 号：R364.5[医药卫生—病理学]