lncHAGLR通过抑制miR-26a激活NF-κB促进骨关节炎大鼠软骨细胞的炎症反应和细胞凋亡  

LncHAGLR Promotes Inflammatory Response and Apoptosis of Chondrocytes in Osteoarthritis Rats by Targeting miR-26a and Activation of NF-κB

作　　者：孟晓源[1] 乌尔坎·叶尔肯 王志刚[1] 马乐[1] MENG Xiaoyuan;Wuerkan Yeerken;WANG Zhigang;MA Le(Department of Joint Surgery and Sports Medicine,Orthopedic Center,People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi,830001,China)

机构地区：[1]新疆维吾尔自治区人民医院骨科中心关节运动病区,乌鲁木齐市830001

出　　处：《医学分子生物学杂志》2024年第2期115-123,共9页Journal of Medical Molecular Biology

基　　金：新疆维吾尔自治区自然科学基金(No.2022D01C507)。

摘　　要：目的探讨长链非编码RNA(long non-coding RNA,lncRNA)HOXD反义生长相关长链非编码RNA(HOXD antisense growth-associated long non-coding RNA,lncHAGLR)对大鼠膝关节退行性软骨细胞C518细胞的炎症反应和凋亡的作用与潜在机制。方法采用StarBase和荧光素酶报告基因法预测和确认ln-cHAGLR和微小RNA(microRNA,miR)-26a之间的相互作用。为研究lncHAGLR对miR-26a表达的调控作用,将C518细胞分为沉默lncHAGLR的小干扰RNA(small interfering RNA,siRNA)载体质粒(si-ln-cHAGLR)组、siRNA的阴性对照(negative control of siRNA,si-NC)组、miR-26a抑制剂阴性对照(nega-tive control of miR-26a inhibitor,inhibitor-NC)组、miR-26a的抑制剂(miR-26a-inhibitor)组。此外,为研究lncHAGLR是否通过调控miR-26a对C518细胞的炎症和凋亡具有调控作用,将C518细胞分为5组:Control组、白细胞介素(interleukin,IL)-1β组、IL-1β+si-NC组、IL-1β+si-lncHAGLR组和IL-1β+si-lncHA-GLR+miR-26a-inhibitor组。采用实时定量PCR(real-time quantitative PCR,qRT-PCR)分析lncHAGLR和miR-26a的水平。采用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)、乳酸脱氢酶(lactate dehydro-genase,LDH)和流式细胞术(flow cytometry,FCM)检测C518细胞的增殖、细胞毒性和凋亡情况。蛋白质印迹检测切割模式的半胱天冬酶3(CLEAVED-CASPASE3)、核因子κB P65(nuclear factor-κB P65,NF-κB P65)、磷酸化的NF-κB P65(phosphorylated NF-κB P65,P-NF-κB P65)的表达。ELISA法检测白细胞介素(interleukin,IL)-6和肿瘤坏死因子(tumor necrosis factor,TNF)-α的释放。结果lncHAGLR直接靶向miR-26a。在IL-1β组,lncHAGLR水平明显较Control组增高,miR-26a水平较Control组降低(P均<0.05)。此外,IL-1β+si-lncHAGLR组减轻了IL-1β诱导的C518细胞的炎症并抑制了凋亡,而且细胞的活力增加,LDH释放减少,CLEAVED-CASPASE3的表达被抑制,TNF-α和IL-6的分泌减少,且p-NF-κB P65表达减少(P均<0.05)。IL-1β+si-lncHAGLR+miR-26a-inhibitor组逆转了IL-1β+si-lncHAGLR�Objective To investigate the effect and potential mechanism of long non-coding RNA HAGLR on the inflammatory response and apoptosis of chondrocytes in rats with osteoarthritis.Methods StarBase and luciferase gene reporter assay were used to predict and verify the interaction between lncHAGLR and miR-26a.To investigate the regulatory role of lncHAGLR on miR-26a expression,C518 cells were divided into 4 groups:si-lncHAGLR group,si-NC group,miR-26ainhibitor group,and inhibitor-NC group.Furthermore,to study whether lncHAGLR regulates inflammation and apoptosis in C518 cells by modulating miR-26a,C518 cells were divided into 5 groups:Control group,IL-1β group,IL-1β+si-NC group,IL-1β+si-lncHAGLR group,and IL-1β+si-lncHAGLR+miR-26a-inhibitor group.Real-time quantitative PCR(qRT-PCR)was used to analyze the expression levels of lncHAGLR and miR-26a.The proliferation,cytotoxicity and apoptosis of C518 cells were detected by MTT,lactate dehydrogenase(LDH)Kit and flow cytometry(FCM).Western blotting assay was used to detect the protein expression levels of cleaved-CASPASE3,NF-κB P65,and phosphorylated NF-κB P65(P-NF-κB P65).The levels of released inflammatory factors(TNF-α and IL-6)were detected by ELISA.Results lncHAGLR directly targeted miR-26a.The expression level of lncHAGLR in the IL-1βgroup was significantly higher than that in the Control group,and the expression level of miR-26a was significantly lower than that in the Control group(all P<0.05).In addition,cells in the IL-1β+si-lncHAGLR group had reduced IL-1β-induced chondrocyte inflammation,inhibited apoptosis,increased cell viabilities,decreased release of LDH,inhibited expression of cleaved-CASPASE3,and decreased secretions of TNF-αand IL-6(all P<0.05).Moreover,the expression level of P-NF-κB P65 was decreased(P<0.05).Adding of miR-26a-inhibitor(IL-1β+si-lncHAGLR+miR-26a-inhibitor group)reversed the performances of cells in the IL-1β+si-lncHAGLR group(all P<0.05).Conclusion lncHAGLR promotes the inflammatory response and apoptosis of chondrocytes i

关 键 词：lncHAGLR 骨关节炎 C518细胞 细胞凋亡 miR-26a NF-κB P65 

分 类 号：R684.3[医药卫生—骨科学]