摘要
目的探讨骨关节炎(OA)发生过程中微小RNA-210(microRNA,miR-210)及细胞周期蛋白依赖性激酶7(CDK7)的调控作用。方法SD大鼠随机分为正常对照组、模型组、AgomiR-210组(注射miR-210激动剂,2 nmol/只)、AgomiR-210 NC组(注射miR-210阴性对照试剂,2 nmol/只),每组10只。采用膝关节内侧韧带离断与半月板切除术建立OA模型,术后1周经关节腔注射相应药物8周。观察大鼠软骨大体形态;苏木精-伊红染色(HE)观察软骨组织学形态并进行Mankin’s评分;蛋白免疫印迹法(Western blot)测软骨组织中CDK7、核因子-κB活性因子p65(NF-κB p65)及磷酸化NF-κB p65(p-NF-κB p65)、白细胞介素-6(IL-6)、肿瘤坏死因子(TNF-α)表达水平;实时荧光定量聚合酶链反应(RT-qPCR)法检测软骨组织中miR-210表达。双荧光素酶报告试验验证miR-21与CDK7的靶向关系。取人SW1353软骨细胞系,设置为:正常组、OA模型组(10 ng/ml IL-1β诱导建立OA模型)、miR-210 mimic组(转染miR-210过表达试剂)、miR-210 mimic NC组(转染miR-210过表达阴性对照试剂)、miR-210 mimic+pcDNA-CDK7(转染miR-210过表达试剂及CDK7过表达序列)、miR-210 mimic+pcDNA组(转染miR-210过表达试剂+CDK7过表达阴性对照序列);RT-qPCR及Western blot检测miR-210及CDK7、p-NF-κB p65/NF-κB p65、IL-6、TNF-α蛋白表达。结果与正常对照组相比,模型组及AgomiR-210 NC组大鼠关节面糜烂、软骨缺损等病理损伤严重;AgomiR-210组大鼠关节及软骨病变缓解。与正常对照组相比,模型组及AgomiR-210 NC组大鼠Mankin’s评分、CDK7、p-NF-κB p65/NF-κB p65、IL-6、TNF-α蛋白表达升高(P<0.05),miR-210表达降低(P<0.05)。AgomiR-210组大鼠上述指标变化趋势与模型组及AgomiR-210 NC组相反(P<0.05)。双荧光素酶报告试验结果显示,与miR-210 NC+CDK7-3′UTR-WT组比较,miR-210 mimics+CDK7-3′UTR-WT组荧光素酶活性降低(P<0.05)。miR-210 mimic与pcDNA-CDK7共转染,可削弱miR-210 mimic下调CDK7-NF-κB p65通路表达及缓解软骨细胞炎性反应的作用(P<0.05)。结论miR-210可能通过靶向抑制CDK7表达,抑制NF-κB通路活化,进而缓解OA炎症损伤及软骨病变。
Objective To investigate the regulatory role of microRNA-210(miR-210)and cyclin dependent kinase 7(CDK7)in osteoarthritis(OA).Methods SD rats were randomly divided into normal control group,model group,AgomiR-210 group(injected with 2 nmol miR-210 agonist),AgomiR-210 NC group(injected with 2 nmol miR-210 negative control reagent),with 10 rats in each group.OA models were established by interruption of the medial ligament of the knee and meniscus resection,and the corresponding drugs were injected into articular cavity one week after operation for eight weeks.The gross morphology of cartilage was observed.Hematoxylin-eosin(HE)staining was used to observe the histological morphology of cartilage and calculate Mankin’s score.Western blot was used to detect the expression levels of CDK7,nuclear factor-κB active factor p65(NF-κB p65),phosphorylated NF-κB p65(p-NF-κB p65),interleukin-6(IL-6)and tumor necrosis factor(TNF-α)in cartilage tissue,and real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-210 in cartilage tissue.The dual luciferase reporter test was used to verify the targeting relationship between miR-21 and CDK7.The human SW1353 chondrocyte cell line was taken and set as normal group,OA model group(10 ng/ml IL-1β),miR-210 mimic group,miR-210 mimic NC group,miR-210 mimic+pcDNA-CDK7(transfected with miR-210 overexpression reagent and CDK7 overexpression sequence)and miR-210 mimic+pcDNA group(transfected with miR-210 overexpression reagent and CDK7 overexpression negative control sequence).RT-qPCR and Western blot were used to measure the expression of miR-210 and CDK7,p-NF-κB p65/NF-κB p65,IL-6,TNF-αproteins.Results Compared with normal control group,the pathological damages such as articular surface erosion and cartilage defects of rats in model group and AgomiR-210 NC group became severe,and the articular and cartilage lesions were relieved in AgomiR-210 group.Compared with normal control group,the Mankin’s score and the protein expression levels of CDK7,p-NF-κB p65/NF-κB p65,IL-6 and TNF-αwere increased in model group and AgomiR-210 NC group(P<0.05),while the expression of miR-210 was decreased(P<0.05).The change trends of the above indicators in AgomiR-210 group were opposite to those in model group and AgomiR-210 NC group(P<0.05).The results of dual luciferase report test showed that the luciferase activity in miR-210 mimics+CDK7-3′UTR-WT group was lower than that in miR-210 NC+CDK7-3′UTR-WT group(P<0.05).Co-transfection of miR-210 mimic and pcDNA-CDK7 attenuated the effects of miR-210 mimic down-regulating the expression of CDK7-NF-κB p65 pathway and alleviating the inflammatory response of chondrocytes(P<0.05).Conclusion MiR-210 may inhibit the activation of NF-κB pathway by targetedly inhibiting the expression of CDK7,thereby alleviating the inflammatory injury and the cartilage lesions of OA.
作者
徐志强
李晗
XU Zhiqiang;LI Han(Fifth Department of Orthopedics,General Hospital of Jizhong Energy Fengfeng Group Co.,Ltd,Handan 056200,China;Third Department of Orthopedics,Third Hospital of Hebei Medical University,Shijiazhuang)
出处
《山西医科大学学报》
CAS
2021年第9期1195-1201,共7页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(81501911)。
作者简介
徐志强,男,1970-07生,硕士,主治医师,E-mail:speed0032@163.com;通讯作者:李晗,E-mail:723119356@qq.com。
