miR-146a-5p过表达对椎间盘退变大鼠髓核损伤的影响及其机制  

Effect of miR⁃146a⁃5p overexpression on nucleus pulposus injury in intervertebral disc degeneration rats and its mechanism

作　　者：张腾 李培坤 丁广江 杨雯 陈营 张立创 ZHANG Teng;LI Peikun;DING Guangjiang;YANG Wen;CHEN Ying;ZHANG Lichuang(Department of Orthopedics,People's Hospital of Jiawang District of Xuzhou City,Xuzhou 221011,China)

机构地区：[1]徐州市贾汪区人民医院骨科,徐州221011

出　　处：《山西医科大学学报》2024年第12期1516-1526,共11页Journal of Shanxi Medical University

基　　金：徐州市卫生健康委科技项目(XWKYHT20230083)。

摘　　要：目的 探讨miR-146a-5p过表达对椎间盘退变(IDD)大鼠髓核损伤的影响及其机制。方法 采用生物信息学分析方法筛选并预测miR-146a-5p下游靶基因,并与GEO数据库筛选得到的差异基因取交集得到与NF-κB信号通路相关的差异靶基因;双荧光素酶报告基因实验验证miR-146a-5p与PTGS2之间的靶向调控关系。将SD大鼠随机分为假手术(sham)组、模型(model)组和miR-146a-5p agomir组,每组6只。采用椎间盘穿刺法构建IDD大鼠模型,miR-146a-5p agomir组大鼠于术后立即在穿刺部位注射10μL miR-146a-5p agomir(10 nmol/L),而sham组和model组注射等量生理盐水。术后4周,采用HE染色观察大鼠椎间盘髓核组织形态;TUNEL法检测大鼠椎间盘髓核组织细胞凋亡情况;ELISA法检测大鼠血清炎症细胞因子水平;qRTPCR检测大鼠椎间盘髓核组织中miR-146a-5p表达水平;Western blot法检测大鼠椎间盘髓核组织中凋亡相关蛋白Bcl-2、Bax和NF-κB p65、p-NF-κB p65、环加氧酶2(PTGS2)蛋白表达水平。结果 生物信息学分析结果显示,miR-146a-5p在IDD中下调;GEO数据库筛选得到的差异基因与miRDB数据库预测的miR-146a-5p下游靶基因取交集,得到miR-146a-5p与NF-κB信号通路相关的靶基因PTGS2;双荧光素酶报告基因实验证实,PTGS2是miR-146a-5p下游靶基因。与model组比较,miR-146a-5p agomir组大鼠椎间盘髓核组织损伤明显改善,髓核组织细胞凋亡率和血清中IL-6、TNF-α水平降低(P<0.05),髓核组织中miR-146a-5p表达水平和Bcl-2蛋白表达水平升高(P<0.05),而Bax、PTGS2蛋白表达水平和NF-κB p65蛋白磷酸化水平降低(P<0.05)。结论 miR-146a-5p过表达可改善IDD大鼠髓核损伤,其机制可能与靶向PTGS2调控NF-κB信号通路有关。Objective To investigate the effect of miR‐146a‐5p overexpression on nucleus pulposus injury in rats with intervertebral disc degeneration(IDD)and its mechanism.Methods Bioinformatics analysis was used to screen and predict the downstream target genes of miR‐146a‐5p,and the differential genes screened by GEO database were intersected to obtain the differential target genes re‐lated to NF‐κB signaling pathway.Dual luciferase reporter gene experiment was used to verify the targeted relationship between miR‐146a‐5p and PTGS2.SD rats were randomly divided into sham group,model group,and miR‐146a‐5p agomir group,with 6 rats in each group.The IDD rat model was established by intervertebral disc puncture,and the rats in miR‐146a‐5p agomir group were in‐jected with 10μL miR‐146a‐5p agomir(10 nmol/L)at the puncture site immediately after surgery,while the rats in sham group and model group were injected with the same amount of normal saline.At week 4 after operation,HE staining was used to observe the mor‐phology of nucleus pulposus tissue of rat intervertebral disc,TUNEL method was used to detect the apoptosis of nucleus pulposus tis‐sue of rat intervertebral disc,ELISA method was used to detect the levels of inflammatory cytokines in serum of rats,qRT‐PCR was used to detect the expression level of miR‐146a‐5p in nucleus pulposus tissue of intervertebral disc,and Western blot was used to de‐tect the expression levels of apoptosis‐related proteins Bcl‐2,Bax,NF‐κB p65,p‐NF‐κB p65 and prostaglandin‐endoperoxide syn‐thase 2(PTGS2)in the nucleus pulposus tissue of rat intervertebral disc.Results Bioinformatics analysis showed that miR‐146a‐5p was downregulated in IDD.After the differentially expressed genes screened by GEO database were intersected with the downstream target genes for miR‐146a‐5p predicted by miRDB database,the target gene PTGS2 related to miR‐146a‐5p and NF‐κB signaling path‐way was obtained.Dual luciferase reporter gene assay confi

关 键 词：miR-146a-5p 椎间盘退变 髓核损伤 凋亡 核因子ΚB 环加氧酶2 大鼠 

分 类 号：R864[医药卫生—临床医学]