LncRNA NEAT1通过miR-195-5p/TXNIP轴对人骨髓间充质干细胞成骨分化的影响  

Effect of LncRNA NEAT1 on osteogenic differentiation of human bone marrow mesenchymal stem cells through miR-195-5p/TXNIP axis

作　　者：罗鹏 吴钒 陈佳伟 LUO Peng;WU Fan;CHEN Jiawei(Dept.of Orthopedics,Hubei Hospital of Integrated Traditional Chinese and Western Medicine,Wuhan 430015,Hubei,China)

机构地区：[1]湖北省中西医结合医院骨科,湖北武汉430015

出　　处：《武汉大学学报（医学版）》2024年第11期1297-1303,共7页Medical Journal of Wuhan University

摘　　要：目的:探讨LncRNA NEAT1通过miR-195-5p/TXNIP轴对人骨髓间充质干细胞(HBMSCs)成骨分化的影响。方法:观察HBMSCs成骨分化前、后的NEAT1、TXNIP mRNA、miR-195-5p表达;根据处理方式将HBMSCs分为沉默NEAT1(si NEAT1)组、阴性对照(si NC)组、miR-195-5p模拟物(miR-195-5p mimics)组、阴性对照(mimicsNC)组、siNEAT1+阴性对照(inhibitorNC)组、siNEAT1+miR-195-5p抑制剂(miR-195-5p inhibitor)组,以未处理的HBMSCs为对照组,转染后进行成骨诱导,qRT-PCR、CCK-8法、茜素红染色、碱性磷酸酶(ALP)试剂盒、Western Blot分别检测各组HBMSCs中NEAT1、miR-195-5p、TXNIP mRNA表达水平、细胞活力、矿化能力、ALP活性以及TXNIP、骨形态发生蛋白2(BMP2)、骨钙素(OCN)蛋白表达水平;双荧光素酶实验验证miR-195-5p与NEAT1、TXNIP的靶向关系。结果:NEAT1、TXNIP mRNA在成骨分化后的表达水平较分化前显著降低,miR-195-5p表达较分化前显著增加(P<0.05);与si NC组、对照组相比,si NEAT1组有明显结节产生,NEAT1、TXNIP蛋白及mRNA表达显著降低,miR-195-5p表达、24 h、48 h OD_(450)值、ALP活性、BMP2、OCN蛋白表达显著增加(P<0.05);与mimics NC组相比,miR-195-5p mimics组有明显结节产生,TXNIP蛋白及mRNA表达显著降低,miR-195-5p表达、24 h、48 h OD_(450)值、ALP活性、BMP2、OCN蛋白表达显著增加(P<0.05),NEAT1表达无统计学意义(P>0.05);与si NEAT1+inhibitor NC组相比,si NEAT1+miR-195-5p inhibitor组有少量结节,且颜色较浅,TXNIP蛋白及mRNA表达显著增加,miR-195-5p表达、24 h、48 h OD_(450)值、ALP活性、BMP2、OCN蛋白表达显著降低(P<0.05),NEAT1表达差异无统计学意义(P>0.05)。miR-195-5p与NEAT1、TXNIP分别存在靶向关系。结论:LncRNA NEAT1通过上调miR-195-5p/TXNIP轴促进HBMSCs成骨分化。Objective:To investigate the effect of LncRNA NEAT1 on osteogenic differentiation of human bone marrow mesenchymal stem cells(HBMSCs)through the miR-195-5p/TXNIP axis.Methods:The changes of expression of NEAT1,TXNIP mRNA,and miR-195-5p in HBMSCs before and after osteogenic differentiation were observed.According to the treatment,HBMSCs were assigned into silence NEAT1(si NEAT1)group,negative control(si NC)group,miR-195-5p mimics group,negative control(mimics NC)group,si NEAT1+negative control(inhibitor NC)group,and siNEAT1+miR-195-5p inhibitor(miR-195-5p inhibitor)group,untreated HBMSCs were used as thecontrols and transfected for osteogenic induction.qRT-PCR,CCK-8 method,Alizarin Red staining,Alkaline phosphatase(ALP)assay kit,and Western Blot,respectively,were used to detect the expressionlevels of NEAT1,miR-195-5p,TXNIP mRNA,cell viability,mineralization ability,ALPactivity,the expression levels of TXNIP,BMP2,and OCN proteins of HBMSCs.Dual luciferase assaywas applied to verify the targeting relationship of miR-195-5p with NEAT1 and TXNIP.Results:The expression levels of NEAT1 and TXNIP mRNA obviously decreased after osteogenic differentiation,while the expression of miR-195-5p obviously increased(P<0.05);compared with those in thesi NC group and control group,obvious nodule formation was found in the si NEAT1 group,the expressionof NEAT1 and TXNIP were obviously reduced,the expression of miR-195-5p,OD_(450)valuesat 24 and 48 hours,ALP activity,the expression of BMP2,and OCN proteins were obviously increased(P<0.05).Compared with those in the mimics NC group,obvious nodule formation wasfound in the miR-195-5p mimics group,the expression of TXNIP was obviously reduced,the expressionof miR-195-5p,OD_(450)values at 24 and 48 hours,ALP activity,the expression of BMP2,andOCN proteins were obviously increased(P<0.05),but no obvious changes of NEAT1 expressionwas found(P>0.05).Compared with those in the si NEAT1+inhibitor NC group,the si NEAT1+miR-195-5p inhibitor group had few nodles and lighter color,the expression

关 键 词：LncRNA NEAT1 miR-195-5p/TXNIP轴 人骨髓间充质干细胞 成骨分化 

分 类 号：R580[医药卫生—内分泌]