LncRNA FGD5-AS1调节miR-93-5p/BMP2轴促进人骨髓间充质干细胞成骨分化  

LncRNA FGD5-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating miR-93-5p/BMP2 axis

作　　者：张亮亮 赵程锦[1] 周煜虎[1] 曹博[1] 段明明[1] 冯阳阳[1] ZHANG Liangliang;ZHAO Chengjin;ZHOU Yuhu;CAO Bo;DUAN Mingming;FENG Yangyang(Department of Orthopedics and Trauma,Yan an University Affiliated Hospital,Yan an 716000,China)

机构地区：[1]延安大学附属医院创伤骨科,陕西延安716000

出　　处：《基础医学与临床》2023年第8期1179-1185,共7页Basic and Clinical Medicine

基　　金：国家自然科学基金(82160431)。

摘　　要：目的探讨长链非编码RNA(lncRNA)FGD5-AS1调节微小RNA miR-93-5p/骨形态发生蛋白-2(BMP2)轴对人骨髓间充质干细胞(hBM-MSCs)成骨分化的影响。方法取对数增殖期的hBM-MSCs,分别检测成骨分化前、后lncRNA FGD5-AS1、miR-93-5p、BMP2 mRNA表达水平;将细胞分别转染或共转染pcDNA FGD5-AS1、miR-93-5p inhibitor、miR-93-5p mimics及相应阴性对照,分组为pcDNA NC组、pcDNA FGD5-AS1组、inhibitor NC组、miR-93-5p inhibitor组、pcDNA FGD5-AS1+mimics NC组、pcDNA FGD5-AS1+miR-93-5p mimics组,取未转染细胞作为空白组;CCK-8法检测细胞增殖;碱性磷酸酶(ALP)试剂盒检测其活性;茜素红染色鉴定细胞矿化结节形成;Western blot检测BMP2及成骨相关标志物--骨钙素(OCN)、骨桥蛋白(OPN)、成骨相关转录因子(OSX)水平;双荧光素酶报告基因实验分别验证miR-93-5p与FGD5-AS1、BMP2的靶向关系。结果miR-93-5p分别与FGD5-AS1、BMP2存在靶向关系。与分化前相比,hBM-MSCs成骨分化后lncRNA FGD5-AS1、BMP2 mRNA表达显著增加,miR-93-5p表达显著下降(P<0.05);与pcDNA NC组相比,pcDNA FGD5-AS1组FGD5-AS1表达显著增加(P<0.05),表明转染成功。后续实验发现矿化结节产生、细胞增殖、ALP活性及BMP2、OCN、OPN、OSX表达均显著增加(P<0.05);与inhibitor NC组相比,miR-93-5p inhibitor组miR-93-5p表达降低,表明转染成功。后续实验发现矿化结节产生、细胞增殖、ALP活性及BMP2、OCN、OPN、OSX表达均显著增加(P<0.05);miR-93-5p过表达可抑制FGD5-AS1对细胞成骨分化的促进作用(P<0.05)。结论上调FGD5-AS1可以促进细胞成骨分化,可能与miR-93-5p/BMP2轴有关。Objective To investigate the impact of long non-coding RNA(lncRNA)FGD5-AS1 on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells(hBM-MSCs)through regulation of the microRNA miR-93-5p/bone morphogenetic protein-2(BMP2)axis.Methods hBM-MSCs in logarithmic growth phase were taken,and the expression levels of lncRNA FGD5-AS1,miR-93-5p and BMP2 mRNA were detected before and after osteogenic differentiation;The cells were transfected or co-transfected with pcDNA FGD5-AS1,miR-93-5p inhibitor,miR-93-5p mimics and corresponding negative controls,respectively,then divided into pcDNA NC group,pcDNA FGD5-AS1 group,inhibitor NC group,miR-93-5p inhibitor group,pcDNA FGD5-AS1+mimics NC group,or pcDNA FGD5-AS1+miR-93-5p mimics group and non-transfected cells were taken as blank group.CCK-8 assay was applied to detect cell proliferation ability of each group;Alkaline phosphatase(ALP)kit was applied to detect its activity;Alizarin red staining was applied to identify cellular mineralized nodule formation;Western blot was applied to detect the levels of BMP2,osteogenesis-related markers-osteocalcin(OCN),osteopontin(OPN),and osterix(OSX);Dual-luciferase experiment was applied to verify the targeting relationship of miR-93-5p with FGD5-AS1 and BMP2,respectively.Results lncRNA FGD5-AS1 and BMP2 were found to be targets of miR-93-5p.After osteogenic differentiation,the expression of FGD5-AS1 and BMP2 was increased and the expression of miR-93-5p was decreased(P<0.05).Compared with pcDNA NC group,the expression of FGD5-AS1 in pcDNA FGD5-AS1 group was significantly increased(P<0.05),indicating successful transfection;Mineralized nodules,cell proliferation,ALP activity and the expression of BMP2,OCN,OPN and OSX were obviously higher(P<0.05)in pcDNA FGD5-AS1 group.Compared with the inhibitor NC group,the expression of miR-93-5p in miR-93-5p inhibitor group was significantly decreased(P<0.05),indicating successful transfection;Mineralized nodules,cell proliferation,ALP activity and the expression of BMP2,OCN,

关 键 词：人骨髓间充质干细胞 lncRNA FGD5-AS1 miR-93-5p/BMP2轴 成骨分化 

分 类 号：R394.2[医药卫生—医学遗传学]