摘要
目的探讨微小RNA-543(miR-543)靶向调控转录因子7类似物2(TCF7L2)对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成脂分化的影响。方法分离并鉴定大鼠BMSCs,实时荧光定量PCR(RT-qPCR)检测BMSCs成脂分化诱导过程中miR-543的表达变化;体外培养BMSCs细胞,对BMSCs细胞进行干预,过表达miR-543、抑制TCF7L2表达或同时过表达miR-543与TCF7L2,使用成脂分化诱导培养基培养,油红O染色检测BMSCs成脂分化;免疫印迹法检测BMSCs细胞TCF7L2、脂肪细胞蛋白2(aP2)、CCAAT/增强子结合蛋白α(C/EBPα)、过氧化物酶体增殖物激活受体γ(PPAR-γ)蛋白表达。双荧光素酶报告基因实验验证miR-543与TCF7L2的靶向关系。结果成功分离大鼠BMSCs;BMSCs成脂分化诱导处理1、2、3、4、5 d后miR-543表达水平降低;双荧光素酶报告基因实验证实miR-543与TCF7L2存在靶向关系;过表达miR-543或抑制TCF7L2表达,BMSCs油红O染色后OD值、aP2、PPAR-γ、C/EBPα蛋白表达水平显著降低(P<0.05);过表达TCF7L2可部分逆转过表达miR-543对BMSCs细胞成脂分化的抑制作用(P<0.05)。结论miR-543在大鼠BMSCs细胞成脂分化过程中下调表达,过表达miR-543可通过靶向抑制TCF7L2表达而抑制BMSCs细胞成脂分化。
Objective To investigate the effect of microRNA-543(miR-543)on the adipogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)by targeting and regulating transcription factor 7-like 2(TCF7L2).Methods The rat BMSCs were isolated and identified.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the expression changes of miR-543 during the induction of adipogenic differentiation of BMSCs.BMSCs cells were cultured in vitro,and were intervened with overexpression of miR-543,inhibition of TCF7L2,or overexpression of both miR-543 and TCF7L2.Cells were cultured in the adipogenic differentiation induction medium.Oil red O staining was used to detect the adipogenic differentiation of BMSCs.Western blotting was used to detect the protein expression of TCF7L2,adipocyte protein 2(aP2),peroxisome proliferator-activated receptorγ(PPAR-γ),and CCAAT/enhancer binding proteinα(C/EBPa)in BMSCs cells.Dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-543 and TCF7L2.Results The rat BMSCs were successfully isolated.The expression of miR-543 decreased after 1,2,3,4,and 5-d induction of adipogenic differentiation of BMSCs.The dual luciferase reporter gene experiment confirmed that miR-543 had a targeting relationship with TCF7L2.Overexpressing miR-543 or inhibiting the expression of TCF7L2 led to significantly reduction of the OD value,aP2,PPAR-γ,and C/EBPa protein expression levels of BMSCs(P<0.05).Overexpression of TCF7L2 partially reversed the inhibitory effect of overexpression of miR-543 on the adipogenic differentiation of BMSCs cells(P<0.05).Conclusion The expression of miR-543 is down-regulated during the adipogenic differentiation of rat BMSCs.Overexpression of miR-543 inhibits the adipogenic differentiation of BMSCs by targeting the inhibition of TCF7L2 expression.
作者
封宝红
喻业安
晏莉
伍军
张艳霞
朱戈丽
FENG Baohong;YU Yean;YAN Li;WU Jun;ZHANG Yanxia;ZHU Geli(Department of Nephrology,Guanggu District,Wuhan Third Hospital,Wuhan 430014,China)
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2022年第12期1770-1776,共7页
Chinese Journal of Osteoporosis
基金
武汉市医学科研项目(WX20B22)。
