微小RNA-23a-3p负调控Runx2基因抑制骨髓间充质干细胞成骨分化诱导绝经后骨质疏松的研究

MicroRNA-23a-3p negatively regulates Runx2 gene and inhibits osteogenic differentiation of bone marrow mesenchymal stem cells to induce postmenopausal osteoporosis

目的观察微小RNA(miR)-23a-3p通过调控Runx2基因对骨髓间充质干细胞(BMSCs)成骨分化的影响。方法绝经后骨质疏松症(PMOP)患者及正常的女性志愿者各30例,检测两组血清中miR-23a-3p表达水平。建立雌性小鼠骨质疏松模型后提取BMSCs,将miR-23a-3p inhibitor和miR-阴性对照(NC)分别转染入BMSCs,检测miR-23a-3p表达水平,同时检测成骨基因Runx2、骨钙素(OCN)、Ⅰ型胶原(CollagenⅠ)信使RNA(mRNA),酶联免疫吸附试验(ELISA)试剂盒检测细胞内碱性磷酸酶(ALP)含量。培养HEK293细胞,将miR-23a-3p模拟物和miR-NC分别于野生型Runx23’端非编码区(3’UTR)(WT)或突变型Runx23’UTR(Mut)重组质粒共转染,使用双荧光素酶报告系统检测荧光素酶活性。最后将miR-23a-3p inhibitor和miR-23a-3p inhibitor+siRunx2分别转染入BMSCs,检测Runx2蛋白、OCN、CollagenⅠmRNA含量及ALP含量,两样本两组间比较采用t检验。结果PMOP患者血清中miR-23a-3p表达量高于对照组(0.872±0.307比0.382±0.183,t=7.514,P<0.05)。转染miR-23a-3p inhibitor的BMSCs中miR-23a-3p表达量低于转染miR-NC组(0.480±0.045比1.361±0.113,t=-16.213,P<0.05),差异有统计学意义。转染miR-23a-3p inhibitor的BMSCs中Runx2蛋白、OCN、CollagenⅠmRNA表达量及ALP含量高于转染miR-NC组[0.608±0.065比0.234±0.041,0.523±0.053比0.229±0.046,0.652±0.060比0.381±0.030,(5.569±0.377)U/L比(2.513±0.308)U/L,t=9.409、9.036、10.846、14.036,P<0.05],差异有统计学意义。生物信息学分析及双荧光素酶报告基因检测进一步证实了Runx2是miR-23a-3p的直接靶点。转染miR-23a-3p inhibitor+siRunx2的BMSCs中Runx2蛋白、OCN、CollagenⅠmRNA表达量及ALP含量低于转染miR-23a-3p inhibitor组[0.373±0.051比0.600±0.083,0.321±0.015比0.645±0.030,0.417±0.027比0.617±0.065,(3.733±0.056)U/L比(6.751±0.091)U/L,t=5.188、21.491、6.414、63.040,P<0.05],差异有统计学意义。结论miR-23a-3p通过负调控Runx2基因的表达抑制BMSCs成骨分化。

Objective To observe the effect of miR-23a-3p on osteogenesis differentiation of bone marrow mesenchymal stem cells(BMSCs)by regulating Runx2 gene.Methods The expression level of miR-23a-3p in serum of patients with postmenopausal osteoporosis(PMOP)(30 cases)and normal female volunteers(30 cases)was detected.After the establishment of female mouse osteoporosis model,BMSCs were extracted,and miR-23a-3p inhibitor and miR-NC were transfected into BMSCs respectively to detect the expression level of miR-23a-3p.The expression levels of Runx2,Osteocalcin(OCN)and CollagenⅠmRNA were detected,and the contents of alkaline phosphatase(ALP)in cells were detected by enzyme linked immunosorbent assay(ELISA)kit.HEK293 cells were cultured,and miR-23a-3p mimics and miR-NC were co-transfected into wild-type Runx23′untranslated regions(3′UTR)(WT)or mutant Runx23′UTR(Mut)recombinant plasmids,respectively.The lucifase activity was detected using dual-luciferase reporter systerm.Finally,miR-23a-3p inhibitor and miR-23a-3p inhibitor+siRunx2 were transfected into BMSCs respectively,and the expression level of Runx2 protein,OCN,CollagenⅠand ALP contents were detected.Thet test was used to compare the two samples between the two groups.Results The expression level of miR-23a-3p in serum of PMOP patients was significantly higher than that of control group(0.872±0.307 vs.0.382±0.183,t=7.514,P<0.05).The expression level of miR-23a-3p in BMSCs transfected with miR-23a-3p inhibitor was significantly lower than that in the Mir-NC group(0.480±0.045 vs.1.361±0.113,t=-16.213,P<0.05).The expression levels of Runx2 protein,OCN,CollagenⅠmRNA and ALP content in BMSCs transfected with miR-23a-3p inhibitor were increased as compared with those in BMSCs with mir-NC transfection[0.608±0.065 vs.0.234±0.041,0.523±0.053 vs.0.229±0.046,0.652±0.060 vs.0.381±0.030,(5.569±0.377)U/L vs.(2.513±0.308)U/L,t=9.409,9.036,10.846,14.036,P<0.05].Bioinformatics analysis and dual-luciferase reporter gene assay further confirmed that Runx2 was a direct target of miR-23a-3p.The expression levels of Runx2 protein,OCN,CollagenⅠmRNA and ALP contents in BMSCs transfected with miR-23a-3p inhibitor+siRunx2 were significantly lower than those in BMSCs transfected with miR-23a-3p inhibitor[0.373±0.051 vs.0.600±0.083,0.321±0.015 vs.0.645±0.030,0.417±0.027 vs.0.617±0.065,(3.733±0.056)U/L vs.(6.751±0.091)U/L,t=5.188,21.491,6.414,63.040,P<0.05].Conclusion miR-23a-3p inhibits the osteogenic differentiation of BMSCs by negatively regulating the expression of Runx2 gene.