下调microRNA-10b通过靶向调控CEBPα表达促进脂肪干细胞的成骨分化

Down-regulation of microRNA-10b promotes osteogenic differentiation of adipose-derived stem cells through target regulation of CEBPαexpression

目的探讨下调microRNA-10b(miR-10b)对小鼠脂肪干细胞(ADSCs)成骨分化的影响。方法分离培养小鼠ADSCs,对其进行细胞形态学及流式细胞术鉴定;应用脂质体稳定转染法转染小鼠ADSCs,将转染miR-10b inhibitor、si-CEBPα及miR-10b inhibitor+si-CEBPα的ADSCs依次设为miR-10b inhibitor组、si-CEBPα组及miR-10b inhibitor+si-CEBPα组;同时将上述对应的阴性对照序列转染入ADSCs中,分别记为miR-NC组、si-NC组及miR-NC+si-NC组,正常对照组(control组)为无任何特殊处理的ADSCs;采用RT-PCR法检测转染后ADSCs中miR-10b的表达;生物信息学预测miR-10b与CEBPα的靶向关系,并采用双荧光素酶基因报告实验、实时荧光定量PCR及Western blot验证两者的靶向关系;利用茜素红染色及碱性磷酸酶(ALP)活性检测实验明确miR-10b对小鼠ADSCs成骨分化的影响及CEBPα在其中的作用。结果与control组和miR-NC组相比,miR-10b inhibitor组小鼠ADSCs中miR-10b的表达显著较低(P<0.05);双荧光素酶基因报告实验、RT-PCR和Western blot实验结果表明,miR-10b可在小鼠ADSCs中靶向调控CEBPα基因的表达,且与miR-NC组相比,miR-10b inhibitor组ADSCs中CEBPα的mRNA及蛋白表达均显著增加(P<0.05);茜素红染色及ALP活性检测结果表明,与control组和miR-NC组相比,miR-10b inhibitor组ADSCs细胞形成的矿化结节及ALP活性均显著增加(P<0.05)。功能挽救实验证实,在小鼠ADSCs细胞中同时转染沉默CEBPα的si-CEBPα及miR-10b inhibitor后,miR-NC+si-NC组和miR-10b inhibitor+si-CEBPα组ADSCs细胞形成的矿化结节与ALP活性均较si-NC+miR-10b inhibitor组低(P<0.05),且前2组之间无明显差异(P>0.05)。结论在小鼠ADSCs中,下调miR-10b通过靶向调控CEBPα的表达促进脂肪干细胞的成骨分化。

Objective To investigate the effect of down-regulation of microRNA-10b(miR-10b)on osteogenic differentiation of mice adipose-derived stem cells(ADSCs).Methods ADSCs were isolated and cultured from mice and identified by cell morphology and flow cytometry.Mice ADSCs were transfected with liposome stable transfection method,and the ADSCs transfected with miR-10b inhibitor,si-CEBPαand miR-10b inhibitor+si-CEBPαwere divided into the miR-10b inhibitor group,the si-CEBPαgroup and the miR-10b inhibitor+si-CEBPαgroup according to different transfections.At the same time,the above-mentioned corresponding negative control sequences were transfected into ADSCs,which were marked as the miR-NC group,the si-NC group and the miR-NC+si-NC group,and the normal control group(the control group)was the ADSCs without any special treatment.The expression of miR-10b in transfected ADSCs was detected by RT-PCR.The targeting relationship between miR-10b and CEBPαwas predicted by bioinformatics,and verified by double luciferase gene reporter experiment,RT-PCR and Western blot.Alizarin red staining and alkaline phosphatase(ALP)activity detection experiments were used to determine the effect of miR-10b on osteogenic differentiation of mice ADSCs and the role of CEBPαin it.Results The expression of miR-10b in ADSCs in the miR-10b inhibitor group was significantly lower than that of the control group or the miR-NC group(P<0.05).The results of double luciferase gene reporter experiment,RT-PCR and Western blot showed that miR-10b could target the expression of CEBPαgene in mice ADSCs.And compared with the miR-NC group,the mRNA and protein expression of CEBPαin the miR-10b inhibitor group increased significantly(P<0.05).The results of alizarin red staining and ALP activity detection experiments showed that mineralized nodules formed by ADSCs cells and ALP activity in miR-10b inhibitor group were significantly increased compared with the control group or the miR-NC group.The function rescue experiment confirmed that the mineralized nodules formed by ADSCs cells and ALP activity in the si-NC+miR-NC group and the miR-10b inhibitor+si-CEBPαgroup were significantly lower than those in the si-NC+miR-10b inhibitor group after the mice ADSCs were transfected with si-CEBPαand miR-10b inhibitor of silencing CEBPαat the same time(P<0.05),and there was no significant difference between the si-NC+miR-NC group and the miR-10b inhibitor+si-CEBPαgroup(P>0.05).Conclusion Down-regulation of miR-10b can promote the osteogenic differentiation of mice ADSCs through target regulation of CEBPαexpression.