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目的探讨mi R-93-5p是否通过靶向调控其预测靶基因Smad5的表达,从而抑制小鼠MSCs C3H10T1/2细胞的成骨分化。方法构建Smad5 3’-UTR-荧光素酶报告载体(pmi R-RB-REPORTTM),通过双荧光素酶报告基因检测,观察mi R-93-5p对Smad5 3’-UTR荧光素酶活性的影响,鉴定Smad5是否为mi R-93-5p的靶基因。将mi R-93-5p mimics(M组)与mi R-93-5p inhibitor(In组)及其对应的阴性对照组mi R-93-5p mimics阴性对照(MC组)与mi R-93-5p inhibitor阴性对照(In C组)分别转染至C3H10T1/2细胞中,并进行成骨诱导培养,48 h后分别采用实时荧光定量PCR(real-time fluorescent quantitative PCR,q RT-PCR)及Western blot检测各组细胞Smad5在m RNA及蛋白水平的相对表达量;14 d后通过茜素红染色检测各组细胞外钙盐的沉积情况,了解mi R-93-5p对C3H10T1/2细胞成骨分化的调控效应。结果双荧光素酶报告基因检测结果显示,mi R-93-5p能与Smad5 m RNA3’-UTR特异性结合,抑制其荧光素酶活性(P<0.05)。q RT-PCR检测示,M组及In组Smad5的m RNA相对表达量与对应阴性对照组MC组及In C组比较差异均无统计学意义(P>0.05);Western blot检测示,M组及In组Smad5蛋白相对表达量与对应阴性对照组比较差异均有统计学意义(P<0.05),其中M组较MC组表达量下调,而In组则较In C组表达量上调。茜素红染色示,M组钙盐沉积较MC组明显减少,而In组则较In C组钙盐沉积明显增多。结论Smad5是mi R-93-5p的靶基因,mi R-93-5p可通过靶向调控Smad5的表达,抑制小鼠C3H10T1/2细胞成骨分化。
Objective To investigate whether mi R-93-5p can regulate osteoblast differentiation of mouse MSCs C3H10T1 / 2 cells by regulating the expression of target Smad5. Methods Smad5 3’-UTR-luciferase reporter vector (pmiR-RB-REPORTTM) was constructed and the effect of mi R-93-5p on the luciferase activity of Smad5 3’-UTR was observed by dual luciferase reporter assay , To identify whether Smad5 mi R-93-5p target gene. Mi R-93-5p mimics (group M) and mi R-93-5p inhibitor (group In) and its corresponding negative control group, mi R-93-5p mimics negative control group (MC) 5p inhibitor negative control group (In C) were transfected into C3H10T1 / 2 cells respectively and cultured in osteogenic medium. After 48 h, real-time fluorescent quantitative PCR (q RT-PCR) and Western blot blot was used to detect the relative expression of Smad5 mRNA and protein in each group. The deposition of extracellular calcium in each group was detected by alizarin red staining 14 days later, and the effect of mi R-93-5p on the osteosynthesis of C3H10T1 / 2 cells Differentiation regulation effect. Results The results of dual luciferase reporter assay showed that mi R-93-5p specifically bound to Smad5 m RNA 3’-UTR and inhibited its luciferase activity (P <0.05). q RT-PCR showed that there was no significant difference in the relative expression level of Smad5 mRNA in M group and In group compared with that in MC group and InC group (P> 0.05). Western blot showed that M group (P <0.05). The expression of Smad5 in group M was significantly lower than that in group MC (P <0.05), while the expression of Smad5 in group In was significantly higher than that in group In. Alizarin red staining showed that the calcium deposition in M group decreased significantly compared with that in MC group, while that in In group was significantly higher than that in In group. Conclusion Smad5 is a target gene of mi R-93-5p. Mi R-93-5p can inhibit the osteogenic differentiation of mouse C3H10T1 / 2 cells by regulating the expression of Smad5.