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目的探讨微小RNA-451(micro RNA-451,miRNA-451)靶向调节钙结合蛋白39(calcium binding protein 39,CAB39)表达,促进MSCs向成骨细胞分化的效应。方法选择pMIR-report质粒及pRL-TK质粒,通过双荧光素酶基因报告系统,鉴定CAB39是否为miRNA-451的靶基因。将pre-miRNA-451(A组)及anti-miRNA-451(C组)分别转染到C3H10T1/2细胞中,同时设置相应的pre-miRNA阴性对照组(B组)和anti-miRNA阴性对照组(D组);成骨诱导培养7、14 d,采用实时荧光定量PCR检测成骨标志物Runx2、ALP mRNA相对表达量;诱导培养14 d,采用Western blot检测细胞中CAB39蛋白表达,茜素红染色观察细胞外钙基质沉积情况。结果经鉴定,CAB39是miRNA-451的靶基因。实时荧光定量PCR检测示,成骨诱导培养7、14 d,A组Runx2、ALP mRNA相对表达量显著高于B组,C组显著低于D组,差异均有统计学意义(P<0.05)。成骨诱导培养14 d,Western blot检测示A组CAB39蛋白表达量为0.55±0.05,较B组1.00±0.07明显降低;而C组为1.21±0.05,较D组1.00±0.04明显增高;差异均有统计学意义(P<0.05)。茜素红染色示A组细胞外钙基质沉积较B组明显增多,而C组细胞外钙基质沉积较D组明显减少。结论 miRNA-451可通过抑制CAB39表达,促进MSCs成骨分化。
Objective To investigate the effect of microRNA-451 (miRNA-451) on the regulation of osteoblast differentiation by regulating the expression of calcium binding protein 39 (CAB39). Methods The pMIR-report and pRL-TK plasmids were selected to determine whether CAB39 was the target gene of miRNA-451 by dual luciferase gene reporter system. The pre-miRNA-451 (group A) and anti-miRNA-451 (group C) were transfected into C3H10T1 / 2 cells respectively and the corresponding pre-miRNA negative control group (group B) and anti-miRNA negative control Group D (group D). After osteogenic induction for 7,14 days, the relative expression of Runx2 and ALP mRNA was detected by real-time fluorescence quantitative PCR. The expression of CAB39 protein was detected by Western blot for 14 days. Red staining to observe extracellular calcium matrix deposition. Results It was identified that CAB39 is the target gene of miRNA-451. Real-time fluorescent quantitative PCR showed that the relative expression of Runx2 and ALP mRNA in group A was significantly higher than that in group B at 7 and 14 days after osteogenic induction, and the difference was statistically significant (P <0.05) . Western blot showed that the expression of CAB39 protein in group A was 0.55 ± 0.05, which was significantly lower than 1.00 ± 0.07 in group B, while it was 1.21 ± 0.05 in group C, which was significantly higher than 1.00 ± 0.04 in group D There was statistical significance (P <0.05). Alizarin red staining showed that the deposition of extracellular calcium matrix in group A was significantly increased than that in group B, while the deposition of extracellular calcium matrix in group C was significantly lower than that in group D. Conclusion miRNA-451 can promote the osteogenic differentiation of MSCs by inhibiting the expression of CAB39.