miR-218-5p下调SD大鼠成骨细胞骨硬化蛋白的表达

miR-218-5p down-regulates the expression of sclerostin in osteoblasts of SD rats

目的:探讨SD大鼠成骨细胞中微小RNA-218-5p(miR-218-5p)对骨硬化蛋白(SOST)表达的影响及其作用机制。方法:用生物信息学数据库RNAhybrid和TargetScan预测miR-218-5p靶向SOST基因;构建携带靶基因野生型及突变型3’-非翻译区的双萤光素酶报告基因质粒,采用脂质体Lipofectamine 2000包裹双萤光素酶重组质粒及miR-218-5p模拟物(mimic)或抑制剂(inhibitor),共转染293T细胞,应用双萤光素酶报告基因检测试剂盒测定萤光素酶活性;分离并培养SD大鼠成骨细胞,茜素红染色鉴定,设miR-218-5p mimic组、miR-218-5p inhibitor组及空白和阴性对照组,转染SD大鼠成骨细胞,RT-PCR检测SOST的mRNA表达,Western blot法检测SOST的蛋白表达水平。结果:双萤光素酶报告基因实验验证SOST是miR-218-5p的潜在靶基因;RT-PCR和Western blot实验结果示,SD大鼠成骨细胞转染miR-218-5p mimic后,SOST mRNA及蛋白表达均明显下调,转染miR-218-5p inhibitor后均明显上调,与空白及阴性对照组比较差异有统计学意义(P<0.05)。结论:miR-218-5p靶向SOST基因,从而下调SD大鼠成骨细胞SOST蛋白的表达。

AIM: To investigate the effect of microRNA-218-5 p(miR-218-5 p) on the expression of sclerostin(SOST) in the osteoblasts of SD rats. METHODS: The bioinformatic prediction of miR-218-5 p targeting the SOST gene was performed using the databases of RNAmibrid and TargetScan. Dual-luciferase reporter plasmids carrying wild-type and mutant 3’-untranslated region of the target gene were constructed. The Lipofectamine 2000-encapsulated recombinant plasmids and miR-218-5 p mimic or inhibitor were co-transfected into the 293 T cells. Dual-luciferase reporter assay was used to measure luciferase activity. The osteoblasts of SD rast were isolated and cultured. The rat osteoblasts was identified by ali-zarin red staining. miR-218-5 p mimic, miR-218-5 p inhibitor, and blank and negative controls were used to transfect the osteoblasts of SD rats. The expression of SOST at mRNA and protein levels was determined by RT-PCR and Western blot. RESULTS: Dual-luciferase reporter assays validated that SOST was a potential target gene of miR-218-5 p. Compared with the blank and negative control groups, after transfection of miR-218-5 p mimic in the osteoblasts, the expression of SOST at mRNA and protein levels was significantly down-regulated, and after transfection of miR-218-5 p inhibitor in the osteoblasts, the expression of SOST at mRNA and protein levels was significantly up-regulated(P<0.01). CONCLUSION: miR-218-5 p targets SOST gene, and down-regulates the expression of SOST in the osteoblasts of SD rats.