微小RNA-345-5p抑制Smad1蛋白翻译调节成骨细胞功能

MicroRNA-345-5p inhibit osteoblast function by inhibiting the translation of Smad1 protein

目的探索微小RNA(miRNA,miR)-345-5p对成骨细胞的调控机制。方法利用miRNA芯片寻找差异表达miRNA。pLVX-IRES-ZsGreen1质粒构建miR-345-5p过表达载体,建立过表达miR-345-5p大鼠成骨细胞、过表达变异miR-345-5p大鼠成骨细胞和空白对照组。通过细胞计数试剂盒(CCK-8)及生长曲线实验对比3组细胞增殖能力,双荧光素酶报告基因实验验证miR-345-5p与Smad1基因相互作用。构建miR-345-5p+空质粒、对照RNA+空质粒、对照RNA+Smad1质粒和miR-345-5p+Smad1质粒组,在大鼠成骨细胞内验证miR-345-5p与Smad1基因的相互作用。采用Student-t检验和ANOVA分析。结果生信分析提示miR-345-5p在骨折中的高表达,Smad1为其调控分子。将miR-345-5p质粒导入大鼠成骨细胞,CCK-8提示miR-345-5p质粒较miR-345-5p变异体质粒(48 h:q=7.453,P<0.01,72 h:q=15.34,P<0.01)及空白质粒(48 h:q=6.963,P<0.01,72 h:t=12.68,P<0.01)成骨细胞的活性显著增强。生长曲线显示转入miR-345-5p质粒的成骨细胞48 h时细胞数量显著低于空白对照组(q=3.814,P<0.05),72 h时细胞数量显著低于miR-345-5p变异(q=11.010,P<0.01)和对照组(q=10.410,P<0.01)。双荧光素酶报告基因实验显示miR-345-5p可降低野生型Smad1-3’端非编码区(3’UTR)质粒组荧光素酶的活性,细胞实验证实miR-345-5p显著降低大鼠成骨细胞的活性和生长曲线,过表达Smad1后成骨细胞的活动和生长曲线得到恢复。结论miR-345-5p通过调控Smad1分子调控大鼠成骨细胞的增殖参与骨折的愈合。

Objective To explore the regulation of microRNA(miRNA,miR)-345-5p on osteoblast.Methods MiRNA expression data sets were analyzed to find differentially expressed miRNAs.The pLVX-IRES-ZsGreen1 plasmid was used to construct overexpression vectors.The proliferation ability of the three groups of cells were tested by cell counting kit-8(CCK-8)and growth,the dual luciferase assay verified the interaction of miR-345-5p and Smad1 gene.Cellular studies were established to verify the interaction between miR-345-5p and Smad1 genes in rat osteoblasts.Student-t test and ANOVA were used for analysis.Results The bio-information analysis showed miR-345-5p and its target molecular Smad1.After the miR-345-5p plasmid was transformed into rat osteoblasts,CCK-8 showed miR-345-5p plasmid significantly enhanced the activity of osteoblasts(48 h:q=7.453,P<0.01,72 h:q=15.34,P<0.01).Growth assay showed the number of osteoblasts with miR-345-5p plasmid was significantly lower than that of the control group at 48 h(q=3.814,P<0.05),miR-345-5p mutation(P<0.01)and the control group(P<0.01)at 72 h.The dual-luciferase assay showed miR-345-5p reduced the fluorescence activity of wild-type Smad1-3′untranslated regions(3′UTR)plasmid.MiR-345-5p significantly reduced the activity of rat osteoblasts through modulating Smad1 in cellular experiment.Conclusion MiR-345-5p regulates osteoblasts through Smad1,thus participating in fracture healing.