microRNA-23a对氟暴露大鼠成骨肉瘤细胞成骨活性的影响及靶基因验证

Effects of microRNA-23a on osteogenic activity of fluoride-exposed UMR-106 cells and target gene verification

目的探讨miR-23a对染氟大鼠成骨肉瘤(UMR-106)细胞成骨活性的影响并验证其靶基因。方法体外培养UMR-106细胞,分别转染miR-23a的激动剂(mimics)和抑制剂(inhibitor),再以0、80μmol/L NaF对细胞进行染毒,培养24 h后利用实时荧光定量PCR(qRT-PCR)检测miR-23a、Ⅰ型胶原(COL1)、骨桥蛋白(OPN)、Runt相关转录因子2(Runx2)mRNA的表达水平;培养96 h后利用免疫蛋白印记法(Western blot)检测COL1、OPN、Runx2蛋白的表达水平;体外培养人胚胎肾细胞(293T),利用双荧光素酶报告基因法验证miR-23a可能的靶基因。结果与空白对照组相比,NaF组UMR-106细胞miR-23a、COL1、OPN、Runx2 mRNA表达水平明显升高(P<0.05);与激动剂对照组比较,过表达组miR-23a mRNA表达水平明显上升、COL1 mRNA表达明显降低(P<0.05),Runx2、OPN mRNA表达变化不显著(P>0.05);与抑制剂对照组相比,抑制组miR-23a、COL1、OPN、Runx2 mRNA表达明显降低(P<0.05)。与激动剂对照组比较,过表达组Runx2、OPN蛋白表达水平明显升高(P<0.05),COL1蛋白表达水平变化不显著(P>0.05);与抑制剂对照组相比,抑制组COL1、OPN、Runx2蛋白表达水平均明显降低(P<0.05)。与过表达组比较,过表达+NaF组OPN mRNA和蛋白表达水平明显升高(P<0.05),COL1、Runx2变化不显著;与抑制组比较,抑制+NaF组COL1、OPN、Runx2 mRNA和蛋白表达水平明显升高(P<0.05)。过表达miR-23a后的293T细胞中双特异性磷酸酶5(DUSP5 mRNA)和蛋白表达水平均明显降低(P<0.05),抑制mi R-23a后DUSP5表达水平均明显升高(P<0.05);双荧光素酶报告确定DUSP5为miR-23a的靶基因。结论miR-23a能促进染氟UMR-106细胞中成骨活性基因的表达;DUSP5是miR-23a的靶基因。

Objective To understand the effects of miR-23 a on the osteogenic activity of fluoride-exposed osteosarcoma(UMR-106)cells and to verify its target gene.Methods UMR-106 cells were exposed to NaF at 0 and 80μmol/L after transfecting miR-23 a agonist(mimics)and inhibitors(inhibitor)in vitro.qRT-PCR was used to detect the gene of miR-23 a,COL1,OPN and Runx2 after 24 h of culture.Western blot was used to detect the expression levels of COL1,OPN and Runx2 proteins after 96 h-culture.Dual luciferase reporter gene method was used to verify the possible target genes of miR-23 a.Results Compared with the control,the expression levels of miR-23 a,COL1,OPN,Runx2 gene in NaF group increased significantly(P<0.05).Compared with the mimics control group(miR-NC group),the level of miR-23 a gene increased significantly(P<0.05)and COL1 reduced(P<0.05),Runx2 and OPN revealed no significantly variation in miR-23 a overexpression group.Compared with inhibitor control(NC)group,the levels of miR-23 a,COL1,OPN,Runx2 gene were reduced in miR-23 a inhibitor group(P<0.05).Compared with miR-NC group,the levels of Runx2 and OPN protein increased(P<0.05),COL1 protein revealed no significant variation(P>0.05)in miR-23 a over-expression group;Compared with inhibitor NC group,protein expression levels of COLI,OPN,Runx2 were lower in miR-23 a inhibitor group(P<0.05).Compared with miR-23 a overexpression group,the levels of OPN mRNA and protein increased(P<0.05),and COL1 and Runx2 revealed no significant variation in mi R-23 a over-expression+NaF group;The mRNA and protein expression levels of COL1,OPN,Runx2 in miR-23 a inhibitor+NaF group were significantly higher than those in miR-23 a inhibitor group(P<0.05).The expression levels of DUSP5 mRNA and protein decreased in miR-23 a over-expression 293 T cells(P<0.05),and increased in miR-23 a inhibitor 293 T cells(P<0.05).The dual luciferase reporter identified DUSP5 as the target gene of miR-23 a.Conclusion miR-23 a can promote the expression of osteogenic activity genes in fluoride-exposed UMR-106 cells;DUSP5 is a target gene of miR-23 a.