miR-199a调控IGF1表达对机械刺激下成骨细胞分化的影响

Effect of miR-199a regulating IGF1 expression on differentiation of osteoblasts under mechanical stimulation

目的:探讨微小RNA(miR)-199a在机械牵张力刺激下MC3T3-E1细胞中的表达变化及其对牵张力刺激MC3T3-E1细胞成骨分化的作用机制。方法:对体外培养的MC3T3-E1细胞加载12%牵张力0、3、6、12和24 h后,利用碱性磷酸酶(ALP)活性检测试剂盒检测ALP活性,实时荧光定量PCR检测骨钙素(OCN)、成骨细胞特异性转录因子Osterix(OSX)、Runt相关转录因子2(Runx2)mRNA和miR-199a的表达。将MC3T3-E1细胞分为对照组、牵张力组、牵张力+miR-NC组和牵张力+miR-199a组,加载12%牵张力和转染miR-199a模拟物后,观察miR-199a和OCN、OSX、Runx2 mRNA及蛋白表达以及ALP活性。茜素红S(ARS)染色观察钙结节形成能力。采用双荧光素酶报告基因实验检测miR-199a与胰岛素样生长因子1(IGF1)的靶向关系,实时荧光定量PCR法和免疫印迹法检测miR-199a模拟物对IGF1 mRNA和蛋白表达的影响。采用SPSS 24.0软件包对数据进行统计学分析。结果:与0 h时间点相比,以机械牵张力刺激3、6、12和24 h后,MC3T3-E1细胞ALP活性和OCN、OSX、Runx2 mRNA表达水平均显著升高,而miR-199a表达水平显著降低(P<0.05),12 h时变化最为显著。与对照组相比,牵张力组细胞中miR-199a表达水平显著降低,而细胞ALP活性、OCN、OSX、Runx2 mRNA及蛋白表达水平、钙结节形成水平均显著升高(P<0.05);与牵张力组相比,牵张力+miR-NC组细胞中上述各指标差异均无统计学意义(P>0.05);与牵张力+miRNC组相比,牵张力+miR-199a组细胞中miR-199a表达水平显著升高,而细胞ALP活性、OCN、OSX、Runx2 mRNA及蛋白表达水平、钙结节形成水平均显著降低(P<0.05)。miR-199a可与IGF1靶向结合,miR-199a模拟物可使MC3T3-E1细胞中IGF1 mRNA和蛋白表达水平显著降低(P<0.05)。结论:miR-199a可抑制机械牵张力刺激诱导的MC3T3-E1细胞成骨分化,其作用机制可能与靶向调控IGF1表达有关。

PURPOSE:To investigate the expression change of microRNA(miR)-199a in MC3T3-E1 cells stimulated by mechanical stretch and its mechanism of osteogenic differentiation.METHODS:MC3T3-E1 cells cultured in vitro were loaded with 12%stretch for 0,3,6,12 and 24 hours,alkaline phosphatase(ALP)activity was detected by ALP activity test kit,the expressions of osteocalcin(OCN),osteoblast specific transcription factor osterix(OSX),Runt related tran-scription factor 2(Runx2)mRNA and miR-199a were detected by real-time fluorescence quantitative PCR.MC3T3-E1cells were divided into control group,stretch group,stretch+miR-NC group and stretch+miR-199a group,and the expressions of miR-199a,OCN,OSX,Runx2 mRNA and protein and ALP activity were observed after 12%stretch and transfection of miR-199a.Alizarin red S(ARS)staining was used to observe calcium nodule formation ability.The target relationship between miR-199a and insulin like growth factor-1(IGF1)was detected by double luciferase reporter gene assay;in addition,the effect of miR-199a mimic on IGF1 mRNA and protein expression was detected by real-time fluo-rescent quantitative PCR and Western blot.SPSS 24.0 software package was used to analyze the data.RESULTS:Compared with those at the time point of 0 h,ALP activity and expression level of OCN,OSX and Runx2 mRNA of MC3T3-E1 cells at 3,6,12 and 24 hours after mechanical stretch stimulation were significantly higher,while the expression level of miR-199a was significantly lower(P<0.05),and the change was most significant at 12 h.Compared with those in the control group,the expression level of miR-199a was significantly lower in the stretch group,while ALP activity,the expression level of OCN,OSX and Runx2 mRNA and protein,calcium nodule formation level were significantly high in the stretch group(P<0.05);there was no significant difference in the above indexes between the stretch group and stretch+miR-NC group(P>0.05).Compared with stretch+miR-NC group,the expression level of miR-199a in stretch+miR-199a group was significantly higher;while ALP activity,OCN,OSX,Runx2 mRNA and protein expression level,calcium nodule formation level were significantly lower(P<0.05).miR-199a could targetedly bind to IGF1,and the expression level of IGF1 mRNA and protein in MC3T3-E1 cells was significantly reduced by miR-199a mimic(P<0.05).CONCLUSIONS:MiR-199a can inhibit the osteogenic differentiation of MC3T3-E1 cells induced by mechanical stretch stimulation,and its mechanism may be related to the targeted regulation of IGF1 expression.