miR-27b/PPARγ2轴对小鼠胚胎成骨前体细胞增殖和成骨细胞分化的意义

Significance of miR-27b/peroxisome proliferators-activated receptor gamma 2 axis for proliferation and osteoblast differentiation of mouse embryonic osteogenic precursor cells

背景:先前的研究证实了miR-27b在脂质水平的作用,可以控制多个对血脂异常有重要影响的基因,推测miR-27b可能通过靶向PPARγ2在骨质疏松症中发挥作用。目的:探讨miR-27b/PPARγ轴在地塞米松诱导小鼠胚胎成骨细胞前体细胞MC3T3-E1细胞建立骨质疏松症细胞模型中的影响及相关作用机制。方法:体外地塞米松诱导MC3T3-E1细胞后,通过Lipofectamine^(■)2000转染miR-27b模拟物、miR-27b抑制剂、NC-模拟物、NC-抑制剂、siPPARγ2、siNC,使用二甲基亚砜处理作为对照。细胞诱导培养后,检测细胞活性、碱性磷酸酶活性以确定细胞成骨、分化水平;实时荧光定量PCR法和Western blot法检测成骨分化基因miR-27B、PPARγ2、Runx2、骨形态发生蛋白2和骨钙素的mRNA及蛋白表达水平;生信分析预测miR-27b下游靶基因,并用双荧光素酶基因报告实验验证。结果与结论:①地塞米松处理显著降低了MC3T3-E1细胞的活力和miR-27b表达水平;与二甲基亚砜相比,地塞米松处理在24和48 h显著抑制MC3T3-E1细胞的活力并显著下调了miR-27b的表达水平;②miR-27b可直接调控PPARγ2;与相应的NC相比,miR-27b模拟物显著上调了miR-27b的表达水平,而miR-27b抑制剂显著下调了miR-27b的表达水平;与相应的NC相比,PPARγ2 mRNA和相应蛋白质表达被miR-27b模拟物抑制,并被miR-27b抑制剂显著增强;③miR-27b过表达减弱了地塞米松抑制MC3T3-E1细胞的增殖和成骨分化;与二甲基亚砜+NC-mimic组相比,地塞米松显著抑制成骨细胞分化;而地塞米松介导的抑制作用则被miR-27b模拟物所逆转,细胞碱性磷酸酶活性和骨形态发生蛋白2、Runx2和骨钙素蛋白表达水平部分恢复;④抑制miR-27b通过上调PPARγ2抑制MC3T3-E1细胞的增殖和成骨分化;miR-27b的敲除明显增加了PPARγ2的表达,并降低了细胞活力、成骨细胞分化、碱性磷酸酶活性以及骨形态发生蛋白2、Runx2和骨钙素的表达水平;PPARγ2可能是miR-27b的直接作用靶点。

BACKGROUND:Previous studies have confirmed that miR-27b at the lipid level can control multiple genes that have an important impact on dyslipidemia.It is speculated that miR-27b may function in osteoporosis by targeting peroxisome proliferators-activated receptorsγ2(PPARγ2).OBJECTIVE:To investigate the effect of miR-27b/PPARγ2 axis in the dexamethasone-induced osteoporosis model in mouse embryonic osteogenic precursor cells MC3T3-E1 and related mechanisms of action.METHODS:After MC3T3-E1 cells were dexamethasone-induced and cultured in vitro,miR-27b mimic,miR-27b inhibitor,NC-mimic,NC-inhibitor,siPPARγ2,and siNC were transfected with Lipofectamin^(■)2000,and dimethyl sulfoxide was used as a control.After the cells were induced and cultured,the cell viability and alkaline phosphatase activity were detected to determine the osteogenesis and differentiation levels of the cells.The mRNA and protein expression levels of osteogenic differentiation genes miR-27B,PPARγ2,Runt-related transcription factor 2(Runx2),bone morphogenetic protein 2 and osteocalcin were detected by real-time fluorescence quantitative PCR and western blot methods.Bioinformatics analysis predicted the downstream target genes of miR-27b followed by verification with dual luciferase gene reporter experiments.RESULTS AND CONCLUSION:Dexamethasone treatment significantly reduced cell viability and miR-27b expression levels in MC3T3-E1 pre-osteoblasts.Compared with dimethyl sulfoxide,dexamethasone significantly inhibited MC3T3-E1 cell viability and downregulated miR-27b expression levels at 24 and 48 hours.miR-27b could directly regulate PPARγ2.Compared with the corresponding NC-mimic,the miR-27b mimic significantly upregulated the expression level of miR-27b,while the miR-27b inhibitor significantly downregulated the expression level of miR-27b.Furthermore,PPARγ2 miRNA and protein expression levels were inhibited by the miR-27b mimic and significantly enhanced by the miR-27b inhibitor.miR-27b overexpression attenuated the inhibitory effect of dexamethasone on the proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts.Dexamethasone significantly inhibited osteoblast differentiation compared with the dimethyl sulfoxide+NC-mimic group.In contrast,the dexamethasone-mediated inhibition was reversed by the miR-27b mimic.Cellular alkaline phosphatase activity and bone morphogenetic protein 2,Runx2 and osteocalcin protein expression levels were partially restored.Inhibition of miR-27b suppressed proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts through the upregulation of PPARγ2.Knockout of miR-27b significantly increased PPARγ2 expression and decreased cell viability,osteoblast differentiation,alkaline phosphatase activity,and expression levels of bone morphogenetic protein 2,Runx2 and osteocalcin.PPARγ2 may be a direct target of miR-27b.