miR-98-5p靶向HMGA2通过PI3K/Akt/GSK-3β通路调控骨再生的机制研究

Mechanism of miR-98-5p Targeting HMGA2 in Regulation of Bone Regeneration through PI3K/Akt/GSK-3βPathway

目的研究miR-98-5p靶向高迁移率族蛋白A2(HMGA2)通过PI3K/Akt/GSK-3β通路调控骨再生的机制。方法构建细胞培养模型,根据成骨细胞诱导分化中细胞类别不同分为mBMMSCs组、hBMMSCs组及MC3T3-E1组,并根据对成骨细胞质粒不同转染方法分为正常组、A组(MC3T3-E1细胞诱导分化)、B组(MC3T3-E1细胞诱导分化后转染mimic control)、C组(MC3T3-E1细胞诱导分化后转染miR-98-5p mimic)及D组(MC3T3-E1细胞诱导分化后转染miR-98-5p mimic和HMGA2 plasmid)。检测各组碱性磷酸酶(ALP)、miR-98-5p、Runt相关转录因子2(RUNX2)和Osterix mRNA、HMGA2 mRNA表达水平及HMGA2、Bax、Bcl-2、Caspase-3、PI3K/Akt/GSK-3β通路蛋白相对表达水平。比较各组MC3T3-E1细胞增殖和凋亡情况。结果与0 d时比较,mBMMSCs组、hBMMSCs组、MC3T3-E1组7、14 d时ALP、RUNX2及Osterix mRNA水平均明显上升,miR-98-5p mRNA水平下降(P<0.05)。miR-98-5p minic组wt-HMGA2荧光素酶活性低于mimic control转染组(P<0.01)。C组HMGA2 mRNA及HMGA2蛋白表达水平显著低于于A组、B组和D组(P<0.01)。与正常组比较,A、B、C、D组MC3T3-E1细胞内ALP、RUNX2、Osterix mRNA水平均显著增高(P<0.01)。与A组和B组比较,C组和D组MC3T3-E1细胞内ALP、RUNX2、Osterix mRNA水平降低,但D组上述指标显著高于C组(P<0.01)。与A、B组比较,C、D组MC3T3-E1细胞增殖活性显著降低,凋亡率及Bax、Bcl-2、Caspase-3蛋白相对表达水平显著升高,且D组上述指标较变化C组更显著(P<0.01)。与A组、B组比较,C、D组MC3T3-E1细胞p-PI3K、p-Akt蛋白相对表达水平升高,且C组高于D组(P<0.01)。与A组、B组比较,C、D组MC3T3-E1细胞PI3K、Akt、p-GSK-3β及GSK-3β蛋白相对表达水平降低,且C组低于D组(P<0.01)。结论miR-98-5p可促进成骨细胞增殖分化及抑制其凋亡,作用机制可能与靶向作用HMGA2进行结合激活PI3K/Akt/GSK-3β通路,上调ALP磷酸化等有关。

Objective To study the mechanism of miR-98-5p targeting high mobility group protein A2(HMGA2)in regulation of bone regeneration through PI3K/Akt/GSK-3βpathway.Methods Cell culture models were constructed.According to different cell types in induction and differentiation of osteoblasts,they were divided into mBMMSCs group,hBMMSCs group and MC3T3-E1 group.According to different transfection methods of osteoblast plasmids,they were divided into normal group,group A(induced differentiation of MC3T3-E1),group B(induced differentiation of MC3T3-E1 and transfection with mimic control),group C(induced differentiation of MC3T3-E1 and transfection with miR-98-5p)and group D(induced differentiation of MC3T3-E1 and transfection with miR-98-5p and HMGA2 plasmid).The mRNA expressions of alkaline phosphatase(ALP),miR-98-5p,Runt related transcription factor 2(RUNX2),Osterix and HMGA2,and protein relative expressions of HMGA2,Bax,Bcl-2,Caspase-3 and PI3K/Akt/GSK-3βpathway were detected in all groups.Conditions of proliferation and apoptosis of MC3T3-E1 cells were compared among groups.Results Compared with those on the 0 d,mRNA expressions of ALP,Runx2 and Osterix were significantly higher,while mRNA expressions of miR-98-5p were significantly decreased in mBMMSCs,hBMMSCs and MC3T3-E1 groups on the 7 th and 14 th d(P<0.05).The luciferase activity of wt-HMGA2 in miR-98-5p mimic group was significantly lower than that in mimic control group(P<0.01).Expressions of HMGA2 mRNA and HMGA2 proteins in group C were significantly lower than those in group A,B and D(P<0.01).The mRNA expressions of ALP,RUNX2 and Osterix in MC3T3-E1 cells in group A,B,C and D were significantly increased compared with those in normal group(P<0.01).The mRNA expressions of ALP,RUNX2 and Osterix in MC3T3-E1 cells in group C and D were significantly decreased compared with those in group A and B,but the above indexes in group D were significantly higher than those in group C(P<0.01).In groups C and D,proliferation activities of MC3T3-E1 cells were significantly decreased,while apoptosis rates and relative expressions of Bax,Bcl-2 and Caspase-3 proteins were significantly increased compared with those in group A and B,and changes of the above indexes in group D were more significant than those in group C(P<0.01).Relative expressions of p-PI3k and p-Akt proteins in MC3T3-E1 cells in group C and D were significantly higher than those in group A and B,and the expressions in group C were significantly higher than those in group D(P<0.01).Relative expressions of PI3K,Akt,p-GSK-3βand GSK-3βof MC3T3-E1 cells in group C and D were significantly lower than those in group A and B,and the relative expressions in group C were significantly lower than those in group D(P<0.01).Conclusion The miR-98-5p may promote proliferation and differentiation of osteoblasts and inhibit their apoptosis.The mechanism of action may be related to the targeting of HMGA2 to activate PI3K/Akt/GSK-3βpathway and to up-regulate ALP phosphorylation.