Abstract:Objective To investigate the effect of miR-429 on the osteogenic differentiation of MC3T3-E1 cells in a hypoxic environment.Methods The hypoxia environment was simulated by Cobalt chloride (CoCl-2)in vitro and in vivo,the expression of hypoxia-inducible factor-1α (HIF-1α)was detected by Western blotting to verify the hypoxia condition.The expression of miR-429 in cell tissues and the fracture site was observed by real time-quantitative polymerase chain reaction,(RT-qPCR).The proliferation and apoptosis of MC3T3-E1 cells under hypoxia were detected.miR-429 was overexpressed in MC3T3-E1 cells by lentiviral transfection technology as the experimental group and the control group was transfected with empty plasmid cells,and alkaline phosphatase (ALP)activity detection,alizarin red staining,western blotting and other methods were used to observe the ALP activity,mineral content and protein expression of various osteogenic markers in cells to verify the osteogenic differentiation of MC3T3-E1 cells.The fracture model was injected locally with a lentiviral vector overexpressing miR-429,and the fracture healing was observed by Micro-CT and qRT-PCR.Results The high expression of HIF-1α in MC3T3-E1 cells in a hypoxic environment up-regulated the expression of miR-429.After MC3T3-E1 cells were transfected with miR-429 overexpressing virus,the activity of ALP in the cells,the mineralization of cell matrix and the levels of osteogenic markers (RUNX2 and OC)were higher than those in the control group.After injection of lentiviral reagents overexpressing miR-429 in vivo,the Micro-CT of fracture healing were better and the expression of miR-429 in bone tissue were higher than those in the control group.Conclusion MiR-429 is highly expressed in hypoxic environment and plays a role in inducing osteogenic differentiation of MC3T3-E1 cells.
[1]李宁,李新萍,杨明辉,等.老年髋部骨折的骨质疏松症诊疗专家共识[J].中华骨与关节外科杂志,2021,14(8):657-663.
[2]中华医学会骨科学分会青年骨质疏松学组;中国老年学和老年医学学会老年病分会骨科专家委员会;中国医师协会急救复苏专业委员会创伤骨科与多发伤学组;上海市中西医结合学会骨质疏松专业委员会.中国骨质疏松性骨折骨修复策略专家共识(2019)[J].中华创伤杂志,2019,(9):769-775.
[3]Huang J,Liu L,Feng M,et al.Effect of CoCl-2 on fracture repair in a rat model of bone fracture[J].Mol Med Rep,2015,12(4):5951-5956.
[4]Qiao J,Huang J,Zhou M,et al.Inhibition of HIF-1alpha restrains fracture healing via regulation of autophagy in a rat model[J].Exp Ther Med,2019,17(3):1884-1890.
[5]Bartel DP.MicroRNAs:Genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[6]Ge L,Wang Y,Cao Y,et al.MiR429 improved the hypoxia tolerance of human amniotic cells by targeting HIF-1alpha[J].Biotechnol Lett,2018,40(11-12):1477-1486.
[7]周萌,黄江,安帅,等.远隔缺血后适应对老龄大鼠骨折愈合的影响及研究[J].实用骨科杂志,2018,24(8):715-720.
[8]Kim Y,Brodt Md,Tang Sy,et al.MicroCT for scanning and analysis of mouse bones[J].Methods in molecular biology (Clifton,NJ),2021(2230):169-198.
[9]Saliminejad K,Khorshid HRK,Fard SS,et al.An overview of microRNAs:Biology,functions,therapeutics,and analysis methods[J].J Cell Physiol,2019,234(5):5451-5465.
[10]Hong L,Sun H,Amendt BA.MicroRNA function in craniofacial bone formation,regeneration and repair[J].Bone,2021(144):115789.
[11]Wang C,Zheng GF,Xu XF.MicroRNA-186 improves fracture healing through activating the bone morphogenetic protein signalling pathway by inhibiting SMAD6 in a mouse model of femoral fracture:An animal study[J].Bone Joint Res,2019,8(11):550-562.
[12]Deng J,Wu J,Zhu Y.Inhibition of microRNA-9 improves fracture healing by modulating the bone morphogenetic protein-7 pathway[J].Pharmacology,2019,104(5-6):352-358.
[13]Jia HL,Zhou DS.Downregulation of microRNA-367 promotes osteoblasts growth and proliferation of mice during fracture by activating the PANX3-mediated Wnt/β-catenin pathway[J].J Cell Biochem,2019,120(1):8247-8258.
[14]Yang C,Liu X,Zhao K,et al.miRNA-21 promotes osteogenesis via the PTEN/PI3K/Akt/HIF-1α pathway and enhances bone regeneration in critical size defects[J].Stem Cell Res Ther,2019,10(1):65.
[15]Harrison Js,Rameshwar P,Chang V,et al.Oxygen saturation in the bone marrow of healthy volunteers[J].Blood,2002,99(1):394.
[16]Hannah Ss,Mcfadden S,Mcneilly A,et al."Take My Bone Away?" Hypoxia and bone:A narrative review[J].J Cell Physiol,2021,236(2):721-740.
[17]Serocki M,Bartoszewska S,JanaszakJasiecka A,et al.miRNAs regulate the HIF switch during hypoxia:A novel therapeutic target[J].Angiogenesis,2018,21(2):183-202.