LncRNA (Tcons＿00072170)-miRNA143-KRAS Axis Modulates The Neovasculogenesis Of Endothelial Progenitor Cells In Distraction Osteogenesis Induced By HIF-1α

ObjectiveThe osteogenesis rate of distraction osteogenesis（DO）is 4 to 6 times the growth rate of infants,far beyond fracture healing.The osteogenesis mechanism of DO is complicated and inconclusive,becoming one of the research hotspots at home and abroad.Bone is a highly vascularized tissue and neovasculogenesis serves as a prerequisite for new bone formation.In the sequence study on the pre-DO osteogenic mechanism,our team found rapid formation of blood vessels in the DO tissue,in which hypoxic microenvironment,endothelial progenitor cells（EPCs）,and non-codingRNA played a pivotal role.Therefore,the purpose of this study was to investigate the mechanism of lncRNA（TCONS₀0072170）-miRNA143-KRAS axis modulating the neovasculogenesis of EPCs in DO induced by HIF-1α,providing new ideas for clarifying the biological mechanism of DO rapid osteogenesis.Methods1.Six healthy dogs were randomly divided into 2 groups.Animal models of canine mandible DO and bone fracture（BF）were constructed,and the animals were killed and harvested immediately after the end of distraction.The structure of neovascularization in the distraction area was observed by HE staining.The levels of CD133,CD34,HIF-1α,ZEB1,β-catenin and beclin1 were detected by immunofluorescence staining.The mRNA expression levels of HIF-1α,VEGF,b FGF,E-cadherin,ZEB1,β--catenin and beclin1 in tissues were detected by RT-PCR.2.Bone marrow EPCs were isolated and cultured by density gradient centrifugation combined with special induction medium adherent culture method,and Co Cl2 was used to construct cell hypoxia model in vitro.The effect of hypoxia on the proliferation and migration of EPCs was detected by CCK-8 and transwell analysis.The mRNA and protein expression levels of HIF-1α,VEGF,b FGF,E-cadherin,ZEB1,β-catenin and beclin1 were detected by RT-PCR and WB.The levels of ZEB1,β-catenin and beclin1 were detected by immunofluorescence staining.3.Novel lncRNAs associated with HIF-1α were screened by RT-PCR.Bioinformatics methods were used for prediction of target miRNA of lncRNA and target mRNA of miRNA.The prediction results were validated via luciferase experiment.4.The lncRNA（TCONS₀0072170）overexpression and knockdown were constructed using Lentiviruses.The miRNA143 mimics and inhibitor were constructed using Lentiviruses.The effect of lncRNA（TCONS₀0072170）and miRNA143 on the proliferation,migration,and tube formation of EPCs was detected by CCK-8,transwell analysis,and matrigel tube formation.The mRNA expression levels of HIF-1α,VEGF,b FGF,lncRNA（TCONS₀0072170）,miRNA143,KRAS,PI3 K and AKT were detected by RT-PCR.The expression levels of HIF-1α,VEGF,b FGF,KRAS,PI3 K and AKT were detected by WB.5.Twenty-four healthy dogs were randomly divided into 4 groups: control group（Con）,EPC group（EPC）,virus control group（NC）and lncRNA（TCONS₀0072170）overexpressed group（TCONS₀0072170）.The animal models of dog mandible distraction osteogenesis were constructed,and the corresponding animals were killed and collected from each group at D14（immediately after distraction）and D28（2 weeks after distraction）.The formation and reconstruction of new bone were observed by general observation,CBCT and micro CT.The structures of new vessels and trabeculae in the distraction area were observed by HE staining.The amount of angiogenesis was quantitatively measured by CD31 immunohistochemistry and counting of new microvessels.The expression of alkaline phosphatase in the distraction gap was evaluated by alkaline phosphatase detection.Results1.The result of HE staining showed that the new tissues in the DO group contained a large number of dense fibrous tissues,the direction of which was consistent with the direction of distraction forces,and large amounts of red blood cells could be seen in the fibrous tissue space,which was consistent with the histological manifestations in the early stage of angiogenesis.The new tissue of BF group contained disordered and scattered fibrous tissue,and a lot of inflammatory cells infiltrated into it.The immunofluorescent analysis results revealed more positive to CD133,CD34,β-catenin,beclin1,HIF-1α and less positive to ZEB1 in the new tissues in DO group than in the BF group.The RT-PCR results revealed that DO group significantly induced the mRNA expression of HIF-1α,E-cadherin,β-catenin,beclin1,VEGF,FGF compared with the BF group.ZEB1 mRNA were decreased in DO group.2.The results showed that 0.1 and 0.2 m M Co Cl2 could promote EPCs proliferation and migration,however 0.5 m M Co Cl2 inhibits the processes in 24 h and 48 h.The results showed that 0.1 m M Co Cl2 significantly increased the mRNA expression of HIF-1α,E-cadherin,β-catenin,beclin1,VEGF,FGF compared with the control.ZEB1 mRNA were decreased in cells treated with 0.1 m M Co Cl2.The results of western blot revealed that the Co Cl2 could improve the levels of HIF-1α,E-cadherin,β-catenin,beclin1,VEGF,FGF,but decrease the levels of ZEB1 of EPCs.The immunofluorescent results revealed more positive to β-catenin,beclin1,and less positive to ZEB1 in EPCs treated with or without 0.1 m M Co Cl2.3.RT-PCR results showed that lncRNA（TCONS₀0072170）was correlated with HIF-1α.The miRDB website predicted that lncRNA（TCONS₀0072170）might bind to miRNA143,and the target gene of miRNA143 was KRAS.Luciferase experiments verified the targeting relationship between lncRNA（TCONS₀0072170）and miRNA143,miRNA143 and KRAS.4.The lncRNA（TCONS₀0072170）overexpression improved proliferation,migration,and tube formation of EPCs,while the lncRNA（TCONS₀0072170）knockdown declined.The lncRNA（TCONS₀0072170）overexpression increased the expression of HIF‐1α,VEGF,b FGF,and KRAS/PI3K/AKT,while the lncRNA（TCONS₀0072170）knockdown decreased.The miRNA143 mimics declined proliferation,migration,and tube formation of EPCs,while the miRNA143 inhibitor improved.The miRNA143 mimics decreased the expression of HIF‐1α,VEGF,b FGF,and KRAS/PI3K/AKT,while miRNA143 inhibitor increased.5.By CBCT and micro CT examination,it was found that the bone mineral density in the distraction space of the all the experimental groups at D28 was higher than that at D14,the bone mineral density in the stretch space of the control group was the lowest,the bone mineral density in the lncRNA（TCONS₀0072170）overexpressed group was the highest,and there was no significant difference in the bone mineral density between the EPC group and the virus control group.HE staining,CD31 immunohistochemical staining and new microvessel counting results at D14 show that the control group had the lowest vascular maturity and numbers of microvessels in the distraction space,and the lncRNA（TCONS₀0072170）overexpressed group had the highest vascular maturity and numbers of microvessels,while the EPC group and the virus control group had no significant difference.HE staining results at D28 showed that the control group had slender trabecula,while the lncRNA（TCONS₀0072170）overexpressed group had thicker trabecula.The highest activity of alkaline phosphatase was found in the lncRNA（TCONS₀0072170）overexpressed group.Conclusions1.Our studies showed that HIF‐1α promoted vasculogenesis in DO and EPCs,and the mechanism may involve Mesenchymal-Epithelial transition（MET）,Wnt/β-catenin signaling pathway,and autophagy.2.LncRNA（TCONS₀0072170）-miRNA143-KRAS axis modulates the neovasculogenesis of endothelial progenitor cells in distraction osteogenesis induced by HIF-1α.3.The EPCs modified by lncRNA（TCONS₀0072170）gene promoted the neovasculargenes and osteogenesis in DO.