The Role And Mechanism Of LncRNA H19 Involved In The Process Of Promoting Angiogenesis In HAMSC

Objective: Our previous studies showed that the human amniotic mesenchymal stem cells(HAMSC)can promote bone regeneration to repair bone defects through their ability of inducing angiogenesis.However,the related mechanism is still unclear.According to gene microarray tests,the expression of LncRNA H19 has been significantly increased in the process of angiogenesis caused by HAMSC.Therefore,our study aimed to investigate whether H19 was involved in the process of promoting angiogenesis in HAMSC,which provided theoretical basis that HAMSC can to promote new bone formation through inducing angiogenesis.Methods: To elucidate the important role of HAMSC in promoting the sprouting and tube formation of human umbilical vein endothelial cell(HUVEC),we constructed the HAMSC with H19 stably low-expressed(HAMSC-siH19),and 3D co-cultured with HUVEC in vitro and in vivo to investigate the function of H19 on the early stage of promoting angiogenesis.CCK-8 and transwell assays were conducted to find out whether the conditioned medium secreted by HAMSC altered proliferation and migration of HUVEC after down-regulating H19.Angiogenic cytokine array and ELISA were used to analyze the different expression profiles between the conditioned medium of HAMSC-siH19 and HAMSC-NC.RT-PCR and Western Blot were conducted to compare the expression of angiogenic markers between HAMSC-siH19 and HAMSC-NC on protein and RNA levels.In the mechanism research,we performed CHIP and RIP assays to test whether LncRNA H19 could bind to methyltransferase EZH2,and recruit methyl to the promoter region of angiogenesis inhibitor gene,thus decrease its expression.Results: HAMSC-siH19 and HAMSC-NC were respectively 3D co-cultured with HUVEC in Fibrinogen gels in vitro and injected in nude mice subcutaneously.Compared with the control group,the sprouting length and angiogenesis capacity of HUVEC reduced in the HAMSC-siH19 group.Then,we collected the conditioned medium of HAMSC-siH19 and HAMSC-NC to culture HUVEC.The results of CCK-8 and transwell assays demonstrated that the conditioned medium secreted by HAMSC reduced proliferation and migration of HUVEC after down-regulating H19.Compared with the control conditioned medium,the concentrations of the angiogenesis factors in HAMSC-siH19 were decreased,and the angiogenesis inhibitors increased.The results of RT-PCR and Western Blot showed that the expressions of angiogenesis factors in HAMSC were lower expressed and the angiogenesis inhibitors were elevated after down-regulating H19.Next,CHIP and RIP experiments were performed to demonstrate that the expression of an angiogenesis inhibitor Vasohibin1(VASH1)was regulated by methyltransferase EZH2,which mediated histone methylation modification.H19 could bind to EZH2,then,enhances the epigenetic regulation of EZH2.Conclusion: In the 3D co-cultured models in vitro and in vivo,LncRNA H19 played an important role in the process of promoting angiogenesis in HAMSC.The concentrations of the angiogenesis factors as ANG and PDGF in the conditioned medium secreted by HAMSC were decreased after down-regulating H19,and the angiogenesis inhibitors such as VASH1 and PF4 were increased.Meanwhile,the expressions of angiogenesis factors in HAMSC were lower expressed and the angiogenesis inhibitors were elevated after down-regulating H19.H19 inhibits the expression of angiogenesis inhibitor gene VASH1 by binding to methyltransferase EZH2,and recruiting methyl to the promoter region of VASH1.Then,the concentration of VASH1 secreted by HAMSC was decreased,promoting angiogenesis.