Long Noncoding RNA H19 Knockdown in Human Amniotic Mesenchymal Stem Cells Suppresses Angiogenesis by Associating with Enhancer of Zeste Homolog 2 and Activating Vasohibin-1

Human amniotic mesenchymal stem cells (HAMSCs) are promising seed cells with great advantages in promoting angiogenesis. However, the mechanisms underlying angiogenesis facilitated by HAMSCs are still unclear. Long noncoding RNA H19 is involved in many biological processes, such as enhancing angiogenesis and proliferation, invasion, and migration of cancer cells. In this study, we constructed HAMSCs of stable low-expression H19 (HAMSC-shH19) and the scramble control (HAMSC-shNC) using lentiviral vectors, and in a three-dimensional coculture with human umbilical vein endothelial cells (HUVECs) to investigate the effect of H19 knockdown in HAMSCs on angiogenesis. Our results demonstrated that H19 knockdown significantly inhibited the angiogenic function of HAMSCs at an early stage in vitro and in vivo. The results of CCK8 and transwell assays demonstrated that the conditioned medium secreted by HAMSCs reduced proliferation and migration of HUVECs after downregulating H19. The angiogenesis factors expressed and secreted by HAMSC-shH19 were decreased compared with those secreted by the control, while angiogenesis inhibitors were elevated. Furthermore, we conducted chromatin immunoprecipitation and RNA-binding protein immunoprecipitation assays and found that H19 could interact with the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) and that H19 knockdown inhibited the ability of EZH2 to recruit methyl groups to the promoter region of the angiogenesis inhibitor gene vasohibin-1 (VASH1), thus increasing VASH1 expression and secretion of HAMSCs, suppressing angiogenesis. In summary, our study identified H19 as an important regulator in HAMSCs for promoting angiogenesis, which would help to construct ideal gene-modified seed cells to enhance angiogenesis in regenerative medicine.