Long non-coding RNA H19 facilitates bone marrow mesenchymal stem cell survival and vascularization in hypoxic-ischemic conditions in vitro

Hou Jing-ying; Wang Lei; Long Hui-bao; Wu Hao; Wu Quan-hua; Zhong Ting-ting; Zhou Chang-qing; Guo Tian-zhu; Chen Xu-xiang; Wang Tong

doi:10.3969/j.issn.2095-4344.0430

Abstract：BACKGROUND: Our previous study demonstrated that bone marrow mesenchymal stem cells (MSCs) presented with a low survival rate and newly formed vascular-like structures was sparsely distributed in the local infarct tissues after cell transplantation, which certainly impaired the therapeutic efficacy. Long non-coding RNA-H19 (lncRNA-H19) has been confirmed to be associated with MSCs differentiation and mediate vascularization. OBJECTIVE:To observe the influence of lncRNA-H19 on the survival and vascularization potential of MSCs in vitro and to explore the possible mechanism. METHODS:MSCs were obtained and cultured in vitro.Cells were divided into five groups:MSCs+H19,MSCs+H19 negative control (MSCs+H19 NC), MSCs+si-H19, MSCs+si-H19 negative control (MSCs+si-H19 NC) and MSCs groups. MSCs+H19 and MSCs+H19 NC groups were transfected with lncRNA-H19 and lncRNA-H19 scramble RNA respectively, while MSCs+siH19 and MSCs+si-H19 NC groups were transfected with lncRNA-H19 siRNA and lncRNA-H19 siRNA scramble respectively. Cells were cultured under hypoxic-ischemic condition (serum-free medium, 1% O2) for 24 hours. Then, cell proliferation and apoptosis were evaluated using MTS and TUNEL, respectively. Cell supernatant from each experimental group was further co-cultured with human umbilical vein endothelial cells to induce vascularization. The expression of vascular endothelial growth factor A (VEGFA) was thereafter detected using western blot assay. RESULTS AND CONCLUSION: Compared with MSCs+H19 NC and MSCs groups, MSCs+H19 group presented with significantly higher proliferation rate, lower apoptosis percentage and a larger number of vascular branches on matrigel (P < 0.01). There was a significantly higher expression of VEGFA in the MSCs+H19 group than MSCs+H19 NC and MSCs groups. Compared with the MSCs and MSCs+si-H19 NC groups, MSCs+H19 group presented with significantly lower proliferation rate, higher apoptosis percentage and a less number of vascular branches on matrigel (P < 0.01). In addition, VEGFA expression was distinctly downregulated in the MSCs+si-H19 group in comparison with the MSCs+si-H19 NC and MSCs groups. These findings indicate that lncRNA-H19 effectively promotes MSCs survival and vascularization under hypoxic-ischemic condition in vitro,and this effect may be associated with the upregulation of VEGFA.