长链非编码RNA-H19对缺血缺氧条件下骨髓间充质干细胞生存和血管再生能力的影响

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

背景:课题组前期研究发现骨髓间充质干细胞(mesenchymal stem cells,MSCs)移植后其在局部梗死组织中的生存力低下,形成新生血管的密度较低,在一定程度上影响了其治疗的效率。长链非编码RNA-H19(lncRNA-H19)被证实与骨髓间充质干细胞分化及血管形成密切相关。目的:体外观察lncRNA-H19对骨髓间充质干细胞在缺血缺氧条件下生存和血管再生能力的影响,并探讨其可能机制。方法:体外培养的骨髓间充质干细胞分为5组:MSCs+H19组、MSCs+H19对照组(MSCs+H19 NC组),MSCs+si-H19组、MSCs+si-H19对照组(MSCs+si-H19 NC组)和MSCs组。其中MSCs+H19组和MSCs+H19NC组分别予以转染lncRNA-H19及lncRNA-H19 scramble RNA阴性对照,MSCs+si-H19组和MSCs+si-H19NC组分别转染lncRNA-H19 siRNA及lncRNA-H19 siRNA scramble阴性对照,体外缺血缺氧条件(无血清,体积分数为1%O_2)下培养24 h,MTS法检测细胞增殖情况,TUNEL法检测细胞凋亡情况。取细胞培养基上清液分别刺激人脐静脉内皮细胞,观察血管形成情况。Western blot检测各组血管内皮生长因子A表达。结果与结论:(1)与MSCs组及MSCs+H19 NC组相比,MSCs+H19组增殖能力显著增强,凋亡显著减少,血管管腔样结构明显增多(P<0.01);(2)与MSCs组及MSCs+si-H19 NC组相比,MSCs+si-H19组增殖能力显著减弱,凋亡显著增加,血管管腔样结构明显减少(P<0.01);(3)与MSCs组及MSCs+H19 NC组相比,MSCs+H19组血管内皮生长因子A表达显著增高;(4)与MSCs组及MSCs+si-H19 NC组相比,MSCs+si-H19组血管内皮生长因子A表达显著减少;(5)结果表明,lncRNA-H19促进骨髓间充质干细胞在体外缺血缺氧环境下的生存并增强其血管形成能力,此作用可能与其上调血管内皮生长因子A表达相关。

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.