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

doi:10.3969/j.issn.2095-4344.0430

Abstract

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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Cell Hypoxia, Vascular Endothelial Growth Factor A, Tissue Engineering

CLC Number:

R349.2

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. Long non-coding RNA H19 facilitates bone marrow mesenchymal stem cell survival and vascularization in hypoxic-ischemic conditions in vitro[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 1969-1975.

Figures/Tables 2

2.1 pcDNA3.1-H19载体酶切鉴定和测序结果 pcDNA3.1-H19酶切鉴定及测序结果均显示，目的基因H19已成功转入pcDNA3.1载体中(图1)。H19(2 288 bp)在相应的位置切出1条目的条带(箭头所指)，pcDNA3.1-H19载体构建成功。 2.2 各组lncRNA-H19表达情况 MSCs+H19组的lncRNA-H19表达量较MSCs+H19 NC组和MSCs组明显增高(P < 0.01，图2)，而 MSCs+si-H19组较MSCs+si-H19 NC和MSCs组明显降低(P < 0.01，图2)，表明骨髓间充质干细胞成功转染了lncRNA-H19和lncRNA-H19siRNA。 2.3 lncRNA-H19对骨髓间充质干细胞增殖的影响 MTS检测结果显示：MSCs+H19组在体外培养24，48，72 h的细胞增殖率均明显高于MSCs+H19 NC组和MSCs组(P < 0.01，图3A)，A490值均高于MSCs+H19 NC组和MSCs组(P < 0.01，图3B)，MSCs+si-H19组上述不同时间点细胞增殖率明显低于MSCs+si-H19 NC组和MSCs组(P < 0.01，图3A)，A490值均低于MSCs+si-H19 NC组和MSCs组(P < 0.01，图3B)，表明lncRNA-H19能够促进骨髓间充质干细胞的增殖。 2.4 lncRNA-H19 对骨髓间充质干细胞凋亡的影响 TUNEL检测结果显示：MSCs+H19组、MSCs+H19 NC组、MSCs+si-H19组、MSCs+si-H19 NC组和MSCs组的凋亡比例分别为(1.90±0.01)%，(2.56±0.06)%，(4.50±0.04)%，(2.62± 0.02)%和(2.50±0.09)%，MSCs+H19组的凋亡比例低于MSCs+H19 NC组和MSCs组(P < 0.01，图4)，而MSCs+ si-H19组凋亡比例高于 MSCs+si-H19 NC组和MSCs组(P < 0.01，图4)。 2.5 lncRNA-H19对骨髓间充质干细胞血管形成能力的影响人脐静脉内皮细胞分别与各组骨髓间充质干细胞培养基上清液共培养后，在matrigel中均形成血管腔样结构(图5)。MSCs+H19组、MSCs+H19 NC组、MSCs+si-H19组、MSCs+si-H19 NC组和MSCs组血管腔样结构的数量分别为22.40±2.02，13.40±1.30，6.8±0.98，13.80±1.21和14.4±1.20。与MSCs+H19 NC组和MSCs组相比较，MSCs+H19组血管腔样结构的数量明显增多(P < 0.01)；与 MSCs+si-H19 NC组和MSCs组相比较，MSCs+si-H19组血管腔样结构数量显著减少(P < 0.01)，以上表明 lncRNA-H19对骨髓间充质干细胞的成管能力具有促进作用。 2.6 各组骨髓间充质干细胞血管内皮生长因子A的表达与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，而与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，差异有显著性意义(P < 0.01，图6)。"

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