中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (7): 1005-1011.doi: 10.12307/2022.136

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

缺氧预处理通过激活MALAT1靶向抑制miR-195促进骨髓间充质干细胞的生存和血管形成

侯婧瑛,郭天柱,于萌蕾,龙会宝,吴  浩   

  1. 中山大学孙逸仙纪念医院急诊科,广东省广州市   510120
  • 收稿日期:2020-06-05 修回日期:2020-06-06 接受日期:2020-06-20 出版日期:2022-03-08 发布日期:2021-10-29
  • 通讯作者: 侯婧瑛,博士,副主任医师,中山大学孙逸仙纪念医院急诊科,广东省广州市 510120
  • 作者简介:侯婧瑛,女,1985年生,安徽省庐江县人,汉族,2012年中山大学孙逸仙纪念医院毕业,博士,副主任医师,主要从事干细胞与心血管疾病方面的研究。
  • 基金资助:
    国家自然科学基金项目(81700242),项目负责人:侯婧瑛;广东省科技计划项目(2017A020215176),项目负责人:侯婧瑛;广东省医学科研基金项目(A2017001),项目负责人:侯婧瑛;广州市科技计划项目(202102080386,202002020004),项目参与人:侯婧瑛

Hypoxia preconditioning targets and downregulates miR-195 and promotes bone marrow mesenchymal stem cell survival and pro-angiogenic potential by activating MALAT1 

Hou Jingying, Guo Tianzhu, Yu Menglei, Long Huibao, Wu Hao   

  1. Emergency Department, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China
  • Received:2020-06-05 Revised:2020-06-06 Accepted:2020-06-20 Online:2022-03-08 Published:2021-10-29
  • Contact: Hou Jingying, MD, Associate chief physician, Emergency Department, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China
  • About author:Hou Jingying, MD, Associate chief physician, Emergency Department, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81700242 (to HJY); the Science and Technology Project of Guangdong Province, No. 2017A020215176 (to HJY); the Guangdong Medical Research Fund, No. A2017001 (to HJY); the Guangzhou Science and Technology Project, No. 202102080386, No. 202002020004 (to HJY)

摘要:

文题释义:
长链非编码RNA-MALAT1:MALAT1促进骨髓间充质干细胞生存和血管形成,其受缺氧因素的调控,在缺氧条件下表达上调。研究显示MALAT1能够通过竞争性内源性RNA调控微小RNA(miRNA)的表达,进而影响骨髓间充质干细胞的生物学行为。
微小RNA-195(miR-195):miRNA与骨髓间充质干细胞生存和血管形成密切相关。miR-195被证实是骨髓间充质干细胞生存和血管形成的一个负向调控因子,它能够促进骨髓间充质干细胞的衰老,削弱其生存和抗凋亡能力,并对血管形成具有抑制作用。
背景:研究表明,缺氧预处理能够促进骨髓间充质干细胞生存和血管形成,但具体机制未完全明确。长链非编码RNA-MALAT1(MALAT1)和microRNA-195(miR-195)均被证实与骨髓间充质干细胞生存和血管形成密切相关,MALAT1促进骨髓间充质干细胞生存和血管形成,而miR-195是骨髓间充质干细胞生存和血管形成的负向调节因子。
目的:体外探讨缺氧预处理是否通过激活MALAT1靶向抑制miR-195进而提高骨髓间充质干细胞生存和促血管形成能力。
方法:将体外培养的骨髓间充质干细胞分为以下6组:常氧组(体积分数为20% O2)、缺氧组(体积分数为1% O2)、缺氧+si-MALAT1组、缺氧+
si-MALAT1对照组、缺氧+miR-195组和缺氧+miR-195对照组,其中缺氧+si-MALAT1组和缺氧+si-MALAT1对照组细胞分别转染MALAT1 siRNA以及scramble阴性对照,再进行缺氧处理,缺氧+miR-195组和缺氧+miR-195对照组细胞分别转染miR-195模拟物及阴性对照,再进行缺氧处理。各组培养24 h后检测细胞增殖、凋亡以及血管腔样结构形成情况,qRT-PCR检测MALAT1和miR-195的表达。在上述基础上,通过生物信息学网站RegRNA 2.0预测MALAT1 与miR-195的潜在结合位点,将MALAT1野生型和突变型质粒分别与miR-195模拟物以及阴性对照在293T细胞中共转染,通过检测各组萤光素酶活性对MALAT1与miR-195的靶向关系进行验证。
结果与结论:①与常氧组相比,缺氧组细胞生存力显著增强,凋亡率明显减少,血管腔样结构数量增多(P < 0.01),MALAT1表达升高,miR-195表达降低(P < 0.01);②与缺氧组和缺氧+si-MALAT1对照组比较,缺氧+si-MALAT1组细胞生存力显著降低,凋亡率明显增加,血管腔样结构数量减少(P < 0.01),MALAT1表达下降,miR-195表达升高(P < 0.01);与缺氧组和缺氧+miR-195对照组比较,缺氧+miR-195 组细胞生存力显著降低,凋亡率明显增加,血管腔样结构数量减少(P < 0.01),MALAT1表达明显下降,miR-195表达升高(P < 0.01);③双萤光素酶检测结果显示:与miR-195对照组和空白对照组相比,miR-195组MALAT1野生型报告基因的萤光素酶活性显著降低,下降约63%(P < 0.01),而miR-195组、miR-195对照组以及空白对照组的萤光素酶活性在MALAT1突变型中均无明显差异性(P > 0.05);④结果表明,缺氧预处理能够通过激活MALAT1靶向抑制miR-195提高骨髓间充质干细胞的生存和促血管形成能力。
https://orcid.org/0000-0003-0492-2216(侯婧瑛) 

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


关键词: 干细胞, 骨髓间充质干细胞, 缺氧, 预处理, 血管形成, miR-195

Abstract: BACKGROUND: Previous studies demonstrated that hypoxia preconditioning promotes bone marrow mesenchymal stem cell survival and angiogenesis. However, specific mechanism remained unclear. Both RNA-MALAT1 (MALAT1) and microRNA-195 (miR-195) are associated with bone marrow mesenchymal stem cell survival and angiogenesis. MALAT1 promotes bone marrow mesenchymal stem cell survival and angiogenesis, whereas miR-195 is a negative regulator of  bone marrow mesenchymal stem cell survival and angiogenesis.
OBJECTIVE: To investigate whether hypoxia preconditioning could activate MALAT1 to target and downregulate miR-195 to further promote bone marrow mesenchymal stem cell survival and pro-angiogenic potential in vitro. 
METHODS:  Bone marrow mesenchymal stem cells cultured in vitro were divided into six groups: normoxia (20% O2), hypoxia preconditioning (1% O2), hypoxia preconditioning+si-MALAT1, hypoxia preconditioning+si-MALAT1 negative control, hypoxia preconditioning+miR-195 and hypoxia preconditioning+miR-195 negative control. MALAT1 siRNA and relevant scramble were transfected into the hypoxia preconditioning+si-MALAT1 and hypoxia preconditioning+si-MALAT1 negative control groups respectively prior to hypoxia preconditioning, and miR-195 mimics and relevant negative control were transfected into the hypoxia preconditioning+miR-195 and hypoxia preconditioning+miR-195 negative control groups respectively prior to hypoxia preconditioning. Mesenchymal stem cells in different groups were cultured for 24 hours and cell proliferation, apoptosis and vascularization were evaluated. The expression levels of MALAT1 and miR-195 were detected using qRT-PCR. The potential complementary binding sites of MALAT1 and miR-195 were predicted by RegRNA 2.0. miR-195 mimics and miR-195 mimics negative control were co-transfected into 293T cells with the luciferase reporters containing MALAT1 or MALAT1-mut. Luciferase activity was detected in different groups of cells in order to verify the relationship between MALAT1 and miR-195. 
RESULTS AND CONCLUSION: (1) Compared with the normoxia group, the hypoxia group presented more rapid growth; cells apoptosis percentage was significantly declined; and number of vascular lumen like structures increased (P < 0.01). The expression of MALAT1 was upregulated, while the level of miR-195 was significantly decreased in the hypoxia group (P < 0.01). (2) Compared with the hypoxia preconditioning and hypoxia preconditioning+si-MALAT1 negative control groups, hypoxia preconditioning+si-MALAT1 group presented much lower growth rate, and cells apoptosis percentage was significantly higher; number of vascular lumen like structures decreased (P < 0.01). The expression of MALAT1 was decreased, while the level of miR-195 was increased (P < 0.01). Compared with the hypoxia preconditioning and hypoxia preconditioning+miR-195 negative control groups, hypoxia preconditioning+miR-195 group also presented much lower growth rate, and cells apoptosis percentage was significantly higher; number of vascular lumen like structures decreased (P < 0.01). The expression of MALAT1 was decreased, while the level of miR-195 was increased (P < 0.01). (3) The dual luciferase reporter assay indicated that compared with miR-195 control and blank control groups, the MALAT1 reporter gene luciferase activity was decreased significantly in miR-195 mimics group, down-regulating about 63% (P < 0.01). However, miR-195 mimics, miR-195 mimics negative control and blank control showed no effect on MALAT1-mut reporter gene (P > 0.05). (4) Hypoxia preconditioning could activate MALAT1 to target and downregulate miR-195 to further promote bone marrow mesenchymal stem cell survival and pro-angiogenic potential.


Key words: stem cells, bone marrow mesenchymal stem cells, hypoxia, preconditioning, angiogenesis, miR-195

中图分类号: