miR-15b通过靶向ABCG2信号通路抑制胶质瘤干细胞迁移及侵染  

miR-15b suppresses glioma stem cell migration and invasion by targeting ABCG2 signaling pathway

作　　者：刘义锋[1] 张保朝[1] 温昌明[1] 闻公灵[1] 周国平[2] 张敬伟[3] 贺海发 汪宁[1] 李巍[5] Liu Yi-feng;Zhang Bao-chao;Wen Chang-ming;Wen Gong-ling;Zhou Guo-ping;Zhang Jing-wei;He Hai-fa;Wang Ning;Li Wei(Department of Neurology Affiliated Hospital of Zhengzhou University (Nanyang Hospital), Nanyang Central Hospital, Nanyang 473000, Henan Province, China;Zhou Guo-ping Affiliated Hospital of Zhengzhou University (Nanyang Hospital), Nanyang Central Hospital, Nanyang 473000, Henan Province, China;Department of Oncology Affiliated Hospital of Zhengzhou University (Nanyang Hospital), Nanyang Central Hospital, Nanyang 473000, Henan Province, China;Department of Pathology, Affiliated Hospital of Zhengzhou University (Nanyang Hospital), Nanyang Central Hospital, Nanyang 473000, Henan Province, China;Department of Neurology, General Hospital of Shenyang Military Region, Shenyang 110016, Liaoning Province, China)

机构地区：[1]郑州大学附属医院(南阳医院)南阳中心医院神经内科,河南省南阳市473000 [2]郑州大学附属医院(南阳医院)南阳中心医院神经外科,河南省南阳市473000 [3]郑州大学附属医院(南阳医院)南阳中心医院肿瘤内科,河南省南阳市473000 [4]郑州大学附属医院(南阳医院)南阳中心医院病理科,河南省南阳市473000 [5]郑州大学附属医院(南阳医院)南阳中心医院解放军沈阳军区总医院神经内科,辽宁省沈阳市110016

出　　处：《中国组织工程研究》2017年第33期5305-5312,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年科学基金资助项目(81401097)~~

摘　　要：背景:mi R-15b在肿瘤起始和发展过程中发挥重要作用,于是作者推测miR-15b可能参与调解胶质瘤干细胞细胞的迁移和侵染,而这目前尚无相关报道,并且其机制也不清楚。目的:探讨mi R-15b对胶质瘤干细胞迁移和侵染的影响及相关分子机制。方法:实时荧光定量PCR检测胶质瘤组织、正常成人脑组织,胶质瘤干细胞及非胶质瘤干细胞中mi R-15b的表达情况。(1)分别以ABCG2特异性si RNA、对照si RNA转染胶质瘤干细胞;(2)分别以mi R-15抑制物、mi R-15模拟物及mi R-对照分别转染胶质瘤干细胞。转染后48 h,Transwell检测细胞迁移和侵染能力,ELISA法和明胶酶谱实验检测细胞培养上清中基质金属蛋白酶2/9蛋白量及活性变化。将胶质瘤干细胞及非胶质瘤干细胞分别注入裸鼠皮下,30 d内观察成瘤情况。结果与结论:(1)与正常脑组织相比,胶质瘤中mi R-15b的表达明显较低且与胶质瘤所处阶段呈负相关性。与非胶质瘤干细胞相比,胶质瘤干细胞中mi R-15b的表达显著下调;(2)与mi R-对照组比较,mi R-15b模拟物组细胞迁移及侵染能力显著下降(P<0.01),mi R-15b抑制物组细胞迁移及侵染能力显著增加(P<0.01);Target Scan生物学软件预测表明ABCG2为mi R-15b潜在的靶基因;(3)与对照si RNA组比较,ABCG2 si RNA组细胞迁移及侵染能力显著下降(P<0.01);(4)ELISA检测表明,上调mi R-15b基因表达显著抑制基质金属蛋白酶2/9蛋白的表达;(5)ELISA法和明胶酶谱实验检测结果表明,ABCG2 si RNA转染可显著抑制基质金属蛋白酶2/9蛋白的活性,但不影响其表达量;(6)裸鼠体内实验表明,胶质瘤干细胞具有更强的成瘤能力;(7)结果表明,mi R-15b通过靶作用于ABCG2调控胶质瘤干细胞的迁移和侵染,上调mi R-15b表达可抑制胶质瘤干细胞的迁移和侵染。BACKGROUND:miR-15b plays an important role in the initiation and development of tumors,based on which,we speculate that miR-15b may be involved in the migration and invasion of glioma stem cells(GSCs).However,there is no relevant report and the mechanism of action is also unclear.OBJECTIVE:To identify the effects of miR-15b on the migration and invasion of GSCs and the mechanisms involved in this process.METHODS:Quantitative PCR was performed to evaluate the expression of miR-15b in the gliomas tissues,normal brain tissues,GSCs and non-GSCs.After knockdown of ATP-binding cassette superfamily G member2(ABCG2)by ABCG2specific siRNA,Transwell assay was performed to determine the effect of ABCG2on GSCs migration and invasion.Additionally,the GSCs were transfected with miR-15b mimics or inhibitor to up-regulate or down-regulate the expression of miR-15b.At48hours after transfection,Transwell assay was used to detect the effect of miR-15b on GSCs migration and invasion;ELISA and gelatin zymography assays were performed to determine the matrix metalloproteinase-2/-9(MMP-2/-9)expression and activity after treatment with miR-15b.CD133-positive or non-CD133-positive cells were directly injected subcutaneously into nude mice.Tumor formation was observed within30days after injection.RESULTS AND CONCLUSION:miR-15b was significantly down-regulated in gliomas tissues compared with normal brain tissues,which was negatively correlated with the stage of gliomas.In addition,miR-15b was significantly down-regulated in GSCs compared with non-GSCs.Up-regulation of miR-15b significantly reduced the migration and invasion ability of GSCs(P<0.01),and down-regulation of miR-15b significantly enhanced the cell migration and invasion of GSCs(P<0.01).By target prediction analysis,we obtained that ABCG2was a potential target gene of miR-15b.Luciferase assay confirmed that miR-15b targeted ABCG2directly,and migration and invasion of GSCs were dramatically reduced by ABCG2siRNA(P<0.01).ELISA results showed that up-regulation miR-15b significant

关 键 词：干细胞 肿瘤干细胞 胶质瘤干细胞 miR-15b 迁移 侵染 ABCG2 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]