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

doi:10.3969/j.issn.2095-4344.2017.33.010

Abstract

Abstract:

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 member 2 (ABCG2) by ABCG2 specific siRNA, Transwell assay was performed to determine the effect of ABCG2 on 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. At 48 hours 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 within 30 days 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 ABCG2 was a potential target gene of miR-15b. Luciferase assay confirmed that miR-15b targeted ABCG2 directly, and migration and invasion of GSCs were dramatically reduced by ABCG2 siRNA (P < 0.01). ELISA results showed that up-regulation miR-15b significantly inhibited MMP-2/-9 expression. ELISA and gelatin zymography assay results showed that ABCG2 siRNA did not affect MMP-2/-9 expression, but significantly inhibited the activity of MMP-2/-9. In the in vivo tumor model, GSCs were more tumorigenic as compared with non-GSCs from the same tumor in vivo. To conclude, miR-15b regulates the migration and invasion of GSCs by targeting the ABCG2 signaling pathway, and up-regulation of miR-15b can suppress the migration and invasion of GSCs.

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: Stem Cells, MicroRNAs, Cell Migration Assays, Tissue Engineering

CLC Number:

R394.2

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. miR-15b suppresses glioma stem cell migration and invasion by targeting ABCG2 signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(33): 5305-5312.

Figures/Tables 3

2.1 qPCR检测人原发性神经胶质瘤细胞和对应的恶性胶质瘤干细胞中miR-15b和ABCG2的表达研究分析了495例胶质瘤患者的TCGA多行性成胶质细胞瘤数据和10个正常脑组织的信息，以探讨miR-15b表达水平。结果表明多行性成胶质细胞瘤中miR-15b表达量显著低于正常脑组织(P < 0.000 1)，见图1A。为进一步探讨胶质瘤组织样品中miR-15b的表达是否降低，茎环-qPCR检测了30个不同时期的胶质瘤样品中miR-15b的表达情况。结果如图1B所示，胶质瘤样本中miR-15b的表达显著下调，并且其表达水平与胶质瘤所处阶段呈负相关，这表明多行性成胶质细胞瘤(Ⅳ期)患者中miR-15b的表达量是最低的。然后，探讨了miR-15b和ABCG2在非胶质瘤干细胞和对应的胶质瘤干细胞中表达的区别。结果表明，与非胶质瘤干细胞相比，胶质瘤干细胞中的miR-15b表达量更低，见图1C。研究进一步分析了557例胶质瘤患者和10个正常脑组织的TCGA 多行性成胶质细胞瘤数据，以探讨ABCG2的表达情况。结果表明，多行性成胶质细胞瘤中ABCG2的表达量略有上升，但差异无统计学意义(P > 0.05)，见图1D，这与以前的报道相反[11-12]。研究也利用qPCR分析了正常脑组织和神经胶质瘤样本中ABCG2的表达情况。结果表明，胶质瘤样本中ABCG2表达量更高并且与肿瘤所处的阶段呈正相关性，见图1E。体外和体内实验检测结果表明，胶质瘤干细胞中ABCG2的表达水平比非胶质瘤干细胞中分别高2.26倍和3.27倍，见图1F，G。 2.2 miR-15b模拟物和抑制物对原发性胶质瘤干细胞迁移和侵染的影响使用化学合成的miR-15b模拟物和抑制物转染原发性胶质瘤干细胞，以上调和下调胶质瘤干细胞细胞中miR-15b的表达。见图2A所示，转染miR-15b抑制物后，miR-15b表达量显著降低，而转染了模拟物组的miR-15b表达量显著升高。见图2B、D所示，与miR-对照组相比，miR-15b模拟物组细胞迁移能力下降了约50%，而miR-15b抑制物组细胞迁移能力增加了2.1倍。Transwell侵染检测结果表明miR-15b模拟物组细胞的侵染能力下降了约23%，而miR-15b抑制物组的细胞侵染能力增加了约1.5倍，见图2C、E。这些结果明miR-15b在调控人胶质瘤干细胞细胞迁移和侵染方面发挥重要的作用。 2.3 miR-15b调控胶质瘤干细胞中基质金属蛋白酶2/9活性有研究证明，包括胶质瘤干细胞在内的高阶段的神经胶质瘤中基质金属蛋白酶2/9的表达水平较高，其在这些部位的表达有利于胶质瘤的侵染和扩散[13]。因此，胶质瘤干细胞中基质金属蛋白酶2/9表达量的变化可能与miR-15b调控的细胞侵染和扩散有关。为探讨上调或下调miR-15b表达是否影响胶质瘤干细胞中基质金属蛋白酶2/9的表达，胶质瘤干细胞细胞转染miR-15b模拟物或抑制物后，通过ELISA检测了基质金属蛋白酶2/9的表达情况。结果表明，上调miR-15b表达显著抑制胶质瘤干细胞中基质金属蛋白酶2/9的表达(图3A)，而下调miR-15b表达显著增加基质金属蛋白酶2/9的表达(图3B)，上述结果表明miR-15b能显著影响胶质瘤干细胞中基质金属蛋白酶2/9蛋白表达。为验证基质金属蛋白酶2或基质金属蛋白酶9是否是miR-15b的一个靶基因，首先使用TargetScan软件进行分析，发现在基质金属蛋白酶2/9基因的3’UTR区域没有miR-15b预测的结合位点。随后构建基质金属蛋白酶2/9 3’UTR序列的荧光素酶报告载体(野生型：pGL3-WT-MMP-2-3’UTR和pGL3-WT-MMP-9-3’UTR)和miR-15b结合位点突变了的突变质粒(pGL3-MUT- MMP-2-3’UTR和pGL3-MUT-MMP-9- 3’UTR)，共转染后检测结果表明，与突变型对照组相比， miR-15b模拟物没有使野生型质粒转染组的荧光素酶活性显著下降，表明基质金属蛋白酶2和基质金属蛋白酶9均不是miR-15b的靶基因(图3C，D)。这些结果表明miR-15b负调控基质金属蛋白酶2/9，但不直接靶向基质金属蛋白酶2/9 mRNA转录。 2.4 ABCG2是miR-15b的靶基，其能调控胶质瘤干细胞细胞迁移和侵染 ABCG2有助于维持GSC-样特征和固有的药物抗性[14]。过表达ABCG2能促进多种肿瘤迁移及侵染[15-17]。然而，ABCG2在胶质瘤干细胞中的功能尚且未知。为探讨ABCG2对胶质瘤干细胞迁移和侵染的影响，胶质瘤干细胞转染ABCG2特异性siRNA以敲低ABCG2的表达，然后进行细胞迁移检测。结果表明，与对照siRNA相比，ABCG2 siRNA转染后24 h显著抑制胶质瘤干细胞细胞迁移(图4A)。研究利用Transwell检测进一步分析了敲低ABCG2表达对细胞侵染的影响，结果发现与对照siRNA相比，ABCG2 siRNA转染后显著抑制胶质瘤干细胞细胞侵染(图4B)。为探讨miR-15b是否影响ABCG2的表达，胶质瘤干细胞转染miR-15b模拟物或抑制物，然后进行Western blot检测。结果表明上调miR-15b表达抑制ABCG2的表达，而下调miR-15b表达则促进ABCG2的表达(图4C)。这些结果表明miR-15b能调控胶质瘤干细胞中ABCG2的表达。为探讨ABCG2是否是miR-15b的直接的靶基因，本实验根据软件预测结果构建了含有预测的miR-15b靶位点的ABCG2 3’UTR的荧光素酶报告载体。miR-15b和预测的靶基因的序列比对结果如图4D所示。荧光素酶报告质粒和miR-15b模拟物或随机miRNA对照共转染胶质瘤干细胞。检测结果表明，与对照组相比，miR-15b模拟物和荧光素酶报告质粒共转染后，荧光素酶活性显著下降(图4E)。这些结果表明ABCG2是miR-15b的直接靶基因。 2.5 敲低ABCG2表达抑制基质金属蛋白酶2/9蛋白活性而不影响其蛋白表达量上面的结果证明ABCG2是miR-15b的靶基因。接下来的实验试图探讨miR-15b是否通过ABCG2调节基质金属蛋白酶的表达。为明确ABCG2和基质金属蛋白酶2/9蛋白之间的关系，胶质瘤干细胞转染ABCG2 siRNA后，ELISA检测基质金属蛋白酶2/9的表达量。结果表明，ABCG2 siRNA组和阴性对照组相比，基质金属蛋白酶2/9的蛋白量没有显著区别(图5A，B)。进一步通过明胶酶谱检测分析胶质瘤干细胞转染ABCG2 siRNA后基质金属蛋白酶2/9活性变化情况。结果表明，敲低ABCG2表达显著抑制胶质瘤干细胞中基质金属蛋白酶2/9活性(图5C，D)。这些结果表明ABCG2显著影响胶质瘤干细胞中基质金属蛋白酶2/9的蛋白活性，而不影响其表达量。 2.6 体内肿瘤模型实验结果从18 d开始，CD133阳性细胞组肿瘤体积明显大于非CD133阳性细胞组，见图6。"

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