DataSheet_1_Hypoxia-Induced miR-137 Inhibition Increased Glioblastoma Multiforme Growth and Chemoresistance Through LRP6.docx

PurposeGlioblastoma multiforme (GBM) is one of the deadliest tumors, which is involved in numerous dysregulated microRNAs including miR-137. However, the mechanism of how miR-137 suppression associated with cancer progression and chemoresistance still remains to be elucidated.
MethodsQuantitative reverse transcriptase-PCR (qRT-PCR), DNA methylation analysis, cell proliferation assay, flow cytometric analysis, invasion assay, in situ tumor formation experiment were performed to test the expression levels and functions of miR-137 in GBM. Bioinformatics analysis, luciferase reporter assay, qRT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry assay were used to identify and verify the target of miR-137.
ResultsWe found that miR-137 was downregulated in primary and recurrent GBM compared with normal brain tissues. Overexpression of miR-137 inhibited cell invasion and enhanced cell chemosensitivity to temozolomide (TMZ) by directly targeting low-density lipoprotein receptor-related protein 6 (LRP6) in GBM. Forced expression of LRP6 cDNA without its 3’-UTR region partly restored the effects of miR-137 in vitro and in vivo. Hypoxia-induced miR-137 methylation was responsible for the miR-137 suppression, leading to the cell chemoresistance and poor prognosis of GBM.
ConclusionsThese findings demonstrated the detailed molecular mechanism of miR-137 in regulating GBM growth and chemoresistance in hypoxia microenvironment, suggesting the potentiality of miR-137 as a therapeutic target for GBM.

研究目的：多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）是致死率最高的恶性肿瘤之一，其发生发展与包括miR-137在内的多种异常调控的MicroRNA密切相关。然而，miR-137的表达抑制如何与肿瘤进展及化疗耐药相关联，其具体分子机制仍有待进一步阐明。
实验方法：本研究采用定量逆转录聚合酶链反应（Quantitative reverse transcriptase-PCR, qRT-PCR）、DNA甲基化分析、细胞增殖实验、流式细胞术分析、侵袭实验以及原位成瘤实验，检测miR-137在GBM中的表达水平并探究其生物学功能；通过生物信息学分析、荧光素酶报告基因实验、qRT-PCR、免疫印迹、免疫荧光及免疫组化实验，鉴定并验证miR-137的靶基因。
实验结果：研究发现，相较于正常脑组织，miR-137在原发性及复发性GBM组织中均呈显著低表达。在GBM细胞中，过表达miR-137可通过直接靶向低密度脂蛋白受体相关蛋白6（LRP6），抑制细胞侵袭并增强其对替莫唑胺（TMZ）的化疗敏感性。将不含3’-UTR区域的LRP6 cDNA强制表达，可在体内外部分逆转miR-137介导的上述生物学效应。缺氧诱导的miR-137甲基化是其表达下调的关键原因，进而导致GBM细胞产生化疗耐药及不良临床预后。
结论：本研究阐明了miR-137在缺氧微环境中调控GBM生长与化疗耐药的具体分子机制，提示miR-137可作为GBM临床治疗的潜在靶点。