miR-1197抑制MGMT表达对胶质瘤替莫唑胺耐药性的影响  

Efficacy of miR-1197 inhibiting MGMT expression on temozolomide resistance in glioblastoma

作　　者：聂耳 石秦宇 韩正中 张旭 苗发安 NIE Er;SHI Qin-yu;HAN Zheng-zhong(Department of Neurosurgery, Xuzhou Medical University, Xuzhou 221002, China)

机构地区：[1]徐州医科大学附属医院神经外科,徐州221002 [2]徐州医科大学研究生院

出　　处：《临床神经外科杂志》2020年第6期645-651,共7页Journal of Clinical Neurosurgery

基　　金：国家自然科学基金青年项目(81802490)。

摘　　要：目的探讨miR-1197是否通过抑制O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)的表达影响胶质瘤对替莫唑胺的耐药性。方法采用qRT-PCR检测胶质瘤及非肿瘤脑组织中miR-1197的表达水平,CCK-8及单克隆实验检测胶质瘤细胞的增殖能力,流式细胞术检测胶质瘤细胞的凋亡率。应用Western blot检测胶质瘤组织及细胞中MGMT及相关蛋白的表达;用荧光素酶基因报告实验验证miR-1197与MGMT的结合。用裸鼠皮下成瘤实验验证miR-1197对胶质瘤对替莫唑胺耐药性的影响。结果生物医学数据库及胶质瘤组织样本分析显示miR-1197在胶质瘤中低表达。荧光素酶基因报告实验显示,miR-1197可以与MGMT的3′-UTR相结合,过表达miR-1197可抑制MGMT的表达。单克隆及流式细胞术结果表明,miR-1197过表达可促进替莫唑胺诱导的细胞凋亡,但其可以被MGMT所逆转。裸鼠皮下成瘤实验表明miR-1197过表达增加胶质瘤对替莫唑胺的敏感性。结论miR-1197通过与MGMT的3′-UTR相结合,抑制MGMT表达,进而促进胶质瘤对替莫唑胺的敏感性。Objective To investigate whether miR-1197 modulates temozolomide(TMZ)resistance in glioblastoma via reducing the expression of O6-methylguanine-DNA methyltransferase(MGMT).Methods The RNA levels of miR-1197 in glioblastoma(GBM)and non-tumor brain tissues were determine by quantitative real-time PCR(qRT-PCR).Cell proliferation was measured by CCK-8 and colony formation assay.Flow cytometry was used to detected cells apoptosis rates.The expression of MGMT was detected by Western blot and qRT-PCR.Luciferase reporter assay was used to investigate the correlation of miR-1197 and MGMT.Subcutaneous xenograft tumor model were used to determine the function of miR-1197 in temozolomide-resistance.Results The biomedical databases and GBM tissue samples analysis showed that miR-1197 expression levels were significantly decreased in GBM.Luciferase reporter assay indicated that miR-1197 directly targets MGMT by binding its seed region to the 3′-UTR of MGMT.And miR-1197 overexpression decreased the protein and RNA levels of MGMT.The combination treatment of miR-1197 plus TMZ significantly induced cell apoptosis,whereas forced expression of MGMT partially abolished this effect.miR-1197 overexpression enhanced temozolomide-induced growth inhibition in vivo.Conclusion miR-1197 enhances TMZ sensitivity through binding to the 3′-UTR of MGMT and repressing MGMT expression.

关 键 词：胶质瘤 替莫唑胺耐药 miR-1197 O6-甲基鸟嘌呤DNA甲基转移酶 

分 类 号：R739.41[医药卫生—肿瘤]