DNA 甲基化介导的长链非编码 RNA SNHG12 的激活促进胶质母细胞瘤对替莫唑胺的耐药性。

DNA-methylation-mediated activating of lncRNA SNHG12 promotes temozolomide resistance in glioblastoma.

机构信息

Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, Jiangsu, China.

Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Jiangsu Collaborative innovation Center For Cancer Personalized Medicine, Nanjing Medical University, Nanjing, 211166, Jiangsu, China.

出版信息

Mol Cancer. 2020 Feb 10;19(1):28. doi: 10.1186/s12943-020-1137-5.

DOI:10.1186/s12943-020-1137-5

PMID:32039732

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7011291/

Abstract

BACKGROUND

Accumulating evidence shows that long noncoding RNAs (lncRNAs) are important regulator molecules involved in diverse biological processes. Acquired drug resistance is a major challenge in the clinical treatment of glioblastoma (GBM), and lncRNAs have been shown to play a role in chemotherapy resistance. However, the underlying mechanisms by which lncRNA mediates TMZ resistance in GBM remain poorly characterized.

METHODS

Quantitative reverse transcription PCR (qRT-PCR) and fluorescence in situ hybridization assays were used to detect small nucleolar RNA host gene 12 (SNHG12) levels in TMZ-sensitive and TMZ-resistant GBM cells and tissues. The effects of SNHG12 on TMZ resistance were investigated through in vitro assays (western blots, colony formation assays, flow cytometry assays, and TUNEL assays). The mechanism mediating the high expression of SNHG12 in TMZ-resistant cells and its relationships with miR-129-5p, mitogen-activated protein kinase 1 (MAPK1), and E2F transcription factor 7 (E2F7) were determined by bioinformatic analysis, bisulfite amplicon sequencing, methylation-specific PCR, dual luciferase reporter assays, chromatin immunoprecipitation assays, RNA immunoprecipitation assays, immunofluorescence, qRT-PCR, and western blot. For in vivo experiments, an intracranial xenograft tumor mouse model was used to investigate SNHG12 function.

RESULTS

SNHG12 was upregulated in TMZ-resistant cells and tissues. Overexpression of SNHG12 led to the development of acquired TMZ resistance, while knockdown of SNHG12 restored TMZ sensitivity. An abnormally low level of DNA methylation was detected within the promoter region of SNHG12, and loss of DNA methylation made this region more accessible to the Sp1 transcription factor (SP1); this indicated that methylation and SP1 work together to regulate SNHG12 expression. In the cytoplasm, SNHG12 served as a sponge for miR-129-5p, leading to upregulation of MAPK1 and E2F7 and endowing the GBM cells with TMZ resistance. Disinhibition of MAPK1 regulated TMZ-induced cell apoptosis and the G1/S cell cycle transition by activating the MAPK/ERK pathway, while E2F7 dysregulation was primarily associated with G1/S cell cycle transition. Clinically, SNHG12 overexpression was associated with poor survival of GBM patients undergoing TMZ treatment.

CONCLUSION

Our results suggest that SNHG12 could serve as a promising therapeutic target to surmount TMZ resistance, thereby improving the clinical efficacy of TMZ chemotherapy.

摘要

背景

越来越多的证据表明，长非编码 RNA（lncRNA）是参与多种生物学过程的重要调控分子。获得性药物耐药性是胶质母细胞瘤（GBM）临床治疗的主要挑战，lncRNA 已被证明在化疗耐药性中发挥作用。然而，lncRNA 介导 TMZ 耐药性的潜在机制仍知之甚少。

方法

采用定量逆转录 PCR（qRT-PCR）和荧光原位杂交检测 TMZ 敏感和 TMZ 耐药性 GBM 细胞和组织中的小核仁 RNA 宿主基因 12（SNHG12）水平。通过体外实验（western blot、集落形成实验、流式细胞术和 TUNEL 实验）研究 SNHG12 对 TMZ 耐药性的影响。通过生物信息学分析、亚硫酸氢盐扩增测序、甲基化特异性 PCR、双荧光素酶报告基因检测、染色质免疫沉淀检测、RNA 免疫沉淀检测、免疫荧光、qRT-PCR 和 western blot 确定 TMZ 耐药细胞中 SNHG12 高表达的调节机制及其与 miR-129-5p、丝裂原活化蛋白激酶 1（MAPK1）和 E2F 转录因子 7（E2F7）的关系。体内实验采用颅内异种移植肿瘤小鼠模型研究 SNHG12 功能。

结果

SNHG12 在 TMZ 耐药细胞和组织中上调。SNHG12 的过表达导致获得性 TMZ 耐药，而 SNHG12 的敲低则恢复了 TMZ 的敏感性。在 SNHG12 启动子区域检测到异常低水平的 DNA 甲基化，并且 DNA 甲基化的缺失使该区域更容易与 Sp1 转录因子（SP1）结合；这表明甲基化和 SP1 共同调节 SNHG12 的表达。在细胞质中，SNHG12 作为 miR-129-5p 的海绵，导致 MAPK1 和 E2F7 的上调，并赋予 GBM 细胞 TMZ 耐药性。MAPK1 的去抑制作用通过激活 MAPK/ERK 通路调节 TMZ 诱导的细胞凋亡和 G1/S 细胞周期转变，而 E2F7 的失调主要与 G1/S 细胞周期转变有关。临床上，SNHG12 的过表达与接受 TMZ 治疗的 GBM 患者的不良生存相关。