DNA-methylation-mediated activating of lncRNA SNHG12 promotes temozolomide resistance in glioblastoma
DNA-methylation-mediated activating of lncRNA SNHG12 promotes temozolomide resistance in glioblastoma
复制标题
DNA甲基化介导的lncRNA SNHG12激活促进胶质母细胞瘤中替莫唑胺耐药
DOI：
10.1186/s12943-020-1137-5
复制
发表时间：
2020-02-10
期刊：
影响因子：
37.3
通讯作者：
You, Yongping
中科院分区：
文献类型：
--
作者：
Lu, Chenfei;Wei, Yutian;You, Yongping
研究背景越来越多的证据表明，长链非编码RNA（longnoncodingRNA，lncRNA）是参与多种生物过程的重要调节分子。获得性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，潜在的机制，lncRNA介导TMZ耐药GBM仍然不佳characteristic.MethodsQuantitative逆转录PCR（qRT-PCR）和荧光原位杂交检测小核仁RNA宿主基因12（SNHG 12）TMZ敏感和TMZ耐药GBM细胞和组织的水平。SNHG 12对TMZ抗性的影响通过体外测定（蛋白质印迹、集落形成测定、流式细胞术测定和TUNEL测定）来研究。通过生物信息学分析、亚硫酸氢盐扩增子测序、甲基化特异性PCR、双荧光素酶报告基因测定、染色质免疫沉淀测定、RNA免疫沉淀测定、免疫荧光、qRT-PCR、免疫荧光、免疫荧光和免疫荧光等方法，和western blot在体内实验中，采用小鼠颅内移植瘤模型研究SNHG 12的功能。SNHG 12的过表达导致获得性TMZ抗性的发展，而SNHG 12的敲低恢复TMZ敏感性。在SNHG 12的启动子区域内检测到异常低水平的DNA甲基化，DNA甲基化的丧失使该区域更容易接近Sp1转录因子（SP1）;这表明甲基化和SP1共同作用以调节SNHG 12的表达。在细胞质中，SNHG 12充当miR-129- 5 p的海绵，导致MAPK 1和E2 F7的上调，并赋予GBM细胞TMZ抗性。MAPK 1的去抑制通过激活MAPK/ERK通路调节TMZ诱导的细胞凋亡和G1/S细胞周期转换，而E2 F7的失调主要与G1/S细胞周期转换有关。临床上，SNHG 12过表达与接受TMZ治疗的GBM患者的生存率差相关。结论我们的结果表明，SNHG 12可以作为一个有前途的治疗靶点，以克服TMZ耐药，从而提高TMZ化疗的临床疗效。
BackgroundAccumulating 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.MethodsQuantitative 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.ResultsSNHG12 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.ConclusionOur results suggest that SNHG12 could serve as a promising therapeutic target to surmount TMZ resistance, thereby improving the clinical efficacy of TMZ chemotherapy.