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.

中文翻译：

---

DNA甲基化介导的lncRNA SNHG12激活可促进胶质母细胞瘤中替莫唑胺的耐药性。

背景技术越来越多的证据表明，长的非编码RNA（lncRNA）是参与各种生物过程的重要调节分子。获得性耐药是胶质母细胞瘤（GBM）临床治疗中的主要挑战，并且lncRNA已显示在化疗耐药中起作用。但是，lncRNA介导GBM中TMZ抗性的基本机制仍然很差。方法采用定量逆转录PCR（qRT-PCR）和荧光原位杂交法检测TMZ敏感和TMZ耐药的GBM细胞和组织中小核仁RNA宿主基因12（SNHG12）的水平。通过体外测定（Western印迹，菌落形成测定，流式细胞术测定和TUNEL测定）研究了SNHG12对TMZ耐药性的影响。通过生物信息学分析，亚硫酸氢盐扩增子测序确定介导SNHG12在TMZ耐药细胞中高表达的机制及其与miR-129-5p，促分裂原活化蛋白激酶1（MAPK1）和E2F转录因子7（E2F7）的关系。 ，甲基化特异性PCR，双重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和Western印迹。对于体内实验，使用颅内异种移植肿瘤小鼠模型研究SNHG12的功能。结果SNHG12在耐TMZ的细胞和组织中上调。SNHG12的过表达导致获得性TMZ抗性的发展，而SNHG12的敲低恢复了TMZ的敏感性。在SNHG12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Sp1转录因子（SP1）更易于进入该区域；这表明甲基化和SP1共同调节SNHG12的表达。在细胞质中，SNHG12充当miR-129-5p的海绵，导致MAPK1和E2F7上调，并使GBM细胞具有TMZ抗性。MAPK1的抑制通过激活MAPK / ERK途径来调节TMZ诱导的细胞凋亡和G1 / S细胞周期转变，而E2F7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患者生存率低有关。结论我们的结果表明SNHG12可以作为克服TMZ耐药性的有希望的治疗靶点，从而提高TMZ化疗的临床疗效。