Department of Laboratory Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250000, China.
Department of Medical Engineering, Shandong Provincial Hospital Affiliated to Shandong First Medical University; Jinan, Shandong 250000, China.
Life Sci. 2021 Aug 1;278:119590. doi: 10.1016/j.lfs.2021.119590. Epub 2021 May 6.
The expression of antisense lncRNA STXBP5-AS1 and its sense gene STXBP5 were found to be downregulated in glioma by RNA sequencing; however, the function and mechanism of both two genes in the development of glioma have not been studied.
QRT-PCR and western blot were used to determine the transcriptional and translational levels of moleculars. MSP and BSP assays were used to evaluate the methylation status of promoter CpG island. MTT, EdU, flow cytometry, and transwell assays were used to reveal biological effects. The in vivo mice model was used to validate the role of target genes in tumorigenesis.
The mRNA and protein expression of STXBP5 was significantly downregulated in glioma tissues and positively correlated with prognosis. STXBP5-AS1 was downregulated in glioma cells and tissues, and associated with tumor size and clinical stages. Both of two genes were significantly restored in cells treatment with 5-Aza. The promoter CpG island of STXBP5/STXBP5-AS1 was hypermethylated in glioma cells, but partially methylated in NHA cells. We found that promoter methylation frequency was significantly higher in glioma tissues. Functionally, overexpression of STXBP5 and STXBP5-AS1 inhibited cell proliferation, migration, and invasion and promoted apoptosis in vitro, whereas depletion of STXBP5 and STXBP5-AS1 showed opposite effects. Both the mRNA and protein expression of STXBP5 were positively regulated by STXBP5-AS1. Ectopic expression of STXBP5 and STXBP5-AS1 suppressed tumor formation in vivo.
Our findings suggested that epigenetically silenced STXBP5-AS1 and STXBP5 might act as novel tumor suppressors of glioma.
通过 RNA 测序发现抗义 lncRNA STXBP5-AS1 和其有义基因 STXBP5 在胶质瘤中表达下调；然而，这两个基因在胶质瘤发展中的功能和机制尚未研究。
实时荧光定量聚合酶链反应（qRT-PCR）和蛋白质印迹法（western blot）用于测定分子的转录和翻译水平。甲基化特异性聚合酶链反应（MSP）和亚硫酸氢盐测序（BSP）检测用于评估启动子 CpG 岛的甲基化状态。MTT、EdU、流式细胞术和 Transwell 检测用于揭示生物学效应。体内小鼠模型用于验证靶基因在肿瘤发生中的作用。
STXBP5 在胶质瘤组织中的 mRNA 和蛋白表达显著下调，与预后呈正相关。STXBP5-AS1 在胶质瘤细胞和组织中下调，与肿瘤大小和临床分期相关。在细胞处理中，两种基因均与 5-Aza 呈显著恢复。STXBP5/STXBP5-AS1 的启动子 CpG 岛在胶质瘤细胞中呈高度甲基化，但在 NHA 细胞中呈部分甲基化。我们发现，在胶质瘤组织中，启动子甲基化频率明显更高。功能上，过表达 STXBP5 和 STXBP5-AS1 可抑制细胞增殖、迁移和侵袭，并促进体外细胞凋亡，而敲低 STXBP5 和 STXBP5-AS1 则表现出相反的效果。STXBP5 的 mRNA 和蛋白表达均受 STXBP5-AS1 的正向调节。STXBP5 和 STXBP5-AS1 的异位表达可抑制体内肿瘤形成。
我们的研究结果表明，表观遗传沉默的 STXBP5-AS1 和 STXBP5 可能作为胶质瘤的新型肿瘤抑制因子。
