DNA甲基转移酶-1通过甲基化修饰调控脑胶质瘤中迁移调控蛋白1表达的研究

DNA methyltransferase-1 regulates SHTN1 expression in gliomas through DNA methylation

目的探讨DNA甲基转移酶-1(DNMT1)在人脑胶质瘤细胞中的生物学功能及其调控机制。方法利用中国胶质瘤基因组图谱(CGGA)、肿瘤基因组图谱(TCGA)、基因表达综合数据库(GEO)中脑胶质瘤相关转录组测序(mRNA-seq)数据、单细胞测序数据, 对迁移调控蛋白1(SHTN1)进行生存分析、临床相关分析等生物信息学分析;采用慢病毒转染构建DNMT1稳定低表达的人脑胶质瘤细胞株, 以DNMT1-NC为对照, 使用实时定量聚合酶链反应检测各组细胞SHTN1 mRNA水平, 以验证DNMT1与SHTN1的表达相关性;BSAS测序验证DNMT1对SHTN1的甲基化调控作用, 采用Kruskall-Wallis检验比较每个CpG位点甲基化差异。结果 SHTN1高表达组脑胶质瘤患者生存优于低表达组(P<0.05);SHTN1在胶质母细胞瘤(GBM)中的表达水平显著低于正常脑组织, 并且SHTN1在不同脑胶质瘤WHO分级和不同病理亚型中表达差异有统计学意义;人脑胶质瘤中SHTN1与DNMT1的表达水平呈负相关;体外实验DNMT1敲减组中SHTN1的mRNA水平显著高于对照组[U251:敲减组SHTN1表达丰度高于对照组(1.692比1.000, P<0.01);U87:敲减组SHTN1表达丰度高于对照组(1.420比1.002, P<0.05)]。BSAS测序验证SHTN1的3个CpG位点在DNMT1敲减组的甲基化水平下调(P<0.05)。结论 SHTN1的低表达可能与脑胶质瘤患者不良预后相关, DNMT1能通过DNA甲基化作用调控SHTN1的表达。

Objective To investigate the mechanisms regulating the biological function of DNA methyltransferase-1(DNMT1)in human glioma cells.Methods The bioinformatics analysis of SHTN1 was carried out using glioma-related mRNA-seq data and single-cell sequencing data in chinese glioma ge-nome atlas(CGGA),the cancer genome atlas(TCGA)and gene expression omnibus(GEO)databases.SHTN1(Shootin 1)mRNA expression levels were detected by real-time quantitative polymerase chain reac-tion to verify the correlation between DNMT1 and SHTN1 expression in human glioma cell lines with stable low-expression of DNMT1 established by lentiviral transfection and control groups named DNMT-NC(U-87,and U-251 cell lines were purchased from Shanghai Genechem Co.,Ltd.).BSAS sequencing vali-dated the regulation for methylation level of SHTN1 by DNMT1.The Kruskall-Wallis test was used to com-pare the methylation levels of each CpG site.Results The survival of glioma patients in the SHTN1 high expression group was longer than that of the low expression group(P<0.05).The expression of SHTN1 in glioblastoma(GBM)was significantly lower than in normal brain tissue,with differences in expression ob-served across different glioma WHO grades and pathological subtypes.The mRNA expression levels of SHTN1 in the DNMT1 knockdown group in vitro experiments were significantly higher than those in the con-trol group(The expression of SHTN1 in the knockdown group of U251 was 1.7 times higher than that in the control group,P<0.01;The expression of SHTN1 in the knockdown group of U87 was 1.4 times higher than that in the control group,P<0.05).BSAS sequencing verified the down-regulation of methylation levels of the three CpG sites of SHTN1 in the DNMT1 low-expression group(Kruskall-Wallis P<0.05).Conclusion The expression of SHTN1 was significantly down-regulated in high-grade gliomas,and the short-term survival of glioma patients with low expression of SHTN1 was poorer than those with high expres-sion.The low expression of SHTN1 may be related to the poor prognosis of glioma patients,and the expres-sion of SHTN1 can be regulated by DNMT1 through DNA methylation.