基于大样本转录组学数据分析组蛋白1H2BH在脑胶质瘤中的表达及生物学功能

The expression and biological function of histone cluster 1 H2B family member H protein in gliomas based on large sample transcriptomics data

目的探讨影响脑胶质瘤恶性进展、患者生存预后的关键基因及其在脑胶质瘤中的表达特征和临床意义。方法回顾性分析中国脑胶质瘤基因组图谱(CGGA)RNA测序数据库310例、癌症基因组图谱(TCGA)RNA测序数据库699例、GSE16011 mRNA芯片数据库268例和REMBRANDT mRNA芯片数据库中317例脑胶质瘤患者的临床资料及其脑胶质瘤样本的测序数据,筛选出与患者预后相关的共同差异基因,通过文献检索确定目标基因。在上述4个数据库中评价该目标基因的表达特征。采用Kaplan-Meier法绘制生存曲线及单因素和多因素Cox回归分析法评价该目标基因对脑胶质瘤患者预后的作用。通过Pearson相关检验,筛选出CGGA和TCGA数据库中与目标基因表达呈正相关(r>0.5且P<0.05)的基因,进一步利用在线软件DAVID对其进行生物功能聚集分析。采用实时荧光定量聚合酶联链式反应(qRT-PCR)检测星形细胞株和脑胶质瘤细胞株中该基因的表达量。采用细胞免疫荧光染色法检测经药物处理后脑胶质瘤细胞株(U87、U251和LN229)中γ-H2AX蛋白的表达情况。结果经筛选,组蛋白1H2BH(HIST1H2BH)为目标基因。在上述4个数据库中,HIST1H2BH的表达量在世界卫生组织(WH0)Ⅱ、Ⅲ及Ⅳ级间的差异均有统计学意义(均P<0.01),其中在Ⅳ级中最高,Ⅱ级中最低;且HIST1H2BH在异柠檬酸脱氢酶(IDH)野生型脑胶质瘤中的表达量均高于IDH突变型(均P<0.001)。在CGGA和TCGA数据库中,全级别脑胶质瘤和胶质母细胞瘤患者,HIST1H2BH高表达组的总体生存率均低于低表达组(均P<0.05)。多因素Cox回归分析结果显示,HIST1H2BH表达量高是影响脑胶质瘤患者总生存期的危险因素(HR=1.275,95%CI:1.040~1.563,P<0.05)。生物功能富集分析结果显示,与HIST1 H2BH表达量呈正相关的基因主要参与细胞增殖、DNA损伤修复和细胞外基质调控。qRT-PCR结果显示,HIST1 H2BH在HA、U87、U251和LN229细胞株中相对表达量的差异有统计学意义(分别为1.03±0.03、1.62±0.08、3.41±0.27、2.86±0.20,P<0.05)。细胞免疫荧光结果显示,经替莫唑胺处理后,U87细胞株的γ-H2AX蛋白的表达水平较U251和LN229细胞株增加(P<0.05)。结论HIST1 H2BH在脑胶质瘤中的表达随病理学级别的升高而增加,是影响脑胶质瘤患者生存期的危险因素,可能与其参与DNA损伤修复从而引起肿瘤细胞耐药有关。

Objective To explore the key genes that affect the malignant progression of glioma and the survival of patients,and to study their expression characteristics and clinical significance in gliomas.Methods A retrospective analysis was conducted on the clinical data and the sequencing data of 310 cases of Chinese glioma genome atlas(CGGA)RNA sequencing database,699 cases of cancer genome atlas(TCGA)RNA sequencing database,268 cases of GSE16011 database and 317 cases of REMBRANDT mRNA database to screen out the differential genes related to patient prognosis,and the target gene was determined through literature search.The expression characteristics of the gene were evaluated in the above four databases.Kaplan-Meier survival curve and Cox regression analysis were used to evaluate the effect of this gene on the outcomes of patients with glioma.Through the Pearson correlation test,the genes in the CGGA and TCGA databases that were significantly positively correlated with the target gene expression(r>0.5 and P<0.05)were screened out.We used the online software DAVID to analyze the biological function enrichment of the differentially expressed genes.Realtime fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of this gene in astrocytic cell lines and glioma cell lines.Cellular immunofluorescence staining was used to detect the expression of γ-H2AX protein in glioma cell lines(U87,U251 and LN229)after drug treatment.Results After screening,histone cluster 1 H2B family member H(HIST1H2BH)was decided as the target gene.In the above four databases,the expression levels of HIST1H2BH in the WHO Ⅱ,Ⅲ and Ⅳ gliomas were statistically significant(all P<0.01),of which the expression was the highest in level Ⅳ gliomas,and the lowest in level Ⅱ gliomas;and the expression of HIST1H2BH in isocitrate dehydrogenase(IDH)wild-type glioma was higher than that in IDH mutant glioma(all P<0.001).In the CGGA and TCGA databases,the overall survival rates of patients with all grades of glioma and glioblastoma in the high-expression group of HIST1H2BH was lower than those of the low-expression group(all P<0.05).The results of univariate and multivariate Cox regression analysis showed that the expression of HIST1H2BH was an risk factor affecting the overall survival of patients with glioma(HR=1.275,95%CI:1.040-1.563,P<0.05).The results of biological function enrichment analysis showed that genes that were significantly positively related to the expression of HIST1H2BH were mainly involved in cell proliferation,DNA damage repair and extracellular matrix regulation.The qRT-PCR results showed that the relative expression levels of HIST1H2BH in HA,U87,U251 and LN229 cell lines were significantly different(1.03±0.03,1.62±0.08,3.41±0.27,2.86±0.20,respectively,P<0.05).The results of cellular immunofluorescence showed that the expression of γ-H2AX protein in U87 cell line after treatment with temozolomide was higher than those in U251 and LN229 cell lines(P<0.05).Conclusions The expression of HIST1H2BH in glioma increases with the pathological grade of glioma.It is a risk factor affecting the survival of glioma patients,which may be related to its participation in DNA damage repair that causing drug resistance of tumor cell.