eRNA AP003469.2与肝细胞癌患者预后的相关性分析

Correlation analysis of eRNA AP003469.2 and prognosis of patients with hepatocellular carcinoma

目的通过癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库筛选肝细胞癌(hepatocellular carcinoma,HCC)中关键的增强子RNA(enhancer RNA,eRNA),探究其作为HCC预后分子和治疗靶点的潜力。方法从TCGA数据库中检索下载33种癌症类型的基因表达RNA-seq配置文件,应用Kaplan-Meier生存分析和Spearman相关分析筛选与HCC患者生存相关的eRNA和靶基因。评估目的eRNA与临床特征的相关性。进行基因集富集分析以研究目的eRNA潜在的生物学功能。在HCC细胞株Hep 3B中分别构建AP003469.2和靶基因酪氨酸/色氨酸羟化酶激活蛋白ζ(tyrosine and tryptophan hydroxylase activators zeta,YWHAZ)功能缺失细胞模型,定量实时PCR检测AP003469.2敲低后对YWHAZ表达的影响。应用体外增殖实验和transwell实验探究AP003469.2和YWHAZ敲低对细胞增殖和迁移、侵袭能力的影响。在泛癌中验证目的eRNA在其他癌种中的预后潜能。结果在HCC中,AP003469.2和靶基因YWHAZ与患者不良预后相关,且AP003469.2的表达在患者的组织病理学分级、AJCC分期和T分期方面,差异均具有统计学意义(均P<0.05)。基因和通路富集分析结果显示,AP003469.2与参与中性粒细胞脱颗粒、中性粒细胞活化、细胞外基质降解、细胞骨架蛋白重构、细胞伪足和细胞黏附等生物学行为的分子和调控通路相关;进一步分析发现,AP003469.2可能通过中性粒细胞细胞陷阱和细胞伪足调控HCC的发生和发展。定量实时PCR实验证实AP003469.2对靶基因YWHAZ转录的促进作用(P<0.05)。CCK-8和transwell实验表明,AP003469.2和YWHAZ敲低均可抑制HCC细胞株Hep 3B的增殖、迁移和侵袭能力(均P<0.01)。结论AP003469.2可能是HCC中的关键eRNA,有潜力成为HCC早期诊断和预后的标志物和治疗靶点。

Objective To identify the key enhancer RNA(eRNA)in hepatocellular carcinoma(HCC)based on The Cancer Genome Atlas(TCGA)database and explore its potential as a prognostic molecule and therapeutic target for HCC.Methods RNA-seq pro-files of gene expression of 33 cancer types were retrieved and downloaded from TCGA database,and the significant prognostic eRNAs and target genes associated with the survival of HCC patients were identified by Kaplan-Meier survival analysis and Spearman correlation analysis.The key eRNA and target gene were selected for clinical correlation analysis.Gene enrichment analyses were performed to explore the potential biological functions of the key eRNA.Cell models with the loss of function of AP003469.2 and tyrosine/tryptophan hydroxylase activator zeta(YWHAZ)were constructed in HCC cell line Hep 3B.Quantitative real-time PCR was used to detect the expression of YWHAZ in AP003469.2 knockdown cells.The effects of AP003469.2 and YWHAZ knockdown on the cell proliferation,migration and invasion were explored by in vitro proliferation experiment and transwell experiment.The prognostic potential of the key eRNA was validated in pan-cancer.Results In HCC,AP003469.2 and target gene YWHAZ were associated with poor prognosis,and the expression of AP003469.2 had statistical significance in histopathological grade,AJCC stage and T stage in HCC patients(all P<0.05).Gene and pathway enrichment analysis revealed that AP003469.2 was related to molecular and regulatory pathways involved in neutrophil degranulation,neutrophil activation,extracellular matrix degradation,cytoskeletal protein remodeling,cell pseudopodia,and cell adhesion.Further analysis revealed that AP003469.2 might regulate the occurrence and development of HCC through neutrophil extracellular traps and cell pseudopodia.Quantitative real-time PCR confirmed that AP003469.2 promoted the transcription of target gene YWHAZ(P<0.05).CCK-8and transwell assays showed that AP003469.2 and YWHAZ inhibited the proliferation,migration and invasion of HCC cell line Hep 3B(all P<0.01).Conclusion eRNA AP003469.2 may be a potential prognostic biomarker and therapeutic target for HCC.