Zeng Yuyang, He Tao, Liu Juejun, Li Zongyuan, Xie Feijia, Chen Changzheng, Xing Yiqiao
Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, People's Republic of China.
Medicine (Baltimore). 2020 Nov 20;99(47):e23314. doi: 10.1097/MD.0000000000023314.
Retinoblastoma (RB) is the commonest malignant tumor of the infant retina. Besides genetic changes, epigenetic events are also considered to implicate the occurrence of RB. This study aimed to identify significantly altered protein-coding genes, DNA methylation, microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and their molecular functions and pathways associated with RB, and investigate the epigenetically regulatory mechanism of DNA methylation modification and non-coding RNAs on key genes of RB via bioinformatics method.We obtained multi-omics data on protein-coding genes, DNA methylation, miRNAs, and lncRNAs from the Gene Expression Omnibus database. We identified differentially expressed genes (DEGs) using the Limma package in R, discerned their biological functions and pathways using enrichment analysis, and conducted the modular analysis based on protein-protein interaction network to identify hub genes of RB. Survival analyses based on The Cancer Genome Atlas clinical database were performed to analyze prognostic values of key genes of RB. Subsequently, we identified the differentially methylated genes, differentially expressed miRNAs (DEMs) and lncRNAs (DELs), and intersected them with key genes to analyze possible targets of the underlying epigenetic regulatory mechanisms. Finally, the ceRNA network of lncRNAs-miRNAs-mRNAs was constructed using Cytoscape.A total of 193 DEGs, 74 differentially methylated-DEGs (DM-DEGs), 45 DEMs, 5 DELs were identified. The molecular pathways of DEGs were enriched in cell cycle, p53 signaling pathway, and DNA replication. A total of 10 key genes were identified and found significantly associated with poor survival outcome based on survival analyses, including CDK1, BUB1, CCNB2, TOP2A, CCNB1, RRM2, KIF11, KIF20A, NDC80, and TTK. We further found that hub genes MCM6 and KIF14 were differentially methylated, key gene RRM2 was targeted by DEMs, and key genes TTK, RRM2, and CDK1 were indirectly regulated by DELs. Additionally, the ceRNA network with 222 regulatory associations was constructed to visualize the correlations between lncRNAs-miRNAs-mRNAs.This study presents an integrated bioinformatics analysis of genetic and epigenetic changes that may be associated with the development of RB. Findings may yield many new insights into the molecular biomarker candidates and epigenetically regulatory targets of RB.
视网膜母细胞瘤（RB）是婴儿视网膜最常见的恶性肿瘤。除了基因改变外，表观遗传事件也被认为与RB的发生有关。本研究旨在通过生物信息学方法，鉴定与RB相关的显著改变的蛋白质编码基因、DNA甲基化、微小RNA（miRNA）、长链非编码RNA（lncRNA）及其分子功能和通路，并研究DNA甲基化修饰和非编码RNA对RB关键基因的表观遗传调控机制。我们从基因表达综合数据库中获取了蛋白质编码基因、DNA甲基化、miRNA和lncRNA的多组学数据。我们使用R语言中的Limma软件包鉴定差异表达基因（DEG），通过富集分析识别其生物学功能和通路，并基于蛋白质-蛋白质相互作用网络进行模块分析以鉴定RB的枢纽基因。基于癌症基因组图谱临床数据库进行生存分析，以分析RB关键基因的预后价值。随后，我们鉴定了差异甲基化基因、差异表达的miRNA（DEM）和lncRNA（DEL），并将它们与关键基因进行交集分析，以分析潜在表观遗传调控机制的可能靶点。最后，使用Cytoscape构建lncRNA-miRNA-mRNA的ceRNA网络。共鉴定出193个DEG、74个差异甲基化DEG（DM-DEG）、45个DEM、5个DEL。DEG的分子通路在细胞周期、p53信号通路和DNA复制中富集。基于生存分析共鉴定出10个关键基因，发现它们与不良生存结局显著相关，包括细胞周期蛋白依赖性激酶1（CDK1）、有丝分裂纺锤体组装检查点蛋白1（BUB1）、细胞周期蛋白B2（CCNB2）、拓扑异构酶Ⅱα（TOP2A）、细胞周期蛋白B1（CCNB1）、核糖核苷酸还原酶M2亚基（RRM2）、驱动蛋白家族成员11（KIF11）、驱动蛋白家族成员20A（KIF20A）、核分裂纺锤体蛋白80（NDC80）和双特异性酪氨酸磷酸化调节激酶（TTK）。我们进一步发现枢纽基因微小染色体维持蛋白6（MCM6）和驱动蛋白家族成员14（KIF14）存在差异甲基化，关键基因RRM2被DEM靶向，关键基因TTK、RRM2和CDK1受DEL间接调控。此外，构建了具有222个调控关联的ceRNA网络，以可视化lncRNA-miRNA-mRNA之间的相关性。本研究对可能与RB发生发展相关的遗传和表观遗传变化进行了综合生物信息学分析。研究结果可能为RB的分子生物标志物候选物和表观遗传调控靶点提供许多新的见解。
