Identification of Epigenetically Modified Hub Genes and Altered Pathways Associated With Retinoblastoma.

Aditi Karmakar,Senthil Kumar Ganesan,Md Maqsood Ahamad Khan,Nidhi Kumari,Nalini Devarajan

doi:10.3389/fcell.2022.743224

Abstract

Retinoblastoma (Rb) is the most common childhood malignancy initiated by biallelic mutation in RB1 gene and driven by various epigenetic events including DNA methylation and microRNA dysregulation. Hence, understanding the key genes that are critically modulated by epigenetic modifications in RB1 −/− cells is very important to identify prominent biomarkers and therapeutic targets of Rb. In this study, we for the first time have integrated various Rb microarray NCBI-GEO datasets including DNA Methylation (GSE57362), miRNA (GSE7072) and mRNA (GSE110811) to comprehensively investigate the epigenetic consequences of RB loss in retinoblastoma tumors and identify genes with the potential to serve as early diagnostic markers and therapeutic targets for Rb. Interestingly, the GEO2R and co-expression network analysis have identified three genes namely E2F3, ESR1, and UNC5D that are significantly deregulated by modified DNA methylation, mRNA and microRNA expression in Rb tumors. Due to their recognition in all epigenetic, transcriptomic, and miRNA datasets, we have termed these genes as “common genes”. The results of our integrative bioinformatics analysis were validated in vitro by studying the gene and protein expression of these common genes in Y79, WERI-Rb-1, Rb cell lines and non-tumorigenic retinal pigment epithelial cell line (hTERT-RPE). The expression of E2F3 and UNC5D were up-regulated and that of ESR1 was down-regulated in Rb tumor cells when compared to that in non-tumorigenic hTERT-RPE cells. More importantly, UNC5D, a potent tumor suppressor gene in most cancers is significantly up-regulated in Y79 and Weri Rb1 cells, which, in turn, questions its anti-cancer properties. Together, our study shows that E2F3, ESR1, and UNC5D may be crucially involved in Rb tumorigenesis and possess the potential to act as early diagnostic biomarkers and therapeutic targets of Rb.

Highlights

Retinoblastoma (Rb), the most common childhood malignancy that develops in the retina of children under age five causes a great impact on the overall life of the affected child (Dimaras et al, 2015a)
These data consist of genome-scale DNA methylation profiling in Retinoblastoma by using Illumina Human Methylation 450 Bead Chip platform and samples from the normal human eye and five ocular diseases (GSE57362, 25 Samples) (Berdasco et al, 2017), Profiling of miRNAs in human retinoblastoma by using 2k custom array and RNA was extracted from two retinoblastoma and two matched normal retina samples (GSE7072, four Samples) (Huang et al, 2007), Distinct Gene Expression Profiles Define Anaplastic Grade in Retinoblastoma by using Affymetrix Human Gene 2ST Array and RNA was extracted from the 28 retinoblastoma and three matched normal retina samples (GSE110811, 31 Samples) (Hudson et al, 2018)
We identified differentially expressed genes (DEGs) between normal and tumor samples individually for each dataset by LIMMA package using GEO2R

Summary

Introduction

Retinoblastoma (Rb), the most common childhood malignancy that develops in the retina of children under age five causes a great impact on the overall life of the affected child (Dimaras et al, 2015a). Hereditary Rb arises due to bilateral genetic mutations in RB1 gene located in chromosome 13. Sporadic Rb is non-heritable and arises due to unilateral somatic mutations in RB1 gene of a single retinal cell after fertilization (Dimaras et al, 2015b). The major obstacle behind the high cure rate of Rb-affected children in these countries is the lack of awareness and the high cost for conventional Rb diagnostic procedures. Growing investigations on Rb affected children of developing countries state that the lack of diagnostic facilities at the genetic level in these countries is the prime cause for late diagnosis, vision loss, and treatment complications in Rb affected children (Chantada et al, 2010; Chintagumpala 2019; Chawla et al, 2021). The development of novel biomarkers and prominent therapeutic targets for the early diagnosis and precise treatment of Rb is of urgent priority

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