| Objective: In order to explore the potential biomarkers of retinoblastoma,we search for the hub genes related to the development of retinoblastoma(RB)by comprehensive bioinformatics analysis.And then,to verify the expression levels of key genes by molecular biological experiments on RB cells and tissue samples,in order to preliminarily explore the genes that may be related to the occurrence of RB.Methods: In this study,firstly,we obtained 3 gene expression datasets from the Gene Expression Omnibus(GEO)database.After they were merged,the sva package in R software was applied to remove the batch effects of these three datasets.The differentially expressed genes(DEGs)were identified by limma package.Cluster Profiler package was used to analyze GO enrichment and KEGG pathway of DEGs.STRING database and Cytoscape software were used to construct the protein–protein interaction network(PPI).Cyto Hubba was applied to find the hub gene of PPI network.Weighted Gene Co-expression Network Analysis(WGCNA)was utilized to identify key modules associated with clinical information.The key genes of the key modules were further searched.Then,Y79,WERI-RB-1 and ARPE-19 cells were cultured in vitro,and the m RNA expression levels of five key genes were detected by quantitative real-time polymerase chain reaction(q RT-PCR).A total of 20 retinoblastoma tissue samples were collected.Immunohistochemistry(IHC)was used to detect the expression of key proteins.Results: A total of 1254 DEGs were identified,among which 422 were up-regulated and 832 were down-regulated.In GO analysis,DEGs were mainly related to protein heterodimerization activity,cation transmembrane transporter activity,and chromatin binding.In KEGG analysis,DEGs were mainly enriched in cell cycle,Phototransduction,and DNA replication.A total of 79 hub genes in the PPI network were obtained.In the co-expression network,DEGS were divided into 11co-expressed gene modules.According to Pearson correlation coefficient between each module and clinical traits,5 important modules including blue,pink,turquoise,red and brown were identified,among which blue module had the highest correlation coefficient with age at diagnosis.After comprehensive analysis,we obtained 5 hub genes.The q RT-PCR results showed that the m RNA relative expression levels of PRC1 and CENPK in RB cells were higher than those in the control group.IHC results indicated that PRC1 protein was highly expressed in RB tissue samples.Conclusion: SMC4,MCM6,CENPK,KIF15,and PRC1 were identified as hub genes by bioinformatics analysis,among which the increased expression of PRC1 in RB cells and tissue samples may be a potential biomarker affecting the occurrence and development of RB. |
