J Cancer 2019; 10(26):6618-6634. doi:10.7150/jca.33433

Research Paper

Significant prognostic values of differentially expressed-aberrantly methylated hub genes in breast cancer

Lina Qi1,3*, Biting Zhou1*, Jiani Chen1,3*, Wangxiong Hu1,2, Rui Bai1,2, Chenyang Ye1,2, Xingyue Weng1, Shu Zheng1,2✉

1. Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310009, China.
2. Research Center for Air Pollution and Health, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310009, China.
3. Department of Surgical Oncology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310016, China.
*These three authors contributed equally to this work.

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Citation:
Qi L, Zhou B, Chen J, Hu W, Bai R, Ye C, Weng X, Zheng S. Significant prognostic values of differentially expressed-aberrantly methylated hub genes in breast cancer. J Cancer 2019; 10(26):6618-6634. doi:10.7150/jca.33433. Available from http://www.jcancer.org/v10p6618.htm

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Abstract

Introduction: Abnormal status of gene expression plays an important role in tumorigenesis, progression and metastasis of breast cancer. Mechanisms of gene silence or activation were varied. Methylation of genes may contribute to alteration of gene expression. This study aimed to identify differentially expressed hub genes which may be regulated by DNA methylation and evaluate their prognostic value in breast cancer by bioinformatic analysis.

Methods: GEO2R was used to obtain expression microarray data from GSE54002, GSE65194 and methylation microarray data from GSE20713, GSE32393. Differentially expressed-aberrantly methylated genes were identified by FunRich. Biological function and pathway enrichment analysis were conducted by DAVID. PPI network was constructed by STRING and hub genes was sorted by Cytoscape. Expression and DNA methylation of hub genes was validated by UALCAN and MethHC. Clinical outcome analysis of hub genes was performed by Kaplan Meier-plotter database for breast cancer. IHC was performed to analyze protein levels of EXO1 and Kaplan-Meier was used for survival analysis.

Results: 677 upregulated-hypomethylated and 361 downregulated-hypermethylated genes were obtained from GSE54002, GSE65194, GSE20713 and GSE32393 by GEO2R and FunRich. The most significant biological process, cellular component, molecular function enriched and pathway for upregulated-hypomethylated genes were viral process, cytoplasm, protein binding and cell cycle respectively. For downregulated-hypermethylated genes, the result was peptidyl-tyrosine phosphorylation, plasma membrane, transmembrane receptor protein tyrosine kinase activity and Rap1 signaling pathway (All p< 0.05). 12 hub genes (TOP2A, MAD2L1, FEN1, EPRS, EXO1, MCM4, PTTG1, RRM2, PSMD14, CDKN3, H2AFZ, CCNE2) were sorted from 677 upregulated-hypomethylated genes. 4 hub genes (EGFR, FGF2, BCL2, PIK3R1) were sorted from 361 downregulated-hypermethylated genes. Differential expression of 16 hub genes was validated in UALCAN database (p<0.05). 7 in 12 upregulated-hypomethylated and 2 in 4 downregulated-hypermethylated hub genes were confirmed to be significantly hypomethylated or hypermethylated in breast cancer using MethHC database (p<0.05). Finally, 12 upregulated hub genes (TOP2A, MAD2L1, FEN1, EPRS, EXO1, MCM4, PTTG1, RRM2, PSMD14, CDKN3, H2AFZ, CCNE2) and 3 downregulated genes (FGF2, BCL2, PIK3R1) contributed to significant unfavorable clinical outcome in breast cancer (p<0.05). High expression level of EXO1 protein was significantly associated with poor OS in breast cancer patients (p=0.03).

Conclusion: Overexpression of TOP2A, MAD2L1, FEN1, EPRS, EXO1, MCM4, PTTG1, RRM2, PSMD14, CDKN3, H2AFZ, CCNE2 and downregulation of FGF2, BCL2, PIK3R1 might serve as diagnosis and poor prognosis biomarkers in breast cancer by more research validation. EXO1 was identified as an individual unfavorable prognostic factor. Methylation might be one of the major causes leading to abnormal expression of those genes. Functional analysis and pathway enrichment analysis of those genes would provide novel ideas for breast cancer research.

Keywords: Breast cancer, Expression, Methylation, Prognosis, Bioinformatics