Identification of CCNB2 as A Potential Non-Invasive Breast Cancer Biomarker in Peripheral Blood Mononuclear Cells Using The Systems Biology Approach

Abstract

Objective: Breast cancer (BC) still remains an imperative clinical issue, despite advances in the diagnosis, prognosis and treatment modalities of this malignancy. Hence, progress has been made to identify non-invasive, high sensitive and specific biomarkers. Since immune system affects development of breast cancer, peripheral blood mononuclear cells (PBMCs) -a subpopulation of immune cells- can be considered as a promising tool in the field of BC biomarker research. In the current study, we initially attempted to use concept of the present shared biomarkers in solid tumors and systemic immune profile and then evaluate correlation of these biomarkers to clinical use in cancer research.

Materials and methods: In this experimental study, available microarray gene expression datasets of BC as well as the related PBMCs were retrieved and downloaded from the Gene Expression Omnibus (GEO) database, followed by analysis using GEO2R along with affylmGUI, a R-based package, to obtain differentially expressed genes (DEGs). Signature genes from 20 types of cancer were also applied to validate DEGs. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was carried out to assess mRNA level of CCNB2 in PBMC of the BC patients and healthy subjects.

Results: DEGs analysis for the transcription profile of BC cells and PBMCs showed two shared targets, CCNB2 and PGK1. Validation with systems biology using reweighted 20 types of cancer signature genes revealed that CCNB2 is the only common target in BC and its related PBMCs, which was further validated by qRT-PCR implying a significant increase in the level of CCNB2 in the BC patients.

Conclusion: Results of this study demonstrated that PBMCs are affected by BC cells and CCNB2 may be of value as a diagnostic biomarker for breast cancer. However, verification would require future detailed experimental plans.

Keywords: Biomarker; Breast Cancer; Peripheral Blood Mononuclear Cell; Systems Biology.

Fig.1

Strategy of the shared target discovery in cancer ecosystem. Two-cycle procedures were applied in this study. The first cycle reveals the identification of central cancer genes and the second one demonstrates the valuable non-invasive cancer diagnostic markers shared with the central cancer signature genes. PPI; Protein-protein interaction network, BC; Breast cancer, DEGs; Differentially expressed genes, PBMCs; Peripheral blood mononuclear cells, and GEO; Gene expression omnibus.

Fig.2

One hundred and eighty seven cancer signature genes in twenty types of cancer were used to construct the PPI network. A. PPI map of the cancer signature genes involving in 320 nodes and 129 interactions, major hubs are highlighted. B. Analysis of network topology, including degree and betweeness centrality indicates major PPI hubs. PPI; Protein-protein interaction.

Fig.3

Co-expression network of four major hubs in breast cancer tissue; purple nodes are hub genes of the network. Yellow lines indicate interaction between the hub genes.

Fig.4

CCNB2 gene expression analysis in PBMCs from the healthy individuals and patients with primary and metastatic stages of BCs. A. Kaplan-Meier plot using CCNB2 gene shows survival rate of the patients in the study. B. mRNA quantification of CCNB2 level in PBMCs of healthy individuals, primary and metastatic stages of breast cancer patients. Kruskal-Wallis test was performed. PBMCs; Peripheral blood mononuclear cells, BC; Breast cancer, * ; P<0.05, and **; P<0.01.