Despite many recent therapeutic innovations, BRCA remains the leading cancer-related cause of death among women. BRCA remains a highlight heterogeneous disease, with four major BRCA subtypes having been defined based upon tumor expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2, and the proliferative marker Ki67 (MKI67) [50–52]. This classification scheme is typically used to predict BRCA patient prognosis and to define treatment strategies together with information pertaining to tumor histological grade, type, and TNM stage. Given the high degree of BRCA tumor heterogeneity, however, it is difficult to predict tumor response to treatment or associated patient outcomes. Additionally, the cooperative effects of TFs and miRNAs in gene regulation remain largely unclear. Recent molecular studies have highlighted a number of genes and signaling pathways that govern BRCA development and progression[53–56], but further work is needed to fully elucidate the mechanistic basis for this complex disease. This study represents the first attempt to construct a trans-regulatory mRNA-miRNA-TF network in order to identify genes, miRNAs, and TFs related to BRCA, thereby clarifying the pathogenesis of BRCA at the molecular level and improving the detection, treatment, and prognostic assessment of this disease.
In the present study, we conducted an integrated bioinformatics analysis of three microarray datasets, leading us to identify 85 BRCA-related DEGs (54 and 31 down- and up-regulated, respectively). We found that our upregulated DEGs were mainly enriched in the cell cycle, cell differentiation, oocyte meiosis, and the p53 signaling pathway, which plays a significant role in the occurrence and progression of tumors. Mitotic catastrophe/error can also contribute to tumorigenesis [57, 58]. As such, mitosis has become an attractive anti-tumor target, with paclitaxel being one of the most successful drugs targeting this pathway that is commonly used to treat BRCA[59, 60]. Previous studies have shown that the p53 signaling pathway controls cell cycle regulation, mitosis, reproduction, and the inhibition of neoplastic transformation and tumor progression[61, 62]. The down-regulated genes were enriched in the lipid metabolic process, lipid transport, regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ions, positive regulation of cell proliferation, positive regulation of cell-matrix adhesion, tyrosine metabolism, cytochrome P450 drug metabolism, protein digestion and absorption, and PPAR signaling pathway. Previous studies have reported that PPAR, as a ligand-activated transcription factor, regulates lipid metabolism and inflammation and contributes to the survival of breast cancer cells[63, 64]. Six DEGs were enriched in these two signaling pathways, including CDK1, CCNB2, RRM2, and AURKA, which have been proven to be involved in the mitotic process[65]. All in all, our results are consistent with the above theories. BRCA is mostly correlated with cell cycle and cell differentiation, mainly for mitotic cell cycle and enriching p53 signaling pathway.
We then constructed PPI network from identified DEGs and identified 16 DEGs as key genes (degree ≥ 14), of which 14 were found to be significantly associated with poorer BRCA patient survival in the Kaplan-Meier Plotter database. We further confirmed that all 14 of these were significantly upregulated in BRCA tissue samples relative to normal tissue controls in a GEPIA analysis (P < 0.05). Mitotic error is not the only cause of BRCA, but it is an important pathogenic factor and can explain one possible mechanism whereby BRCA can develop. Most of the core genes screened out in this study were related to mitosis, including ASPM[66], NEK2[67], RRM2, CEP55[42], CCNB2, BIRC5[47], KIF2C, AURKA, GLGAP5, and TPX2[68].Of these genes, several were previously correlated with the occurrence, development, and prognosis of BRCA. Accumulating evidence indicates that overexpression of AURKA causes drug resistance, promotes progression, and predicts poor prognosis for breast cancer. Suppression AURKA can accelerate PI3K-pathway inhibitor efficacy in breast cancer resulting in apoptosis and tumor regression[69]. Zheng et al. have also shown that AURKA translocates to the nucleus and causes carcinogenic activities in malignant tumor cells by enhancing breast cancer stem cell phenotypes[70]. Furthermore, UBE2C has been shown to regulate the activity of AURKA by controlling the activity of APC/C[49]. Overexpression of UBE2C in breast microcalcification lesions suggests that UBE2C is also related to the occurrence and development of breast cancer[71]. One study found that AURKA, TPX2, and DLGAP5 were highly upregulated in non-small cell lung cancer and correlated with one another. TPX2 and DLGAP5 are also phosphorylated by AURKA during the cell cycle[68]. Chen et al. found TPX2 silencing negatively regulates PI3K / Akt, activates the p53 signaling pathway, inhibits breast cancer cell proliferation, and promotes cell apoptosis, indicating that TPX2 may be a potential target for the treatment of breast cancer[72]. Another study has also found that TPX2 may be a novel prognostic marker of breast cancer[73].
In one prior analysis, RRM2 was found to be highly expressed in diverse cancers, and inhibiting RRM2 overexpression in retroperitoneal liposarcoma can impede tumor progression via downregulating the Akt/mTOR/4EBP1 pathway[74]. Meanwhile, Koppenhafer et al. found that CDK2-mediated RRM2 downregulation promotes DNA damage and cell apoptosis in Ewing sarcoma via inhibiting the ATR-CHK1 pathway[75]. Increasing evidence suggests that miRNAs and lncRNAs related to RRM2 can modulate tumor progression by regulating the expression of RRM2[76]. For example, in BRCA tissues, lncRNA DSCAM-AS1 directly targets miR-204-5p, stimulates proliferation and invasion, and impedes BRCA cell apoptosis by inhibiting miR-204-5p and facilitating RRM2 expression[77]. Consistent with these reports, we found that RMM2 upregulation was associated with a poorer BRCA patient prognosis. According to a previous study, CEP55 is a cell fate determinant in the context of perturbed mitosis in breast cancer. Loss of CEP55 renders breast cancer cells sensitive to anti-mitotic drugs by prematurely activating CDK1 / cyclin B and CDK1 caspase-dependent mitotic cell death. Furthermore, blocking MEK1/2-PLK1 signaling may reduce the outgrowth of MYC-CEP55-dependent basal-like, triple-negative breast cancers[78]. Our results were consistent with these findings, indicating that patients with higher levels of these genes expression may have poorer prognosis.
miRNAs are endogenous non-coding RNA molecules, that can modulate gene expression at the post-transcriptional level by reducing or inhibiting the translation of target genes[79]. This study screened out mRNA-miRNA target pairs using online prediction tools. RRM2, AURKA, NEK2, and BIRC5 were the top four potential miRNA targets in this study (≥ 28). Recent studies have found that some miRNAs are involved in the development of cancer, including BRCA[17–19].TFs also serve as trans-regulatory factors, much as do miRNAs. To gain a further understanding of key gene functions in this oncogenic context, an mRNA-target miRNA-TF regulatory network was therefore constructed. E2F1 was identified as a co-regulator of KIF2C, TOP2A, RRM2, AURKA, and BIRC5. MED1 was found to regulate AURKA and UBE2C, while SP1 was predicted to regulate KIF2C and BIRC5. Several studies have shown that E2F1 plays a critical role in the regulation of both cell proliferation and apoptosis[80, 81]. AURKA overexpression has also been observed in many cancers. He et al. [82] found that the transcriptional activity of E2F1 was increased by AURKA overexpression. We also found that AURKA was associated with miRNA cluster expression by regulating E2F1. AURKA may thus be a putative therapeutic target in BRCA. In addition, a previous study has reported that silencing RRM2 to inhibit the up-regulation of RRM2 can enhance DNA damage, which could in turn improve the efficacy of topoisomerase I inhibitors[83]. ASA has also been found to promote TRAIL-induced apoptosis by down-regulating BIRC5 gene expression, which was mediated by inhibiting E2F-1 binding to the BIRC5 promoter region[84]. The experiments in vitro further conduced to validate. These results suggest that AURKA, RRM2, BIRC5, and related regulatory factors including E2F1 and miRNAs may be key molecules involved in controlling the onset and progression of BRCA.