Integrated analysis of mRNA and miRNA profiles revealed the role of miR-193 and miR-210 as potential regulatory biomarkers in different molecular subtypes of breast cancer

Abstract

Background: Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated.

Methods: The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively.

Results: The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3.

Conclusions: In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.

Keywords: Breast cancer; Cell migration; Cell proliferation; MiRNA-mRNA interaction; miR-193; miR-210.

Fig. 1

Hierarchical clustering analysis of breast cancer cell lines and normal HB4A cells. Unsupervised hierarchical cluster analysis representing the 92 miRNAs expressed in breast cancer cell lines versus normal HB4A cells

Fig. 2

Validation of the selected microRNAs (hsa-miR-193a-3p and hsa-miR-210) expression in TCGA data. The log of the normalized expression values of miRNA data of TCGA from patients of different molecular subtypes were compared to expression levels from normal breast tissue samples. Mean ± SD are shown; *P ≤0.01

Fig. 3

Real-time proliferation analysis using xCELLigence system. The miRNA inhibition time is indicated by arrows. a Silencing of miR-193 in BT-20 cells; b Silencing of miR-210 in BT-20 cells; c Silencing of miR-193 in MCF-7 cells; d Silencing of miR-210 in MDA-MB-231 cells