MiRNA-513a-5p inhibits progesterone receptor expression and constitutes a risk factor for breast cancer: the hOrmone and Diet in the ETiology of breast cancer prospective study

Abstract

MicroRNAs (miRNAs) might be considered both predictors and players of cancer development. The aim of the present report was to investigate whether many years before the diagnosis of breast cancer miRNA expression is already disregulated. In order to test this hypothesis, we compared miRNAs extracted from leukocytes in healthy women who later developed breast cancer and in women who remain healthy during the whole 15-year follow-up time. Accordantly, we used a case-control study design nested in the hOrmone and Diet in the ETiology of breast cancer (ORDET) prospective cohort study addressing the possibility that miRNAs can serve as both early biomarkers and components of the hormonal etiological pathways leading to breast cancer development in premenopausal women. We compared leukocyte miRNA profiles of 191 incident premenopausal breast cancer cases and profiles of 191 women who remained healthy over a follow-up period of 20 years. The analysis identified 20 differentially expressed miRNAs in women candidate to develop breast cancer versus control women. The upregulated miRNAs, miR-513-a-5p, miR-513b-5p and miR-513c-5p were among the most significantly deregulated miRNAs. In multivariate analysis, miR-513a-5p upregulation was directly and statistically significant associated with breast cancer risk (OR = 1.69; 95% CI 1.08-2.64; P = 0.0293). In addition, the upregulation of miR-513-a-5p displayed the strongest direct association with serum progesterone and testosterone levels. The experimental data corroborated the inhibitory function of miR-513a-5p on progesterone receptor expression confirming that progesterone receptor is a target of miR-513a-5p. The identification of upregulated miR-513a-5p with its oncogenic potential further validates the use of miRNAs as long-term biomarker of breast cancer risk.

Figure 1.

(A) Heatmap of top ranked 20 miRNAs from premenopausal samples. The heatmap provides insight into the data structure for each microRNA and samples. We used red and green colors for defining low and high expression values. The blue represents the cases and yellow represents the controls. The clustering methods partitioned the dataset into three clusters identified on the left side of the figure. Each miRNA is listed on the right side. (B) Principal component analysis of the miRNAs signature from METABRIC breast cancer patient sample analysis. (C) Breast cancer survival data based on miR-513a-5p low and high expression. Higher values of miR-513a-5p were associated to a poor prognosis from Kaplan–Meier method and multivariate Cox proportional-hazards regression. Several clinical variables, such as T, N, stage, age, hystotype, menopause status and ER, PR, HER2 receptors, were included in the multivariate model.

Figure 2.

MiR-513a-5p impairs the expression of PR in luminal breast cancer cell lines. (A) qRT-PCR analysis of miR-513a-5p expression levels in MCF-7 and T47D cell lines. (B, C) Western blot analysis and qRT-PCR analysis of PR expression levels in MCF7 cells transiently transfected with miR-513a-5p mimic or control. (D) Western blot analysis of PR protein expression in ZR-75-1 and BT-474 breast cancer cell lines transiently transfected with miR-513a-5p mimic or control. (E) Western blot analysis of PR protein expression in serum-starved MCF-7 cells treated for 48 h with 10 nM E2 upon miR-513a-5p over-expression. (F, G) qRT-PCR analysis of PR and ATP1B1 expression levels in serum-starved MCF-7 cells treated for 48h with 10nM E2 upon miR-513a-5p over-expression. (H) Western-blot analysis of PR protein expression in serum-starved MCF-7 cells treated for 48 h with 10 nM E2 upon miR-513a-5p inhibition (inh miR-513a-5p). (I, J) qRT-PCR analysis of PR and ATP1B1 expression levels in serum-starved MCF-7 cells treated for 48 h with 10 nM E2 upon miR-513a-5p inhibition (inh miR-513a-5p). *P < 0.05.

Figure 3.

MiR-513a-5p increases MCF-7 cells resistance to serum-starvation stress. (A, B) Western blot analysis of PR, p21 and cleaved PARP proteins expression in serum-starved MCF-7 cells treated for 48 h with 10 nM E2 upon miR-513a-5p over-expression. (C) Western blot analysis of PR and cleaved PARP proteins expression in serum-starved MCF-7 cells treated for 48 h with 10 nM P4 upon miR-513a-5p over-expression.