An induction of microRNA, miR-7 through estrogen treatment in breast carcinoma

Abstract

Background: Estrogen plays an important role in the development of estrogen-dependent breast carcinoma. Recently, several studies demonstrated a possible involvement of several micro RNAs (miRNAs) in the development of resistance to endocrine therapy in breast cancer patients, but the correlation between estrogen actions and miRNA expression in breast carcinoma still remains largely unknown. Therefore, in this study, we examined the in vitro effects of estrogen upon miRNA expression profiles in breast carcinoma.

Methods: We first screened the miRNA expression profiles induced by 17β-Estradiol (E2) using RT2 miRNA PCR Array in the ER-positive breast carcinoma cell line MCF-7. We identified miR-7 as the important miRNA associated with estrogen actions in these cells and further examined the changes of estrogen-dependent EGFR expression by miR-7 in ER-positive or -negative breast carcinoma cell lines including MCF-7. We also evaluated the correlation between miR-7 and EGFR expression in breast carcinoma cells derived from 21 patients using laser capture microdissection combined with quantitative reverse transcriptase-PCR.

Results: Seventeen miRNAs were significantly induced by E2 treatment in the MCF-7 cell line. Among 17 miRNAs induced by estradiol treatment, only miR-7 expression was significantly decreased by subsequent ICI treatment. The expression of miR-7 was up-regulated 2.94-fold by E2 treatment. miR-7 was reported to suppress epidermal growth factor receptor (EGFR) expression in several human malignancies. Transfection of miR-7 significantly suppressed EGFR mRNA levels in MCF-7 cells. Depletion of E2 from cell culture media also increased the expression level of EGFR mRNA in MCF-7 and T-47D cells but not in ER-negative, MDA-MB-231 and SK-BR-3 cells. We also evaluated the status of miR-7 in breast carcinoma tissues, but the correlation between the status of miR-7 and EGFR in carcinoma cells isolated by laser capture microscopy was not detected.

Conclusions: These results suggest that miR-7 may play a role in the development of resistance to endocrine therapy in breast cancer patients through regulating EGFR expression of carcinoma cells.

Figure 1

Results of the status of miRNA expression in MCF-7 cell line treated with E2. Scattered plot analysis of miRNA PCR array in MCF-7 treated for 24 hours. Solid lines, y = x with lines indicating two fold increment or decrement of expression levels.

Figure 2

Effects of miR-7 upon EGFR mRNA abundance in MCF-7 cell line. EGFR mRNA levels in MCF-7 transfected with miR-7 for five days. *, Significantly different from miR-NC. p < 0.05. miR-NC, transfection with scramble RNA.

Figure 3

(A) Effects of E2 upon EGFR mRNA in MCF-7. EGFR mRNA levels were evaluated using quantitative RT-PCR after 5 days of culture in each condition. (B) Alterations of miR-7 expression by depletion of E2 in MCF-7. FBS, Medium supplemented with 10% FBS; DCC-FBS, Medium supplemented with 10% DCC-FBS; E2, treatment of 10 nM E2; ICI, treatment of 1 μM ICI. *, Significantly different from FBS; †, Significantly different from E2.

Figure 4

(A) Alterations of miR-7 expression by depletion of E2 in T-47D. Levels of miR-7 expression were normalized using U6 probe. (B-D): Alterations of EGFR mRNA expression by depletion of E2 in breast carcinoma cells. Values for EGFR mRNA were depicted as percent expression to RPL13A by quantitative RT-PCR. (B) Expression of EGFR mRNA in ER-positive T-47D cells cultured in medium supplemented with 10% FBS or DCC-FBS. (C, D): Expression of EGFR mRNA in ER-negative SK-BR-3 (B) and MDA-MB-231 (C) cells cultured in medium supplemented with 10% FBS or DCC-FBS.

Figure 5

MiR-7 expression in breast cancer tissues. Levels of miR-7 expression were normalized using U6 probe. Correlation between miR-7 and (A) ER or (B) PR LIs (%) in fourteen ER-positive FFPE breast carcinoma tissues. Correlation between miR-7 and (C) ER or (D) EGFR mRNA in twenty fresh frozen breast carcinoma tissues.