miR-7 Reverses Breast Cancer Resistance To Chemotherapy By Targeting MRP1 And BCL2

Abstract

Background: MicroRNAs (miRNAs) are a class of non-coding RNAs that have been linked with breast cancer chemoresistance, which is a major clinical problem causing disease relapse and poor prognosis. miR-7 exerts several tumor suppressive activities.

Purpose: This study was designed to clarify whether and how miR-7 regulates breast cancer chemoresistance.

Methods: miR-7 level in breast cancer was determined by qRT-PCR analysis. Cell viability was assessed by MTS assay to quantify the IC₅₀ value of paclitaxel and carboplatin. The targets of miR-7 were confirmed by luciferase reporter assay.

Results: Higher miR-7 expression predicts better pathological complete response (pCR) of breast cancer patients receiving paclitaxel/carboplatin chemotherapy. In vitro, miR-7 sensitizes breast cancer cell lines (MCF-7 and MDA-MB-231) to paclitaxel and carboplatin, alone and in combination. In addition, we reveal that both the multidrug resistance-associated protein 1 (MRP1) and anti-apoptotic B cell lymphoma 2 (BCL2) are targets of miR-7 in breast cancer cells. Furthermore, miR-7-induced sensitization of breast cancer to paclitaxel/carboplatin is markedly reversed by restoration of MRP1 and BCL2.

Conclusion: These findings show that miR-7 reverses breast cancer chemoresistance through suppressing MRP1 and BCL2, and also suggest that miR-7 may possess a predictive value and represent a therapeutic target in breast cancer chemotherapy.

Keywords: BCL2; MRP1; breast cancer; chemoresistance; miR-7; pathological complete response.

Figure 1

Higher miR-7 expression predicts better pathological complete response of breast cancer patients. (A) Clinical information and demographics of breast cancer patients achieving pathological complete response (pCR, n = 28) or not (non-pCR, n = 32) to paclitaxel plus carboplatin neoadjuvant chemotherapy. Fisher’s exact test. (B) miR-7 expression level in breast cancer patients was determined by quantitative real-time PCR (qRT-PCR). Data were normalized to U6. Each symbol represents the mean value from 3 replicates of each patient. (C) Breast cancer patients were evenly stratified by high (n = 30) and low (n = 30) miR-7 expression level, and disease-free survival was analyzed by Kaplan–Meier method.

Figure 2

miR-7 sensitizes breast cancer to chemotherapy. (A) MCF-7 and MDA-MB-231 cells were transfected with control mimic or miR-7 mimic. After 48 hrs, miR-7 expression level was determined by qRT-PCR analysis (n = 3). (B–C) MCF-7 and MDA-MB-231 cells shown in (A) were further treated with serial dilutions of paclitaxel (B) and carboplatin (C) for 72 hrs. Cell viability was measured by MTS assay and IC₅₀ values were calculated (n = 5). (D) MCF-7 and MDA-MB-231 cells shown in (A) were further treated with 10 µM paclitaxel plus 100 µM carboplatin as indicated for 72 hrs. Cell viability was measured by MTS assay and percentage of cell survival relative to NC mimic treatment was calculated (n = 5). (E–H) MCF-7 and MDA-MB-231 cells were transfected with control inhibitor or miR-7 inhibitor. miR-7 expression level (E), IC₅₀ values (F–G), and cell viability (H) were analyzed as in (A-D). (I) The paclitaxel-resistant MCF-7 cells (MCF-7-PR) were transfected as indicated and further treated with serial dilutions of paclitaxel for 72 hrs. Cell viability was measured by MTS assay and IC₅₀ values were calculated (n = 5). Data were expressed as the mean ± S.E.M. *p < 0.05; **p < 0.01.

Figure 3

MRP1 and BCL2 are both targets of miR-7 in breast cancer. (A) Schematic representation of the putative miR-7 binding site within the 3ʹ-UTR sequence of MRP1 mRNA and BCL2 mRNA. (B) HEK293T cells were transfected control mimic or miR-7 mimic along with wild-type (wt) or mutant (mut) MRP1 or BCL2 3ʹ-UTR luciferase reporter as indicated. After 48 hrs, luciferase activity was measured (n = 4). (C) HEK293T cells were transfected control inhibitor or miR-7 inhibitor along with wt or mut MRP1 or BCL2 3ʹ-UTR luciferase reporter as indicated. After 48 hrs, luciferase activity was measured (n = 4). (D–E) MCF-7 and MDA-MB-231 cells were transfected with control mimic or miR-7 mimic (D), or with control inhibitor or miR-7 inhibitor (E). After 48 hrs, the mRNA levels of MRP1 and BCL2 were determined by qRT-PCR analysis (n = 3). (F) MCF-7 and MDA-MB-231 cells were treated as in (D–E). The protein levels of MRP1 and BCL2 were determined by Western blot analysis. Images from 3 independent experiments are shown. (G) The mRNA levels of MRP1 and BCL2 were determined in 5 representative breast cancer patients stratified by non-pCR and pCR. Data were expressed as the mean ± S.E.M. **p < 0.01; NS, not significant.

Figure 4

Targeted MRP1 and BCL2 are both involved in miR-7-sensitized breast cancer to chemotherapy. (A) MCF-7 and MDA-MB-231 cells were stably infected with lentivirus expressing vector control (Lv-vec) or human MRP1 (Lv-MRP1), and then transfected with control mimic or miR-7 mimic. After 48 hrs, MRP1 protein level was determined by Western blot analysis. Images from 3 independent experiments are shown. (B–C) MCF-7 and MDA-MB-231 cells shown in (A) were further treated with serial dilutions of paclitaxel (B) and carboplatin (C). Cell viability was measured by MTS assay and IC₅₀ values were calculated (n = 5). (D) MCF-7 and MDA-MB-231 cells shown in (A) were further treated with 10 µM paclitaxel plus 100 µM carboplatin as indicated for 72 hrs. Cell viability was measured by MTS assay and percentage of cell survival relative to NC mimic treatment was calculated (n = 5). (E–H) MCF-7 and MDA-MB-231 cells stably infected with Lv-vec or Lv-BCL2 were treated as in (A–D). BCL2 protein level (E), IC₅₀ values (F–G) and cell viability (H) were analyzed as in (A–D). (I–K) MCF-7 cells infected with Lv-MRP1 or Lv-BCL2 were transfected with NC inhibitor or miR-7 inhibitor. Protein expression (I), IC₅₀ values of paclitaxel (J) and carboplatin (K) were analyzed. Data were expressed as the mean ± S.E.M. *p < 0.05; **p < 0.01.