Decreased expression of microRNA-17 and microRNA-20b promotes breast cancer resistance to taxol therapy by upregulation of NCOA3

Abstract

Chemoresistance is a major obstacle to effective breast cancer chemotherapy. However, the underlying molecular mechanisms remain unclear. In this study, nuclear receptor coactivator 3 (NCOA3) was found to be significantly increased in taxol-resistant breast cancer tissues and cells. Moreover, overexpression of NCOA3 enhanced breast cancer cell resistance to taxol, whereas depletion of NCOA3 decreased taxol resistance. Subsequently, we investigated whether NCOA3 expression was regulated by miRNAs in breast cancer. By bioinformatics prediction in combination with the data of previous report, miR-17 and miR-20b were selected as the potential miRNAs targeting NCOA3. By real-time PCR analysis, we found that miR-17 and miR-20b were significantly reduced in taxol-resistant breast cancer tissues and cells. In addition, we provided some experimental evidences that miR-17 and miR-20b attenuated breast cancer resistance to taxol in vitro and in vivo models. Furthermore, by luciferase reporter assays, we further validated that both miR-17 and miR-20b directly binded the 3'-untranslated region of NCOA3 mRNA and inhibited its expression in breast cancer cells. Finally, both miR-17 and miR-20b levels were found to be significantly negatively correlated with NCOA3 mRNA levels in breast cancer tissues. Together, our results indicated that loss of miR-17 and miR-20b enhanced breast cancer resistance to taxol by upregulating NCOA3 levels. Our study suggested miR-17, miR-20b and NCOA3 may serve as some predictive biomarkers and potential therapeutic targets in taxol-resistant breast cancer treatment.

Figure 1

Overexpression of NCOA3 enhances breast cancer resistance to taxol. (a) RT-PCR analysis of relative NCOA3 expression in 22 breast cancer tissues from taxol-sensitive patients and 33 breast cancer tissues from taxol-resistant patients. (b and c) Western blot and RT-PCR were performed to detect the protein and mRNA expression of NCOA3 in taxol-resistant MCF-7 cells (MCF-7/Tax1 and MCF-7/Tax2) and taxol-resistant 231 cells (231/Tax1 and 231/Tax2). Data were from three independent experiments. Actin was used as a loading control. Data represent mean±S.D. ***P<0.001. (d and e) (d) MCF-7/Tax1 (A) and 231/Tax1 (B) cells were transfected with NC and NCOA3 siRNA. (e) MCF-7 (A) and 231 (B) cells were transfected with control (CTR) and NCOA3 vectors. After 8 h, cells were treated with indicated dose of Taxol (Tax) for additional 48 h. Cell apoptosis was assessed by Annexin-V-FITC/PI staining assay by flow cytometry. Columns, means of three determinations; bars, S.D.; **P<0.01; ***P<0.001, compared with control treated cells. (f) MCF-7/Tax1 (A) and 231/Tax1 (B) cells were transfected with NC and NCOA3 siRNA for 48 h. (g) MCF-7 (A) and 231 (B) cells were transfected with CTR and NCOA3 vectors for 48 h. Western blot was performed to detect the indicated protein expression. Data were from three independent experiments

Figure 2

MiR-17 and miR-20b are predicted to target NCOA3 and decreased in taxol-resistant breast cancer. (a) Flowchart for the selection of the miR-17 and miR-20b. (b) RT-PCR was performed to detect the expression of miR-17 and miR-20b in MCF-7, 231, MCF-7/Tax1, MCF-7/Tax2, 231/Tax1 and 231/Tax2 cells. U6 was used as an internal control. Data represent mean±S.D. ***P<0.001. (c and d) RT-PCR were performed to detect relative expression of miR-17 and miR-20b in 22 breast cancer tissues from taxol-sensitive patients and 33 breast cancer tissues from taxol-resistant patients. U6 was used as an internal control. Data represent mean±S.D. P<0.0001

Figure 3

Both miR-17 and miR-20b enhances taxol-induced apoptosis in breast cancer cells. (a and b) (a) 231/Tax1 and MCF-7/Tax1 were transfected with miR-17 mimics (a) or miR-20b mimics (b) and NC for 48 h, respectively. (B) MCF-7 and 231 cells were transfected with miR-17 inhibitors (a) or miR-20b inhibitors (b) and NC for 48 h, respectively. RT-PCR was performed to detect the expression of miR-17 or miR-20b. (c and d) MCF-7/Tax1 (A) and 231/Tax1 (B) were transfected with miR-17 or miR-20b mimics. (e and f) MCF-7 (A) and 231 (B) were transfected with miR-17 or miR-20b inhibitors. After 8 h, cells were treated with indicated dose of taxol (Tax) for additional 48 h. MTT assay was performed to examine cell viability (c and e). Cell apoptosis was assessed by Annexin-V-FITC/PI staining assay by flow cytometry (d and f). Columns, means of three determinations; bars, S.D. *P<0.05; **P<0.01; ***P<0.001, compared with NC-treated cells

Figure 4

Identification of NCOA3 as a direct target of miR-17 and miR-20b. (a) The predicted miR-17 and miR-20b target sites in the 3′-UTR of NCOA3 mRNA and their mutated version. (b and c) Luciferase activity assays in 231/Tax1 and 293T cells showed that miR-17 and miR-20b inhibited the expression of NCOA3. 231/Tax1 and 293T cells were co-transfected with pGL3 vector containing the wild-type or mutated 3′-UTR of NCOA3, or pGL3-control vector, along with miR-17 or miR-20b mimics and NC. After 48 h, luciferase activity was detected. Data were normalized to luciferase activity in the corresponding cells transfected with NC and are represented as the mean±S.D. of three replicates. (d-g) Protein and mRNA levels of NCOA3 were downregulated by miR-17 or miR-20b mimics in breast cancer cells. MCF-7/Tax1 (d and e) and 231/Tax1 (f and g) cells were transfected with miR-17 or miR-20b mimics and NC, respectively. (A) Western blot was performed to detect the protein expression of NCOA3. Actin was used as a loading control. Data were from three independent experiments. (B) RT-PCR was performed to detect the mRNA expression of NCOA3. Actin was used as control. Data are mean±S.D. from three independent experiments. ***P<0.001, compared with the control group

Figure 5

MiR-17 and miR-20b are negatively correlated with NCOA3 mRNA levels in breast cancer. Relative expression of NCOA3 along with miR-17 and miR-20b was determined by RT-PCR in 22 taxol-sensitive and 33 taxol-resistant breast cancer tissues from patients with taxol treatment. (a-d) Relative expression of NCOA3 along with miR-17 and miR-20b were determined by RT-PCR in 22 taxol-sensitive breast cancer tissues (a and c) and 33 taxol-resistant breast cancer tissues (b and d). For NCOA3, β-Actin was used as an internal control; For miR-17 and miR-20b, U6 was used as an internal control. Their expression correlations were analyzed by correlation coefficient and t-test

Figure 6

Overexpression of NCOA3 reverses reduction of cell viability and induction of apoptosis by miR-17 and miR-20b in taxol-treated breast cancer cells. (a-d) MCF-7/Tax1 (a and c) and 231/Tax1 (b and d) cells were co-transfected with NC and miR-17 or miR-20b mimics along with control (CTR) or NCOA3 vectors. After 8 h, cells were treated with indicated dose of taxol (Tax) for additional 48 h. (a and b) MTT assay was performed to examine cell viability. (c and d) Cell apoptosis was assessed by Annexin-V-FITC/PI staining assay by flow cytometry. Columns, means of three determinations; bars, S.D.; **P<0.01; ***P<0.001, compared with cells treated by NC plus CTR

Figure 7

MiR-17 and miR-20b decrease resistance of breast tumor to taxol in xenograft tumor models. MCF-7/Tax1 and 231/Tax1 cells stably expressing miR-17 or miR-20b mimics or NC mimics were injected into nude mice. Nude mice were administered with taxol (20 mg/kg) as indicated at each time point. (a and b) Tumor volume was measured once per three days by using calipers (as indicated at each time point) for 30–36 days. (c and d) Average body weight changes were measured over the course of the study. Data are shown as mean±S.D. (n=6 per group). ***P<0.001