STAT3 is required for MiR-17-5p-mediated sensitization to chemotherapy-induced apoptosis in breast cancer cells

Signal transducer and activator of transcription 3 (STAT3) controls cell survival, growth, migration, and invasion. Here, we observed that STAT3 exerted anti-apoptotic effects in breast cancer cells. On the other hand, miR-17-5p induced apoptosis in breast cancer cells, and overexpression of miR-17-5p sensitized MCF-7 cells to paclitaxel-induced apoptosis via STAT3. Overexpression of STAT3 in MCF-7 cells decreased paclitaxel-induced apoptosis, but STAT3 knockout abolished the miR-17-5p-induced increases in apoptosis. Finally, miR-17-5p promoted apoptosis by increasing p53 expression, which was inhibited by STAT3. These results demonstrate a novel pathway via which miR-17-5p inhibits STAT3 and increases p53 expression to promote apoptosis in breast cancer cells.


INTRODUCTION
Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor [1]. In response to interferon, cytokines, and growth factors, Janus kinases (JAK) phosphorylate STAT3, which then forms homo-or heterodimers that translocate to the cell nucleus and mediate the expression of a variety of genes; STAT3 thus plays a key role in many cellular processes, including cell survival, growth, and apoptosis [2]-5]. Furthermore, STAT3 can act as a tumor suppressor or an oncogenic agent; for example, STAT3 suppresses brain tumor progression via PTEN and promotes progression in various breast cancer types [3,4].
MicroRNAs (miRNAs) are small (approximately 22 nt) regulatory RNAs that base pair with the 3' untranslated region (3'UTR) of target genes, ultimately resulting in the degradation of target mRNAs or inhibition of their translation [5]. Several studies have demonstrated that miRNAs play important roles in human cancer [6][7][8][9][10], and miR-17-5p is particularly important in breast cancer. Specifically, the miR-17-5p cluster acts as a tumor suppressor by directly inhibiting the expression of AIB1 and cyclin D1 in human breast cancer [11,12]. Yu et al. demonstrated that the miR-17-5p cluster mediates migration and invasion in breast cancer cells by affecting the secretion of a heterotypic signal [13]. MiR-17-5p also suppresses MDA-MB-231 cell migration and invasion by inhibiting HBP1 [14,15]. In addition, miR-17/20 regulates p53 and inhibits Akt, in turn mediating breast cancer cell apoptosis [16], and miR-17*92 increases apoptosis by inhibiting the transition of pro-B into pre-B [17]. The onset and progression of breast cancer involves apoptotic signals that are stimulated by miRNAs [17]. Here we investigated the role of miR-17-5p, which may repress the translation of the STAT3 oncogene, in the control of breast cancer cell apoptosis.

MiR-17-5p sensitized breast cancer cells to stress signal-induced apoptosis
Our previous studies demonstrated that miR-17-5p suppressed proliferation in MCF-7 breast cancer cells [18]. To determine the mechanism by which miR-17-5p regulates breast cancer cell apoptosis, MCF-7 cells and MDA-MB-231 cells were transfected with miR-17-5p mimics or negative control (NC). The cells were then treated with 0.1 μM paclitaxel or Taxol for 48 hours and the TUNEL assay was used to analyze cell apoptosis. Transfection of miR-17-5p mimics increased numbers of apoptotic cells in both MDA-MB-231 and MCF-7 cells compared to control cells; this increase was greatest in the MCF-7 cells ( Figure 1A). Thus, miR-17-5p strongly increased the sensitivity of MCF-7 cells to Taxol-induced DNA damage.
The SRB assay was used to test relative cell survival. Overexpression of miR-17-5p attenuated cell survival in the presence of tamoxifen ( Figure 2D). Survival decreased in both control and miR-17-5p-transfected MCF-7 cells after treatment with 15 μM tamoxifen for 36 and 24 hours, respectively, compared to untreated cells ( Figure 2D). Furthermore, survival was lower in miR-17-5p-transfected cells at both the 24 and 36 hour timepoints than in control MCF-7 cells after tamoxifen treatment ( Figure 2D).

MiR-17-5p attenuated Taxol resistance in MCF-7 cells
MCF-7 and MDA-MB-231 cells treated with different concentrations (0-500 nM) of Taxol for 48 hours or 72 hours were used for quantitative analysis of cell survival. Survival decreased in miR-17-5p-transfected MCF-7 cells compared to control cells after treatment with 400 or 500 nM Taxol ( Figure 3A and 3B). Survival also decreased in miR-17-5p-transfected cells (~29% vs. ~39%) after treatment with Taxol for 72 hours ( Figure 3B). MiR-17-5p overexpression reduced the IC50 for Taxol after 48 hours of treatment in MCF-7 cells, and survival decreased in miR-17-5p-transfected MDA-MB-231 cells after 72 hours of treatment with 500 nM Taxol ( Figure  3E). In contrast, miR-17-5p did not affect the sensitivity of MDA-MB-231 cells to 48 hours of Taxol treatment ( Figure 3D). Cell growth curves revealed that sensitivity to 500 nM Taxol increased in miR-17-5p-transfected MCF-7 cells after 24 hours ( Figure 3C). Furthermore, while transfection of miR-17-5p also increased the sensitivity of MDA-MCB-231 cells to 24 hours of treatment with 500 nM Taxol, this effect was weaker than that observed in miR-17-5p-transfected MCF-7 cells in response to the same treatment ( Figure 3F). Taken together, these data indicate that miR-17-5p attenuated resistance to Taxol in breast cancer cell lines.

STAT3 and pSTAT3 expression are elevated, while miR-17-5p expression is decreased, in breast cancer tissue
Since miR-17-5p plays an important role in breast cancer cell apoptosis, we measured miR-17-5p expression in human breast tissues. Compared to normal breast tissue, miR-17-5p expression was lower in breast cancer tissue ( Figure 6A). Western blots confirmed that STAT3 and pSTAT3 expression were higher in breast cancer tissue than in normal breast tissue ( Figure 6B). These clinical data also indicate that the miR-17-5p-STAT3 axis contributes to the development of breast cancer.
In conclusion, our study demonstrated that miR-17-5p directly targets STAT3 and induces apoptosis in breast cancer cells by inhibiting the STAT3/p53 pathway. These findings highlight the potential role of miR-17-5p as a prognostic marker and a therapeutic target for breast cancer.

Cell culture and reagents
The MCF-7 and MDA-MB-231 breast cancer cells were cultured in DMEM containing penicillin and streptomycin (100 mg/L) and supplemented with 10% fetal bovine serum (FBS). Tamoxifen (MP Bio) and paclitaxel (Taxol) (Sigma) were used at the doses and for the times indicated in the individual figure legends.

Quantitative real time RT-PCR for miRNA
Quantitative RT-PCR for mature miR-17 was performed as described previously [51]. The miRNAspecific forward primers from the miScript primer assay (Qiagen, Hilden, Germany) were used. Data are shown as relative expression levels after normalization to U6 (Qiagen).

TUNEL assay
MiR-17-5p-transfected cells and control cells were cultured in medium containing paclitaxel (0.1μM). After 24-48 hours, MCF-7 cells or MDA-MB-231 cells were plated into a 96-well plate in triplicate. Apoptosis assays were performed using the In Situ Cell Death Detection Kit, TMR red (Roche Diagnostics, Mannheim, Germany) following the manufacturer's instructions.

Annex V staining
After treatment with Taxol or transfection with miR-17-5p mimics, STAT3-expressing MCF-7 cells were incubated with fluorochrome-conjugated Annexin V and then stained with propidium iodide. Flow cytometry was used to count apoptotic cells.

Sulphorhodamine B assay
The cytotoxicity of paclitaxel was determined using the sulphorhodamine B (SRB) assay [19]. Cells were plated at 3 × 10 3 cells/well in sextuplicate in 96-well plates. The cells were then cultured overnight to allow them to adhere, after which culture medium containing different concentrations of paclitaxel was added. Cells were harvested 0 (control) or 72 hours after treatment, fixed with 10% Trichloroacetic acid for 1 hour at 4 °C, and washed five times with water. The cells were then stained with 0.4% SRB in 1% acetic acid for 30 min, washed five times with 1 % acetic acid, and allowed to dry. 10 mM Tris-base was added to dissolve SRB and absorbance was measured with a plate reader at 530 nm. IC50 was defined as the concentration that killed 50% of cells compared to the untreated control.

Statistical analysis
Data are presented as means ±SEM. The standard two-tailed student's t-test was used for analysis and p<0.05 was considered significant.

ACKNOWLEDGMENTS AND FUNDING
This work was financially supported by the National Natural Science Foundation of China (No. 31501149, 31570764, 31401117, 31471282, 31440038, and 31270837), Hubei Province health and family planning scientific research project (WJ2017M173) and the Science and Technology Young Training Program of the Wuhan University of Science and Technology (2016) (2016xz035).