Breast cancer is one of the three common cancers

Breast cancer is one of the three common cancers, and the most common malignancy among women worldwide. Breast cancer is still the most common fatal cancer and the second one in less and more developed countries, respectively.1 Breast cancer is a heterogeneous disease and cannot be explained only by clinical parameters or biomarkers such as epidermal growth factor receptor-2 (HER-2), estrogen receptor (ER) and progesterone receptor (PR). However, based on gene expression profiling, breast tumors are grouped into six subtypes including HER-2-enriched, luminal A, luminal B, basal-like, normal breast and claudin-low. Basal-like subtype with lack of hormone receptors and HER-2 expression is also called triple-negative breast cancer (TNBC).2,3 Surgery, radiation therapy, chemotherapy and targeted therapy as treatment methods for breast cancer are chosen based on the type and stage of cancer.4 For certain biological tumor subtypes such as TNBC, Anthracycline and Taxane-containing chemotherapy could be recommended.1 Paclitaxel, a member of Taxane antitumor agents, is a chemotherapeutic agent that is used in treatment of several cancers including breast cancer. Paclitaxel inhibits depolymerization of the microtubules and blocks cell mitosis cycle in G2/M phase and finally leads to apoptotic cell death.5,6
MiRNAs are one of the important classes of regulatory mechanisms that target about 5300 human genes7 and play critical roles in various biological processes and their abnormal expression are related to various human diseases and cancers.8,9 Expression profile of miRNAs in breast cancer was described for the first time in 2005 by Iorio et al.10 Two groups of miRNAs including tumor- suppressor and oncogenic miRNAs (oncomiRs) are involved in breast cancer.11 Moreover, it has been found that some miRNAs are involved in metastasis which called “metastamirs”. Metastasis causes approximately 90% of cancer-related mortality.12 The role for miRNAs in drug resistance is also under vigorous investigations.13
EMT which was initially introduced by Greenburg and Hay in 198214 is identified as an initial step in the metastatic cascade.15 EMT is a process in which cells lose their epithelial characteristics and acquire mesenchymal features through declining in E-cadherin expression and increasing in expression of mesenchymal markers such as Vimentin, Fibronectin, and N-cadherin and eventually leads to cell migration and invasiveness.16 Physiologically, the process of EMT has a critical role in embryonic development, tissue repair and wound healing. In addition, EMT contributes to chemo-resistance, metastasis and fibrosis pathologically.17 Various signaling pathways such as Transforming Growth Factor-Beta (TGF-?), Sonic Hedgehog (SHH) and Wingless-type MMTV integration site family member (WNT) pathways, and effector molecules e.g. Vimentin, MMP-9, MMP-2 are involved in EMT.18 Several miRNAs have been identified to target the EMT regulatory factors in order to inhibit or induce EMT process. The first evidence of linking between miRNAs and metastasis was provided by Ma et al. who demonstrated the initiation of tumor invasion and metastasis by miR-10b in breast cancer.19 MiR-10b which is located between HOXD4 and HOXD8 on 2q3120 is the most well-known metastamir and EMT-promoting miRNA in breast cancer. MiR-199a-5p is down-regulated in breast cancer and has a tumor-suppressor role.21
In this study, we investigated the effect of anticancer agent paclitaxel on the expression level of miR-199a-5p, miR-10b, Vimentin and MMP-9 in breast cancer cell lines.

Materials and Methods
Cell Culture
Human breast cancer cell lines including BT-474, SKBR-3, MDA-MB-231 and MCF-7 were purchased form National Cell Bank of Iran (Pasteur Institute, Tehran, Iran) and cultured in RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, USA), 100 units/ml penicillin and 100 µg/ml streptomycin. Cells were grown at 37°C in a humidified atmosphere with 5% CO2.

MTT assay
In order to determine the IC50 (half maximal inhibitory concentration) of paclitaxel for studied cell lines, MTT assay was performed. First, 15×103 cells of each cell line were seeded with 200 ?l culture medium in 96-well plates. In 70-80% confluency, medium was removed and various concentration of paclitaxel (0.1 nM, 1nM, 10 nM, 100 nM, 1 µM, 10 µM and 100 µM) with 200 ?l of fresh media were added and cells were incubated for 24 hours at 37 oC with 5% CO2. After 24 hours, the medium was removed and after washing by phosphate buffered saline (PBS), 50 ?l of 2 mg/ml MTT solution (Sigma, USA) and 150 ?l medium was added to each well and incubated for 4 hours at 37 oC with 5% CO2. Supernatants were removed and dimethyl sulfoxide (DMSO) and Sorenson,s buffer were added. After 30 minutes, the optical density (OD) of each concentration was read at 570 nm, and IC50 of paclitaxel for each cell line was calculated. In the second step to obtain more accurate results for IC50 of paclitaxel in cell lines, we limited the range of paclitaxel concentrations (Table 1). All assays were run as triplicate.

Cell treatment
5×105 of each cell line were seeded in 6-well plate and incubated overnight at 37oC with 5% CO2. Then, paclitaxel was added to wells in IC50 concentration previously determined by MTT assay and incubated for 24 hours.

RNA extraction and cDNA synthesis
Total RNA of both untreated and treated cells were extracted using RNX-PLUS reagent (CinnaGen, Iran) according to the manufacturer’s instructions. RNA concentration was determined using Nano Drop Spectrophotometer (Thermo Scientific Nanodrop 2000c, USA) and the quality of extracted RNAs was evaluated by agarose gel electrophoresis.
Because of using primers based on Locked Nucleic Acid (LNA) technology (Exiqon, Denmark), cDNA for microRNA detection was synthesized by Universal cDNA Synthesis Kit II (Exiqon, Denmark) according to the manufacturer’s protocol. cDNA for mRNAs was synthesized from extracted RNA as described before.22

Quantitative Real-time PCR
Expression level of miRNAs before and after treatment with paclitaxel in each cell line were evaluated by quantitative real-time PCR using SYBR Green master mix (Yekta Tajhiz Azma, Iran) on a Corbett Rotor-gene 6000 system (Corbett Life Science, Australia). MiRNA primers with LNA technology were purchased from Exiqon and real-time PCR was performed according to the manufacturer’s protocol. U6 snRNA was used as an internal control to normalize miRNA expression level. Expression of Vimentin and MMP-9 were also evaluated by real-time PCR, and ?-actin was used as an endogenous control. The sequences of the primers have been shown in Table1.

Statistical analysis
Analyses of data were performed using Prism Software version 6.01(Irvine, CA). Multiple t-test was used to compare data of two groups (treated and untreated) and p-values less than 0.05 were considered as statistically significant.
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Results

MTT assay results
MTT assay was performed dose dependently for each cell line to determine IC50 concentration of paclitaxel in 24 hours. IC50 concentration of paclitaxel for breast cancer cell lines including BT-474, SKBR3, MDA-MB-231 and MCF-7 were calculated as 19 nM , 4 µM, 0.3 µM and 3.5 µM, respectively (Figure 1).

Paclitaxel alters the expression level of miR-199a-5p and miR-10b in breast cancer cell lines
Our results showed that after treatment with IC50 concentration of paclitaxel, expression level of miR-199a-5p was decreased in MCF-7 and SKBR-3 cell lines, while it was increased in two other cell lines including MDA-MB-231 and BT-474 (Fig 2 A). Also, our results demonstrated decreased expression level of miR-10b in MCF-7, MDA-MB-231 and SKBR-3 and increased expression level in BT-474 cell lines (Fig 2 B). P-values and fold changes of studied miRNAs are shown in Table 2.

Down-regulation of EMT effector molecules in triple-negative metastatic breast cancer cell line, MDA-MB-231 in response to treatment with paclitaxel
Expression level of Vimentin and MMP-9 mRNAs, two EMT effector molecules, in MDA-MB-231 breast cancer cell line was studied before and after treatment with paclitaxel. Our findings indicated that both Vimentin (41.6 fold) and MMP-9 (83.3 fold) were decreased in response to treatment with paclitaxel (Figure 3).
Discussion

MiR-199a-5p (also called miR-199a) plays different oncogenic or tumor suppressor roles in various types of cancer. It has been shown that miR-199a is down-regulated in ovarian cancer,23 breast cancer21 and in advanced small cell carcinoma of the cervix (SCCC).24 Other studies showed that miR-199a suppressed cell growth in renal cancer25 and functions as tumor-suppressor in HCC.26 In contrast, oncogenic role of miR-199a has been shown in gastric cancer.27 In breast cancer, inhibitory role of miR-199a in proliferation via inducing G0/G1 phase arrest and apoptosis in MDA-MB-231 cells, has been shown by Chen et al.28 Our results indicated that after treatment with paclitaxel, the expression level of miR-199a-5p was increased in triple-negative breast cancer cell line, MDA-MB-231. Since paclitaxel causes cell cycle arrest and also miR-199a-5p induces cell cycle arrest, our findings are justifiable and paclitaxel exerts synergic effects with miR-199a-5p in MDA-MB-231 cells. Considering the findings of two previous studies, it could be suggested that miR-199a-5p may sensitize the TNBC to paclitaxel chemotherapy. Because of the unresponsiveness of targeted therapy such as Tamoxifen (an antagonist to the ER) or Tratuzumab (monoclonal antibody against HER2 receptor) in TNBC, due to the lack of the ER, PR and HER2, systemic chemotherapy with paclitaxel and its sensitization by miR-199a-5p mimics could be considered as an approach for TNBC treatment.
Chen et al. also found that miR-199a-5p significantly inhibited cell migration and invasion ability in MDA-MB-231cells through EMT process.28 Another study has demonstrated that miR-199a-5p suppresses invasion in breast cancer by regulating ?1 integrin through Ets-1.29 Also it has been shown that miR-199a-5p inhibits the invasion in human hepatocellular carcinoma by targeting DDR1.30 In our study, up-regulation of miR-199a-5p in breast cancer metastatic cells, MDA-MB-231 was shown after treatment with paclitaxel. These findings are consistent with the usage of paclitaxel in combination with other chemotherapeutic agents as first line chemotherapy in metastatic breast cancer.31,32
Since miR-10b was identified as the starter of tumor invasion and metastasis in 2007,19 other studies reported the involvement of miR-10b in metastasis of cancers including breast cancer. Liu et al. found that miR-10b targets E-cadherin and modulates breast cancer metastasis.33 It was shown that miR-10b targets syndecan-1 and promotes breast cancer cell motility and invasiveness by mechanisms which depend on Rho-GTPase and E-cadherin.34 Also, miR-10b plays a critical role in TGF-?1-induced EMT in breast cancer35 and Up-regulation of miR-10b is related to brain metastasis of breast cancer.36 According to the all mentioned reports miR-10b as the most well-known metastamir plays an important role in EMT process and metastasis in breast cancer. In line with previous studies, our results showed that after treatment with paclitaxel, the expression level of miR-10b was decreased in breast cancer metastatic cell line, MDA-MB-231. Hence, it could be suggested that the down-regulation of miR-10b could be considered as a probable mechanism of paclitaxel effects.
We also investigated the effect of paclitaxel on two EMT effectors, Vimentin and MMP-9 in MDA-MB-231 cell line and showed their significant down-regulation after treatment with paclitaxel. Yang et al. demonstrated that paclitaxel-resistant breast cancer cells displayed EMT phenotype with up-regulation of Vimentin. Also it has been shown that Vimentin was up-regulated in paclitaxel-resistant ovarian cancer cells.37,38 In line with these studies, our results showed that paclitaxel reduces the expression level of Vimentin in MDA-MB-231 breast cancer cell line. Also, our results showed decreased expression level of MMP-9 after treatment with paclitaxel in MDA-MB-231 cell line and this finding is in agreement with the findings of Ruan et al. who showed that paclitaxel down-regulates MMP-9 expression in glioblastoma cells.39 According to these results, it could be suggested that paclitaxel inhibits EMT process in part via reduction of vimentin and MMP-9 expression levels.
Conclusion: Defining the exact molecular mechanism of action of chemotherapeutic agent including paclitaxel improves cancer therapy regimens. According to the increased expression level of EMT-inhibitor, miR-199a-5p and decreased expression level of miR-10b, metastamir and also EMT effector genes including Vimentin and MMP-9 after treatment of triple-negative metastatic MDA-MB-231 breast cancer cell line, it would be noted that altering the expression of EMT-prompting and –inhibiting miRNAs and genes could be part of paclitaxel chemotherapeutic agent.