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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 4  |  Issue : 2  |  Page : 48-53

Cancer stem-like cells have cisplatin resistance and miR-93 regulate p21 expression in breast cancer


1 Department of Pharmacology, School of Medicine, Showa University, Shinagawa-Ku, Tokyo, Japan
2 Kameda Medical Center, Breast Center 929 Higashi-Cho, Kamogawa City, Chiba, Japan
3 Department of Orthopaedic Surgery, Showa University Fujigaoka Hospital, Aoba-Ku, Kanagawa, Japan

Date of Submission12-Dec-2017
Date of Acceptance03-Apr-2018
Date of Web Publication27-Apr-2018

Correspondence Address:
Dr. Akiko Sasaki
Department of Pharmacology, School of Medicine, Showa University, Hatanodai 1-5-8, Shinagawa-Ku, Tokyo 142-8666
Japan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ctm.ctm_41_17

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  Abstract 


Aim: This study aims to examine the role of microRNAs (miRNAs) in regulating the expression of p21, a cyclin-dependent kinase inhibitor, and in inducing resistance to cisplatin, an anticancer drug.
Methods: Human breast cancer cell line MDA-MB231 cells were separated into two subpopulations, cancer stem-like cells (CSCs) and cancer cells, based on the expression of cell surface antigens CD44 and CD24.
Results: p21 protein expression was higher in CSCs than in cancer cells. Exposure of MDA-MB-231 cells to cisplatin increased p21 protein expression. However, p21 expression was significantly lower in cisplatin-treated CSCs than in cisplatin-treated cancer cells, suggesting that p21-dependent cell cycle suppression was lower in CSCs than in cancer cells. Moreover, caspase-3 activity was significantly lower in cisplatin-treated CSCs than in cisplatin-treated cancer cells, indicating that CSCs were more resistant to cisplatin-induced apoptosis than cancer cells. Treatment with miR-93 inhibitors increased p21 expression in CSCs, suggesting that miR-93 suppressed p21 expression.
Conclusion: The results of the present study indicate that CSCs contribute to cisplatin resistance of MDA-MB231 cells and suggest that miR-93 inhibits the expression of p21, a factor involved in drug resistance.

Keywords: Cancer stem-like cells, microRNA, p21, triple-negative breast cancer


How to cite this article:
Sasaki A, Tsunoda Y, Furuya K, Oyamada H, Tsuji M, Udaka Y, Hosonuma M, Shirako H, Ichimura N, Kiuchi Y. Cancer stem-like cells have cisplatin resistance and miR-93 regulate p21 expression in breast cancer. Cancer Transl Med 2018;4:48-53

How to cite this URL:
Sasaki A, Tsunoda Y, Furuya K, Oyamada H, Tsuji M, Udaka Y, Hosonuma M, Shirako H, Ichimura N, Kiuchi Y. Cancer stem-like cells have cisplatin resistance and miR-93 regulate p21 expression in breast cancer. Cancer Transl Med [serial online] 2018 [cited 2018 May 22];4:48-53. Available from: http://www.cancertm.com/text.asp?2018/4/2/48/231377




  Introduction Top


Breast cancer is the most prevalent cancer in women. Triple-negative breast cancer (TNBC) lacks the expression of hormone receptors, namely, estrogen receptor, progesterone receptor, and epidermal growth factor receptor. TNBC accounts for 10%–15% of all breast cancer cases and is associated with poor prognosis, recurrence, and distant metastasis in 30% patients. Cancer stem-like cells (CSCs) contribute to the recurrence and distant metastasis of TNBC.[1]

CSCs are self-replicating pluripotent cells that serve as a source of cancer cells. CSCs are regulated by the cancer microenvironment (niche). The niche is resistant to chemotherapy and radiotherapy targeting progression of the cell cycle because it maintains CSCs in the resting phase through cytokines, calcium ions, and oxygen molecules.[2],[3] In TNBC tissues, CSCs are recognized by the expression of cell surface antigen CD44. CSCs in TNBC tissues show high drug efflux activity, are a part of side population cells, and promote tumor recurrence.[4] Many studies have assessed molecular targets in CSCs for treating different cancers; however, these targets are still under research and are yet to reach clinical application. Therefore, there is an urgent need for developing treatment strategies for eliminating CSCs in TNBC.

The enhanced p21 gene expression was reported as a cause of resistance to CSC treatment in 2015.[5] In CSCs, p21 expression is regulated by MAPK signaling proteins JNK and p38 under the control of the cancer microenvironment.[6],[7],[8] Niche-regulated CSCs show high p21 expression to prevent signal transmission, which induces resistance to chemotherapy that targets cells in the growth phase. To eliminate chemotherapy-resistant CSCs, we focused on p21 and identified microRNAs (miRNAs) that regulated p21 expression. miRNAs bind to specific complementary nucleotides in mRNAs, i.e., 3'-untranslated region (UTR) and inhibit protein expression.[9],[10],[11],[12],[13] In addition, miRNAs can be used as drug delivery systems because they reach the cell nucleus more easily than antibodies and low molecular weight compounds, thus attracting the attention of researchers for developing controllable low molecular weight compounds.[14],[15],[16],[17]

In the present study, we focused on miRNAs strongly expressed in CD44+CD24- cells exposed to an anticancer drug and investigated the possibility of using miRNAs for regulating p21 expression in the cell nucleus.


  Methods Top


Cell lines and culture conditions

Human nonbasal-like type of TNBC cell line MDA-MB-231 (MSL, nonbasal-like type) was obtained from Japanese Cancer Resources Bank (Osaka, Japan). MDA-MB-231 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma Aldrich, Oberhaching, Germany) supplemented with 10% fetal bovine serum (Gibco Life Technologies, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco penicillin-streptomycin liquid; Invitrogen, CA, USA) at 37°C in 5% carbon dioxide and 95% air.

Isolation of CD44+CD24- and CD44- cells

We mimicked the physiological cancer microenvironment by establishing a cell culture medium, in which cancer cells coexisted with CSCs. MDA-MB-231 cells were treated with an anticancer drug and miRNA inhibitors, and were separated into CSCs and cancer cells. CSCs were isolated based on CD44 positivity and CD24 negativity. CD44+CD44- cells were separated by performing magnetic cell sorting (MACS ®; Miltenyi Biotec K. K. Heiden-Westfalen, Germany). The cells were labeled with CD44- biotin, an Fc receptor-blocking reagent, and anti-biotin microbeads in a CSC buffer were given Biotin marker by the magnetism in microbeads (130-095-194, MACS®). Next, CD44- cells lacking the biotin label were separated from CD44+ cells with the biotin label. CD24 microbeads were used to separate CD24- cells from CD44+ cells. Finally, cells lacking CD24 were separated, and the remaining CD44+CD24- cells were used as CSCs.

Immunohistochemical staining

All cell samples were fixed in 10% formalin (WAKO, Osaka, Japan) for 1 h, treated with 0.3% H2O2 for 10 min to remove endogenous peroxidase and blocked with a nonspecific blocking regent (DAKO, Tokyo, Japan) for 5 min. The samples were then treated with primary monoclonal mouse antihuman CD44 antibody, a phagocytic glycoprotein-1 (M7082) (DAKO, CA, USA) for 1 h, followed by treatment with Alexa Fluor 555-conjugated goat anti-mouse IgG (A21422; Molecular Probes, MA, USA). Nuclear staining was performed using bisbenzimide H33342 (Dojindo, Tokyo, Japan). After 30 min, the samples were observed under an inverted microscope (ECLIPSE, Ti-U; Nikon, Tokyo, Japan).

Exposure to anticancer drug

MDA-MB-231 (1 × 105 cells/mL) cells were seeded in 10 cm diameter plates and treated with 1 nM cisplatin (Cisplatin®, Yakult Co., Ltd, Chuo-ku, Tokyo, Japan) for 24 h. The cell count included in 10 cm dish was 0.98 × 105 when exposed to cisplatin for 24 h.

Enzyme-linked immunosorbent assay for determining p21 expression

Isolated CSCs (CD44+CD24-) or cancer cells (CD44-) were added to 1.5 mL tubes and were washed with phosphate buffered saline (PBS). Next, the cells were incubated with cell extraction buffer (FNN0011; Thermo Fisher Scientific K. K., Yokohama, Japan) containing 1 mM phenylmethylsulfonyl fluoride and 0.5 μL/mL protease inhibitor cocktail (P8340; Sigma, Tokyo, Japan) for 15 min and were centrifuged. The supernatant obtained was used to measure p21 expression with an enzyme-linked immunosorbent assay (ELISA) kit (ADI-900-161; Enzo Life Sciences, NY, USA) and a leader fluorospectrophotometer plate (λ = 450 nm).

Apoptosis assay

Ac(N-acetyl)-DEVD-7-amino-4-trifluoromethylcoumarin-(AFC) (Kamiya Biomedical, USA) was used as a substrate for caspase-3. MDA-MB-231 cells (density, 1 × 105 cells/mL) were plated in a 10 cm dish. The dish was divided into 10 sections, and the cells were cultured for 24 h. Next, the cells were exposed to 1 nM cisplatin for 24 h and were separated into CSCs (CD44+CD24-) and cancer cells (CD44-). The isolated CSCs and cancer cells were washed in warm PBS and were treated with cell lysis buffer 4 (80-1339; Enzo Life Sciences) on ice for 10 min. Next, a reaction buffer (0.1 M HEPES buffer [pH = 7.5], 20% glycerol, 0.5 mM EDTA, and 10 mM DTT) was added to the sample, and fluorescence was measured using the leader fluorospectrophotometer plate.

microRNA measurement

MDA-MB-231 cells (density, 1 × 105 cells/mL) were cultured in a 10 cm dish. The dish was divided into 10 sections, and the cells were cultured for 24 h. Next, the cells were treated with 1 nM cisplatin for 24 h. miRNAs in CSCs (CD44+CD24-) and cancer cells (CD44-) (density, 1 × 105 cells/mL) were extracted using miRNeasy Mini kit (Qiagen, Tokyo, Japan). Next, cDNAs were synthesized from 500 ng miRNAs using miScript II RT kit, and five different miRNAs were determined using a PCR array (miScript miRNA PCR Array for human breast cancer, MIHS-109ZA; Qiagen). PCR was performed using miRNA MiScript SYBR Green human breast cancer miRNA PCR kit (Qiagen) and ABI PRISM 7000 sequence System (Applied Biosystems Inc., CA, USA) with the following conditions: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Data were analyzed using ΔΔCt method (original expression level = 2=–ΔΔCt). Data are expressed as a ratio (ΔΔCt) of Ct level in cisplatin-treated CD44+CD24- cells to that in untreated MDA-MB-231 cells. Differential miRNA expression was defined as > 10-fold difference in miRNA expression between cisplatin-treated CD44+CD24- cells and untreated MDA-MB-231 cells.

Transfection

miRNA inhibitors of miR-17, miR-20a, miR-20b, and miR-93 complementary to the 5'-UTR of the p21 gene were generated using miRCURY LNA™ microRNA inhibitors (Exiqon Inc., Vedbaek, Denmark). Sequence of the nucleic acids is as follows (http://www.exiqon.com/mirna-inhibitors):has-miR-17-5p: 5'-CTACCTGCACTGTAAGCAC-3';has-miR-20a-5p: 5'-CTACCTGCACTAAGCAC-3';has-miR-20b-3p: 5'-TGGAAGTGC CCATACTACAG-3'; and has-miR-93-5p: 5'-CAAAGTG CTGTTCGTGCAGGTAG-3'.

MDA-MB-231 cells were transfected with each miRNA inhibitor by performing lipofection. For transient transfection, the cells were seeded at low density (1 × 105 cells/mL) in six-well dishes and were transfected with 100 pmol of each miRNA inhibitor using Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific K. K., MA, USA). At 4 h after transfection, the cells were washed and were incubated in complete DMEM for 48 h.

Flow cytometry

FCM acquisition was performed with a Muse Cell Analyzer (MERCK, Tokyo, Japan). Muse System Check Kit (MCH100101) was used to standardize the flow cytometer setup as per the manufacturer's instructions. Transfer the 1 × 106 cell sample to 1.5 mL conical tube. Centrifuge the tube at 3000 g for 5 min. Remove and discard the supernatant without disturbing the cell pellet, and add of PBS to each tube. Centrifuge the tube at 3000 g for 5 min and remove and discard the supernatant. While vortexing at medium speed, 1 mL ice-cold 70% ethanol washing was performed. Resuspend the Ethanol-fixed cells pellet in 200 μL of muse cell reagent (MCH100106) and incubate for 30 min at room temperature, protected from light. Then, it was measured. PBS buffers were run as a negative control.

Statistical analysis

Significant differences between the groups were determined by performing two-way analysis of variance (ANOVA) with Bonferroni post hoc test. Significance of miRNA analysis was assessed by performing ANOVA with miScript miRNA PCR Array Data analysis software (Qiagen), with significance level set at P < 0.05.


  Results Top


Cancer stem-like cells express CD44 positivity

MDA-MB-231 cells were separated into CSCs and cancer cells. Nuclei stain DAPI of cells was stained with blue. CD44 positive was stained with red. Cancer cells were CD44 negativity. In contrast, CSCs were CD44 positivity [Figure 1].
Figure 1: CD44 expression in Cancer cells (CD44−) and cancer stem-like cells (CD44+CD24−) isolated from MDA-MB-231 cells. (a) MDA-MB-231 cells were treated separated into cancer cells. Nuclei stain DAPI of cells was stained with blue. CD44 positive was stained with red. Cancer cells were CD44 negativity. (b) The cell cycle phases in cancer cells (CD44−) cells of cultured MDA-MB-231 cell lines. (c) MDA-MB-231 cells were treated separated into cancer stem-like cells. CD44 positive was stained with red. Cancer stem-like cells were CD44 positivity. (d) The cell cycle phases in cancer stem-like cells (CD44+CD24−) of cultured MDA-MB-231 cell lines

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Cancer stem-like cells are present in the stationary G0 phase of the cell cycle

The G0/G1 phase of cancer cells (CD44-) was 39.5%, the S phase was present at 8.9%, G2/M phase 39.9%. The G0/G1 phase of CSCs (CD44+CD24-) was 56%, the S phase was present at 7.7%, and G2/M phase was 22.4%. CSCs were present in the G0/G1 phase at a higher level than in the cancer cells and were low in the S phase and the G2/M phase [Figure 1].

p21 protein level was higher in cisplatin-treated cancer stem-like cells

p21 protein levels in untreated cancer cells (CD44-) and CSCs (CD44+CD24-) were 0.02 and 0.043 pg/μg, respectively, and in cisplatin-treated CD44- and CD44+CD24- were 0.43 and 0.069 pg/μg, respectively (P< 0.05)[Figure 2]. These results indicate that p21 protein level was significantly higher in cisplatin-treated CD44- than in cisplatin-treated CD44+CD24-.
Figure 2: Expression levels of p21 in cancer cells (CD44−) and cancer stem-like cells (CD44+CD24-) isolated from MDA-MB-231 cells exposed to cisplatin. p21 levels in cells treated with cisplatin for 24 h compared with those in untreated control cells are shown. Data are expressed as mean ± standard deviation (n = 4) of four assays of the same sample. *P < 0.05 vs. untreated control cells

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Caspase-3 activity was significantly lower in cisplatin-treated cancer stem-like cells

MDA-MB-231 cells were exposed to cisplatin for 24 h. Caspase-3 activity in CSCs (CD44+CD24-) and cancer cells (CD44-) is shown in [Figure 3]. Caspase-3 activity in untreated CD44- and CD44+CD24- were 15.76 and 32.06 mmol·mg protein·h, respectively. However, caspase-3 activity in cisplatin-treated CD44- and CD44+CD24- were 22.57 and 18.41 mmol·mg protein·h, respectively. This result indicates that caspase-3 activity was significantly lower in cisplatin-treated CD44+CD24- than in cisplatin-treated CD44- (P< 0.05) [Figure 3].
Figure 3: Caspase-3 activity in cancer cells (CD44-) and cancer stem-like cells (CD44+CD24-) and cancer cells isolated from MDA-MB-231 cells exposed to cisplatin. Caspase-3 activity in cells treated with cisplatin for 24 h compared with that in untreated control cells is shown. Data are expressed as mean ± standard deviation (n = 4) of four assays of the same sample. *P < 0.05 vs. untreated control cells

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Cisplatin-treated cancer stem-like cells showed higher differential expression of miR-17 and miR-93

We analyzed miRNA expression in MDA-MB-231 cells exposed to cisplatin. We observed that five miRNAs, namely, miR-17, miR-20a, miR-20b, miR-93, and miR-182, were involved in p21 expression in MDA-MB-231 cells. Cisplatin-treated MDA-MB-231 cells showed significantly higher differential expression of these five miRNAs than control cells. In cisplatin-treated CSCs (CD44+CD24-), the ΔΔCT values for miR-17 and miR-93 expression were 5.19 and 2.31, respectively, and those for miR-20a, miR-20b, and miR-182 expression were −1.63, −1.48, and −5.22, respectively, indicating that the cisplatin treatment decreased the expression of miR-20a, miR-20b, and miR-182 compared with that of miR-17 and miR-93 in CD44+CD24-. In cisplatin-treated cancer cells (CD44-), the ΔΔCT values for miR-17, miR-20a, and miR-20b expression were 3.22, 1.41, and 3.6, respectively, and those for miR-93 and miR-182 expressions were −3.56 and −5.25, respectively, indicating that cisplatin treatment decreased the expression of miR-93 and miR-182 compared with that of miR-17, miR-20a, and miR-20b in CD44− [Figure 4].
Figure 4: Changes in microRNA expression in cancer cells (CD44-) and cancer stem-like cells (CD44+CD24-) isolated from MDA-MB-231 cells exposed to cisplatin. ΔΔCT values for the expression of miR-17, miR-20a, miR-20b, miR-93, and miR-182 after cisplatin exposure for 24 h are shown

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p21 protein level increased miR-93 inhibitors in cancer stem-like cells

p21 protein level increased to 0.003 and 0.0033 pg/μg cell protein in cancer cells (CD44-) and CSCs (CD44+CD24-) transfected with miR-17 inhibitors, 0.0026 and 0.0025 pg/μg cell protein in miR-20a inhibitor, 0.003 and 0.003 pg/μg cell protein in miR-20b inhibitor, respectively (P< 0.05). Moreover, p21 protein level in cancer cells (CD44-) transfected with miR-93 inhibitor was 0.003 and 0.0017 pg/μg cell protein, which was significantly higher than that in CD44- (P< 0.05). However, no significant difference in p21 protein level was observed in CD44+CD24- and CD44- transfected with miR-17, miR-20a and miR-20b inhibitors [Figure 5].
Figure 5: p21 expression levels in cancer cells (CD44−) and cancer stem-like cells (CD44+CD24−) isolated from MDA-MB-231 cells transfected with miR-17, miR-20a, miR-20b, or miR-93 inhibitor. Data are expressed as mean ± standard deviation (n = 4) of four assays of the same sample. *P < 0.05 vs. CD44- cells

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  Discussion Top


TNBC tissue contains several CSCs that induce anticancer drug resistance.[17] CSCs are self-replicating, competent, and multipotent cells that serve as a source of cancer cells. CSCs are present in the stationary growth phase and induce resistance to chemotherapy and radiation therapy. However, no effective therapy is available for eliminating CSCs.[18],[19] p21 binds to transcription factor NRF2. NRF2 activation accelerates the synthesis of reduced glutathione that functions downstream of p21 and removes active oxygen. Furthermore, the glutathione conjugation response is accelerated, and the excretory functions of the anticancer drug increase.[20] This induces chemoresistance in CSCs. p21 is a cyclin-dependent kinase (CDK) inhibitor involved in anticancer drug resistance and is activated during stress, starvation, and aging. Intracellular p21 levels decrease during the division of normal cells. p21 inhibits cell cycle progression in G1, S, and G2/M phases, thus inhibiting cell proliferation.[5],[6],[7] Cancer cells multiply endlessly in the absence of cancer-inhibiting p53 downstream signal. In TNBC, many things have mutation in p53. However, TNBC does not show mutations in the p21 gene.[21],[22] In CSCs, miRNAs regulate p21 expression. CSC has treatment resistance. However, the identification of microRNA in CSC of TNBC can lead CSC to the cell death. Because CSCs induce resistance to anticancer treatments, identification of miRNAs in CSCs present in TNBC tissue can help in eliminating these cells to inhibit cancer progression. Thus, CSC targeting is a novel approach for treating cancer. In the present study, we used MDA-MB-231 cells, which are TNBC nonbasal-like type of cells, and isolated CSCs (CD44+CD24-) from cisplatin-treated MDA-MB-231 cells. Because p21 expression is associated with anticancer drug resistance, we identified miRNAs that regulated p21 expression in CSCs. MDA-MB-231 cells not treated with cisplatin were used as control cells. Next, we examined p21 expression level in control and cisplatin-treated cells. p21 expression was significantly higher in CSCs (CD44+CD24-) than in cancer cells (CD44-) [Figure 2]. p21 inhibits CDK indicating that it inhibits cell cycle progression. CSCs are present in the stationary G0 phase of the cell cycle. p21 expression was higher in CSCs, suggesting the inhibition of cell cycle progression in these cells. Next, we examined p21 expression in cisplatin-treated cancer cells and CSCs. p21 expression was significantly higher in cisplatin-treated cells than in control cells. Next, we compared p21 expression in CSCs and cancer cells and found that p21 expression was significantly lower in cisplatin-treated CSCs than in cisplatin-treated cancer cells. Cisplatin covalently bind to purine bases (adenine and guanine) in the DNA of cancer cells. More than 90% tumor cells contain DNA adducts because of the bridging between the purine bases. Cisplatin induces DNA damage and cell death.[23] Cisplatin exposure significantly increased p21 expression in cancer cells compared with that in CSCs. This may be because cell cycle progression is slower in CSCs than in cancer cells, which makes CSCs more resistant to DNA damage-induced cell death. Therefore, CSCs may show low p21 expression to delay cell cycle progression. Cisplatin-treated CSCs showed 72% inhibition of caspase-3 activity compared with cisplatin-treated cancer cells. Thus, CSCs showed lower cisplatin-induced apoptosis than cancer cells. These results suggest that CSCs can survive after treatment with anticancer drugs [Figure 3].

Next, we treated MDA-MB-231 cells with cisplatin and analyzed miRNA expression in CSCs and cancer cells. Moreover, we assessed the expression of five miRNAs involved in p21 expression [Figure 4].[24] miR-17, miR-20a, miR-20b, miR-93, and miR-182 target p21 in a cancer cells. However, it is unclear whether these miRNAs target p21 in CSCs. Cisplatin exposure decreased miR-182 expression, suggesting that it did not regulate p21 expression by complementarily binding to the 5'-UTR of the p21 gene. Next, we examined miR-17, miR-20a, miR-20b, and miR-93 expression but not miR-182 expression and its correlation with p21 protein expression. Moreover, we examined p21 expression in CSCs transfected with the miRNA inhibitors. No significant difference in p21 expression was observed in CSCs transfected with miR-17, miR-20a, and miR-20b inhibitors. However, p21 expression was significantly higher in CSCs transfected with miR-93 inhibitors than in cancer cells [Figure 5].

Increase in the miR-93 expression results in the upregulation of the cell cycle through decreasing p21 protein expression levels. Consequently, there is uptake of an anticancer drug in the nuclei, leading to the inhibition of tumor growth.[24] Results of the present study highlight the novel role of miR-93 in inhibiting p21 expression in CSCs. Moreover, the results of the present study indicate that CSCs induce cisplatin resistance in MDA-MB-231 cells. Furthermore, our results indicate that miR-93-induced inhibition of p21 expression induces cisplatin resistance in CSCs. Thus, CSCs present in TNBC tissues induce cisplatin resistance because they show decreased apoptosis and suggest that miR-93 are novel molecular targets for increasing the anticancer drug susceptibility of TNBC.

Together, our results indicate that CSCs induce anticancer drug resistance and that miR-93 regulates p21 expression in TNBC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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