Sixteen pairs of tissue samples of ductal breast cancer were obtained from the West China Hospital of Sichuan University. All the patients underwent surgical resection to obtain the breast cancerous and corresponding adjacent non-tumorous tissues, without receiving chemotherapy or radiotherapy beforehand. All tissues were preserved in liquid nitrogen. The present study was approved by the local ethical standards of the Institutional Review Board of Sichuan University. Informed consent was obtained from all individual participants included in the present study.

All procedures performed in the present study involving human participants were in accordance with the ethical standards of the Institutional and/or National Research Committee of Sichuan University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

The MCF-7 cell line was purchased from the Shanghai Institutes for Biological Sciences (Shanghai, China) and cultured according to a protocol from the ATCC.

The doxorubicin-resistant MCF-7/ADR cell line (MCF-7/ADR) was induced by continuously culturing MCF-7 cells in medium containing progressive concentrations of doxorubicin (Sigma-Aldrich, St. Louis, MO, USA). MCF-7/ADR cells were cultured in medium with 2 µg/ml doxorubicin, and subsequently transferred into a drug-free medium for at least 2–3 weeks before use in the assays.

Using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, MCF-7 cells were transfected with 50 nM hsa-miR-93-5p mimics or hsa-miR-93-5p negative control mimics (RiboBio Co., Ltd., Guangzhou, China) in order to produce an MCF-7-miR-93 mimics group (MCF-7-miR-93 mimics) and a negative control group (MCF-7-miR-93 mimics NC), respectively. In addition, MCF-7/ADR cells were transfected with 100 nM hsa-miR-93-5p inhibitor (RiboBio Co., Ltd.) to produce an MCF-7/ADR-miR-93 inhibitor group, or an hsa-miR-93-5p inhibitor negative control (RiboBio Co., Ltd.) to produce an MCF-7/ADR-miR-93 inhibitor NC group, following the same procedure as aforementioned.

Total miRNAs were extracted from cells and tissue samples using an miRcute miRNA Isolation kit (Tiangen Biotech Co., Ltd., Beijing, China). The expression of miR-93 was analyzed using the Bulge-Loop™ miRNA qRT-PCR Starter kit (RiboBio Co., Ltd.). The primers for miR-93 and endogenous control U6 were purchased from RiboBio Co., Ltd. (ssD809230675, ssD809231367, ssD809261711, ssDD0904071006, ssDD0904071007 and ssDD0904071008).

TRIzol reagent (Takara, Dalian, China) was used for total RNA extraction. The expression of different genes was analyzed using a SYBR-Green qRT-PCR kit (Takara). The primer sequences for qRT-PCR were as follows: E-cadherin, 5′-tgcccagaaaatgaaaaagg-3′, 5′-gtgtatgtggcaatgcgttc-3′ (product size, 200 bp); vimentin, 5′-gagaactttgccgttgaagc-3, 5′-tccagcagcttcctgtaggt-3′ (product size, 170 bp); N-cadherin, 5′-gac aatgcccctcaagtgtt-3′, 5′-ccattaagccgagtgatggt-3′ (product size, 179 bp); fibronectin, 5′-accaacctacggatgactcg-3′, 5′-gctcatcatc tggccatttt-3′ (product size, 230 bp); Snail, 5′-ggttcttctgcgctactgct-3′, 5′-tagggctgctggaaggtaaa-3′ (product size, 157 bp); Twist, 5′-ggagtccgcagtcttacgag-3′, 5′-tggaggacctggtagaggaa-3′ (product size, 199 bp); MRP, 5′-aggtggacctgtttcgtgac-3′, 5′-cctg tgatccaccagaaggt-3′ (product size, 181 bp); BCRP, 5′-caccttattg gcctcaggaa-3′, 5′-cctgcttggaaggctctatg-3′ (product size, 206 bp); MDR, 5′-gctcctgactatgccaaagc-3′, 5′-tcttcacctccaggctcagt-3′ (product size, 202 bp); PTEN, 5′-ttacagttgggccctgtacc-3′, 5′-atttgatgctgccggtaaac-3′ (product size, 153 bp); GAPDH, 5′-ct ttggtatcgtggaaggactc-3′, 5′-gtagaggcagggatgatgttct-3′ (product size, 132 bp).

Cell apoptosis was detected using an Annexin V-FITC apoptosis detection kit (KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's instructions. The data were analyzed using FlowJo 9.1 software.

Cellular proliferation and growth inhibition were assessed using Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's protocol. For the detection of the proliferation of cells in the MCF-7-miR-93 mimic group, the cells were assessed at 0, 24, 48 and 72 h. In addition, to determine the effect of miR-93 on drug resistance, the cell survival ratio was assessed after cells in the MCF-7-miR-93 mimic group were cultured with 0.08, 0.4, 1 and 5 µg/ml doxorubicin (Sigma-Aldrich) for 24 h.

A bioinformatics analysis using the ‘miRanda’ database was performed to analyze the possibility of miR-93 binding to PTEN. Subsequently, MCF-7 cells were cotransfected with 2.5 µg pGL3 luciferase reporter plasmid (GeneCopoeia) containing either a wild-type (5′-GGAUUAAUAAAGAUGGCACUUUC-3′) or mutated (5′-GGAUUAAUAAAGAUGGCTCUAUC-3′) form of the PTEN 3′-UTR, and 50 nM hsa-miR-93-5p or miR-93 mimics NC in 6-well plates using Lipofectamine 3000. Luciferase activity was assessed consecutively at 24 h post-transfection using a Dual-Luciferase Assay (GeneCopoeia), and normalized to the blank vector control group.

All the experiments were repeated three times independently. Data were calculated as the mean ± SD. The paired t-test was applied for statistical analysis using SPSS software (version 20.0) (SPSS, Inc., Chicago, IL, USA).

qRT-PCR was performed to identify the patterns of miR-93 expression in breast cancer tissues and a doxorubicin-resistant breast cancer cell line (MCF-7/ADR) compared with paired normal breast tissue samples and a doxorubicin-sensitive parental breast cancer cell line (MCF-7). The results revealed that 62.5% of cancer tissue samples (10 out of 16 cases) exhibited a markedly higher expression level of miR-93 compared with their corresponding paired adjacent normal tissue (Fig. 1A; p<0.05). Furthermore, the miR-93 expression level in the MCF-7/ADR cells was significantly higher than that in MCF-7 cells (Fig. 1B; p<0.01).

A CCK-8 assay was performed to evaluate the proliferation and sensitivity to doxorubicin of cells in the MCF-7-miR-93 mimics group. The results revealed that the overexpression of miR-93 markedly upregulated the proliferation rate of MCF-7 cells (Fig. 2A; p<0.01), and also significantly increased the survival ratio of cells treated with doxorubicin for 24 h (Fig. 2B; p<0.05).

qRT-PCR was used to examine the expression levels of the multi-drug resistant-related genes MDR, MRP and BCRP in MCF-7 and MCF-7-miR-93 mimic cells. The results demonstrated that the expression levels of each of these genes were significantly higher in the MCF-7-miR-93 mimic cells than in the MCF-7 cells (Fig. 2C; p<0.01). Furthermore, flow cytometric analysis revealed that the rate of doxorubicin-induced apoptosis was lower in the MCF-7-miR-93 mimic cells than in untransfected MCF-7 cells (Fig. 3A, B and E; p<0.01). By contrast, in the MCF-7/ADR-miR-93 inhibitor cells, the rate of doxorubicin-induced apoptosis was significantly increased compared with that in the untransfected MCF-7/ADR-miR-93 cells (Fig. 3C, D, and F; p<0.01).

To explore the possible relationship between miR-93 expression and cancer cell EMT, the changes in morphological features and the mRNA levels of EMT-related genes were examined after the transfection of MCF-7 cells with miR-93 mimics or NC. The morphological features of the cells are shown in Fig. 4A. MCF-7-miR-93 mimic-transfected cells displayed a cobbleston-like appearance and tight cell-cell junctions. By contrast, the MCF-7-miR-93 mimic NC and untransfected MCF-7 cells appeared to have spindle-cell morphology. Meanwhile, the qRT-PCR results revealed that the expression level of the epithelial marker E-cadherin was markedly decreased, while the levels of the mesenchymal markers N-cadherin, vimentin, Twist, Snail and fibronectin were markedly increased after transfection with the miR-93 mimics (Fig. 4B; p<0.01).

To detect the possible association between miR-93 and PTEN, a ‘miRanda’ bioinformatics analysis was initially performed. The miRNA target prediction program indicated PTEN as one of the possible target genes of miR-93. In particular, the 3′-untranslated region (UTR) of PTEN mRNA contains a binding site for miR-93 (Fig. 5A). To confirm this, a dual-luciferase reporter assay was carried out. The results revealed that, compared with the control group (MCF-7-miR-93 mimic NC), transfection with the miR-93 mimics decreased the luciferase activity of the reporter construct containing the wild-type PTEN 3′-UTR (Fig. 5B; p<0.01), whereas the miR-93 mimics induced no significant change in the activity of the reporter construct containing the mutated PTEN 3′-UTR. This indicated that miR-93 can bind directly to the PTEN 3′-UTR. Furthermore, the qRT-PCR results confirmed that the miR-93 mimics could downregulate the expression level of PTEN in MCF-7 cells compared with the MCF-7-miR-93 mimics NC group (Fig. 5C; p<0.01).