This research was approved by the Institutional Research Ethics Committee of the Third Hospital Affiliated to Qiqihar Medical College, and written informed consent was obtained from all patients. In this study, a total of 37 BC tissues and paired adjacent non-cancer tissues (NC) were collected from patients without the history of radiotherapy or chemotherapy before operation and stored in liquid nitrogen until ready for use. Patients were classified into high (n=22) and low miR-182-5p expression groups (n=15) according to the mean value of miR-182-5p abundance. The 5-year survival rate assay was conducted in all participants. The clinicopathologic characteristics of BC patients are displayed in Table 1.

The normal breast epithelial cell line MCF-10A and BC cell lines MCF-7, MDA-MB-231, BT20, T47D, and SKBR3 were obtained from American Tissue Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in Dulbecco's Modified Eagle Medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco), 1% penicillin, and streptomycin (Invitrogen, Carlsbad, CA, USA) at 37℃ in 5% CO₂ during the study.

PTEN overexpression vectors (PTEN) were cloned into pcDNA by GenePharma (Shanghai, China). MiR-182-5p mimics (miR-182-5p), miR-182-5p inhibitor (anti-miR-182-5p), and negative control (miR-NC or anti-NC) were obtained from GenePharma. To investigate the effect of miR-182-5p and PTEN on cell proliferation and invasion, transfection was performed in MCF-7 and MDA-MB-231 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

TRIzol reagent (Invitrogen) was used to isolate total RNA from tissues or cells following the manufacturer's instructions. The quality of RNA from every group was evaluated with a NanoDrop Spectrophotometer (NanoDrop, Wilmington, DE, USA), followed by synthesis of complementary DNA (cDNA) using a commercial Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). In turn, synthesized cDNA was diluted for quantitative real-time polymerase chain reaction (qRT-PCR) with SYBR Green Real-time PCR Master Mix (Toyobo, Tokyo, Japan) following the amplification instructions. All primers were listed as follows: miR-182-5p (Forward, 5′-TGCGGTTTGGCAATGGTAGAAC-3′; Reverse, 5′-CCA GTGCAGGGTCCGAGGT-3′), U6 (Forward, 5′-CTCGCTTCGGCAGCACA-3′; Reverse, 5′-AACGCTTCACGAATTTGCGT-3′), PTEN (Forward, 5′-CCCTTTG AAGACCATAACCCAC-3′; Reverse, 5′-TTACACCAGTTCGTCCCT-3′), β-actin (Forward, 5′-CAGCCTTCCTTCTTGGGTAT-3′; Reverse, 5′-TGGCATAGAGGTC TTTACGG-3′). The expression of miR-182-5p or PTEN was analyzed by the 2^−ΔΔCt method using U6 or β-actin as housekeeping gene, respectively.

Cell counting kit-8 (CCK-8) assay was performed to assess cell proliferation in MCF-7 and MDA-MB-231 cells. Cells were seeded into 96-well plates at a density of 1×10⁴ cells per well overnight. Then transfection was conducted for 24, 48, 72, and 96 h. At the end point, CCK-8 solution (Sigma, St. Louis, MO, USA) was added into each well and incubated for another 2 h at 37℃. Optical density was measured at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).

Cell invasion was analyzed by trans-well assay. MCF-7 and MDA-MB-231 cells were cultured in the upper chambers (Costar, Corning, NY, USA) pre-coated with Matrigel (BD Biosciences, San Jose, CA, USA) using serum-free medium at 37℃ for 24 h and then carefully removed using a cotton swab. Invasive cells on the basal side of the membrane were fixed with 4% paraformaldehyde for 10 min, stained with crystal violet (Sigma), and then counted under a microscope (Olympus, Tokyo, Japan). Three visual fields were randomly selected.

miRTarBase and miRanda analysis revealed putative binding sites for miR-182-5p and 3′ untranslated regions (3′-UTR) sequences of PTEN. The wild-type plasmid (PTEN-WT) or mutant-type plasmid (PTEN-MUT) was generated using pGL3 luciferase reporter vector (Promega, Madison, WI, USA) based on wild or mutant sequences of 3′-UTR of PTEN, respectively. WT or MUT luciferase reporter plasmids, Renilla luciferase plasmid, and miR-182-5p or anti-miR-182-5p were transfected into MCF-7 and MDA-MB-231 cells for 48 h using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. The luciferase activity analysis was evaluated by using Luciferase Assay Kit (Promega) and normalized by Renilla luciferase activity.

RNA immunoprecipitation (RIP) assay was performed in MCF-7 and MDA-MB-231 cells with transfection of miR-182-5p mimics or miR-NC using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). Transfected cells were lysed in cell lysis buffer and added to magnetic beads bound with anti-argonaute 1 (Ago1) or IgG antibody for 2 h at 4℃. The RNA-protein-beads complexes were isolated for qRT-PCR.

Total proteins were isolated from tumor tissues or cells using cell lysis buffer containing 1% protease inhibitor (Thermo Fisher, Wilmington, DE, USA) and quantified using BCA assay kit (Sigma) according to the instructions. Denatured proteins were separated by SDS-PAGE gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with blocking reagent (Thermo Fisher) for 1 h at room temperature and then incubated with primary antibodies against PTEN or β-actin (Cell Signaling Technology, Danvers, MA, USA) overnight at 4℃, followed by interacting with horseradish peroxidase conjugated secondary antibodies (Cell Signaling Technology) for 2 h at room temperature. The protein blots were detected using enhanced chemiluminescence chromogenic substrate (GE Healthcare, Amersham, UK) and investigated by Image Lab software (Bio-Rad).

All experiments were approved by the Animal Research committee of the Third Hospital Affiliated to Qiqihar Medical College. SPF BALB/c nude mice (female, 6-week-old) were obtained from Vital River Laboratory Animal Technology (Beijing, China) and maintained in specific pathogen-free microisolator cages with free access to water and food during the experiments.

MCF-7 cells were transfected with lentiviral vectors with miR-182-5p inhibitor (anti-miR-182-5p) or control (anti-NC) constructed by GeneCopoeia (Rockville, MD, USA) according to the manufacturer's instructions. Stably transfected cells were subcutaneously injected into nude mice after acclimatization (n=8 per group). Tumors were examined every ten days, and tumor volume was calculated with slide calipers as (length×width×height)/2. Mice were sacrificed at 50 days after cell implantation, and tumor specimens were collected for weight or further molecular analyses.

Data are presented as the mean±SEM from three independent experiments. Student's t test was used to evaluate differences using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). Kaplan-Meier plots were used to analyze survival rates. Multivariate Cox regression analyses were conducted to assess the prognostic value of miR-182-5p and other factors. Statistical significance was considered for p values less than 0.05.

To probe the roles of miR-182-5p in BC progression, its expression was detected in BC tissues and cell lines. A marked increase in miR-182-5p levels was noted in BC tissues, compared with that in para-tumor samples (n=37) (Fig. 1A). Classifying patients into high miR-182-5p expression (n=22) and low miR-182-5p expression (n=15) groups according to the mean value thereof, we found that high miR-182-5p expression was correlated with poor survival rate (p< 0.01) (Fig. 1B). Moreover, high miR-182-5p expression was significantly associated with lymph node metastasis (p=0.0396) and tumor stage (p=0.0237). Similarly, an abundance of miR-182-5p was strong higher in MCF-7, MDA-MB-231, BT20, T47D, and SKBR3 cells than in MCF-10A cells, particularly in MCF-7 and MDA-MB-231 cells (Fig. 1C). Hence, MCF-7 and MDA-MB-231 cells were used for further experiments.

In the light of dysregulation of miR-182-5p, we further investigated the effect of miR-182 depletion on BC cell proliferation and invasion in MCF-7 and MDA-MB-231 cells with transfection of anti-miR-182-5p or anti-NC. As a result, the expression of miR-182-5p was effectively reduced in MCF-7 and MDA-MB-231 cells transfected with anti-miR-182-5p, compared with cells treated with anti-NC (Fig. 2A). Moreover, abrogation of miR-182-5p resulted in a loss of proliferation rate, compared with treatment of anti-NC in MCF-7 and MDA-MB-231 cells (Fig. 2B and C). In addition, invasive ability was obviously impaired in both MCF-7 and MDA-MB-231 cells with the absence of miR-182-5p (Fig. 2D).

Generally, the function of miRNA is known to modulate targeted gene expression in various cancers. Therefore, a promising target of miR-182-5p was required for understanding the pathogenesis of BC. Bioinformatics analysis described the putative binding sites of miR-182-5p and 3′-UTR of PTEN (Fig. 3A). Hence, luciferase activity and Ago1 RIP assays were performed to validate the interaction. Results showed that overexpression of miR-182-5p leads to a marked reduction in luciferase activity in MDA-MB-231 and MCF-7 cells transfected with PTEN-WT, whereas its efficacy was lost in response to that in the PTEN-MUT group (Fig. 3B and C). However, absence of miR-182-5p showed the opposite effect on luciferase activity in MDA-MB-231 and MCF-7 cells (Fig. 3D and E). Moreover, the abundance of Ago1-enriched PTEN was effectively enhanced in MCF-7 and MDA-MB-231 cells transfected with miR-182-5p, compared with that in the miR-NC group; IgG failed to show any efficacy of enrichment (Fig. 3F). The abundance of PTEN was measured in BC tissues and cells by qRT-PCR and Western blot. The results revealed obvious decreases in the expression of PTEN in BC tissues and cells, compared with their corresponding control, respectively (Fig. 3G and H). Meanwhile, accumulation of miR-182-5p resulted in reductions in PTEN protein, while its abrogation induced elevated expression thereof, in MDA-MB-231 and MCF-7 cells (Fig. 3I).

Since PTEN was found to be a target of miR-182-5p, we further investigated whether PTEN was required for the miR-182-5p-mediated effect on proliferation and invasion in BC cells. Unsurprisingly, the abundance of miR-182-5p was markedly elevated in MCF-7 and MDA-MB-231 cells with transfection of miR-182-5p mimics (Fig. 4A). Meanwhile, the mRNA expression of PTEN was evidently increased in cells suffering from transfection of PTEN overexpression vector (Fig. 4B). Moreover, introduction of PTEN overexpression vector reversed the inhibitory effect of miR-182-5p on expression of PTEN in MCF-7 and MDA-MB-231 cells (Fig. 4C). Functional analysis demonstrated that richness of miR-182-5p contributed to cell proliferation in MDA-MB-231 and MCF-7 cells, whereas restoration of PTEN attenuated the effect (Fig. 4D and E). Furthermore, invasive cells were greatly increased in MCF-7 and MDA-MB-231 cells transfected with miR-182-5p, compared with that in miR-NC group, while the presence of PTEN ablated the regulatory effect of miR-182-5p on cell invasion (Fig. 4F).

Given that miR-182-5p deletion was found to protect against cell proliferation and invasion in BC cells, we next investigated whether miR-182-5p knockdown might inhibit tumor growth in vivo. Tumor size was effectively suppressed in anti-miR-182-5p treated mice, compared with the anti-NC group (Fig. 5A). Moreover, miR-182-5p deficiency led to a marked loss of tumor weight, compared with anti-NC treatment (Fig. 5B). Subsequently, the expression levels of miR-182-5p and PTEN were investigated in tumor tissues, revealing an obvious reduction of miR-182-5p in the tumors with anti-miR-182-5p insult (Fig. 5C). Besides, the expression of PTEN was enhanced at mRNA and protein levels in tumors in anti-miR-182-5p group, compared with the anti-NC group (Fig. 5D and E).