This study was approved by the Ethics Committee of The Fourth Affiliated Hospital of Harbin Medical University (Harbin, China) and each patient enrolled provided written informed consent. Pairs of breast cancer tissues and adjacent normal breast tissues (n=51) were obtained at The Fourth Affiliated Hospital of Harbin Medical University between May 2014 and March 2017. Patients had been treated with preoperative adjuvant chemotherapy, radiotherapy or other therapy were excluded from this research. All tissue specimens were snap-frozen in liquid nitrogen and stored at −80°C prior to RNA isolation.

A human breast epithelial cell line (MCF-10A) and four human breast cancer cell lines (MCF-7, BT-474, MDA-MB-231 and SKBR3) were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All the cell lines were cultured at 37°C in a 5% CO₂ incubator using Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mixture (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

miR-511 mimics and negative control miRNA mimics (miR-NC) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The miR-511 mimics sequence was 5′-GUGUCUUUUGCUCUGCAGUCA-3′ and the miR-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. An siRNA targeting SOX9 and negative control siRNA (NC siRNA) were purchased from Guangzhou RiboBio (Guangzhou, China). The SOX9 siRNA sequence was 5′-AGCAAGTCCGCGAGCC AGTAC-3′ and the NC siRNA sequence was 5′-UUCUCCGAA CGUGUCACGUTT-3′. The full-length SOX9 lacking the 3′-UTR sequence was chemically synthesized by the Chinese Academy of Sciences (Changchun, China) and inserted into the pcDNA3.1 vector to produce pcDNA3.1-SOX9 plasmid.

Cells were inoculated into 6-well plates at 60-70% confluence. Following overnight incubation, cells were transfected with miR-511 mimics (100 pmol), miR-NC (100 pmol), SOX9 siRNA (100 pmol), NC siRNA (100 pmol), pcDNA3.1-SOX9 (4 µg) or empty pcDNA3.1 vector (4 µg) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer’s protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was utilized to determine the transfection efficiency of miR-511 mimics. The transfection efficiencies of SOX9 siRNA and pcDNA3.1-SOX9 were evaluated by western blot analysis after 72 h of incubation.

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to isolate total RNA from tissue specimens or cultured cells. For the quantification of miR-511 expression, first-strand cDNA synthesis was performed using the miScript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s protocol. Subsequently, qPCR was performed using the miScript SYBR^®-Green PCR kit (Qiagen GmbH). The thermocycling conditions for qPCR were as follows: 95°C for 15 min, followed by 45 cycles of 94°C for 15 sec, 55°C for 30 sec and 70°C for 30 sec. miR-511 expression was normalized to that of U6 small nuclear RNA. To analyze the mRNA expression of SOX9, RT was conducted using the PrimeScript™ RT reagent kit, and the synthesized cDNA was then subjected to qPCR using a SYBR^® Premix Ex Taq™ kit (both from Takara Biotechnology Co., Ltd., Dalian, China). qPCR for mRNA was performed with the following cycling conditions: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. GAPDH was used as an internal reference for the mRNA expression of SOX9. The primers were designed as follows: miR-511, 5′-GTGTCTTTTGCTCTGCAGTC-3′ (forward) and 5′-AACATGTACAGTCCATGGATG-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); SOX9, 5′-CTGGGAACAACCCGTCTA-3′ (forward) and 5′-GGGTAA TGCGCTTGGATA-3′ (reverse); and GAPDH, 5′-CGGAGTCA ACGGATTTGGTCGTAT-3′ (forward) and 5′-AGCCTTCTCC ATGGTGGTGAAGAC-3′ (reverse). Relative gene expression was analyzed using the 2^−ΔΔCq method (29).

Transfected cells were collected at 24 h post-transfection and seeded into 96-well plates with an initial density of 3×10³ cells per well. Cells were then incubated at 37°C under 5% CO² for 0, 24, 48 or 72 h. At each time point, the CCK-8 assay was performed by adding 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) into each well. After another 2 h incubation with CCK-8 reagent at 37°C, the optical density was detected at 450 nm wavelength using a SpectraMax^® microplate spectrophotometer (Molecular Devices, LLC, Sunnyvale, CA, USA).

The colony formation assay was applied to assess the ability of breast cancer cells to form colonies. Following 24 h incubation, transfected cells were harvested, and single cells were plated into 6-well plates with a density of 1,000 cells per well. Cells were maintained at 37°C supplied with 5% CO₂ for 2 weeks. On day 15, cells were fixed with 4% paraformaldehyde, stained with methyl violet (Beyotime Institute of Biotechnology, Haimen, China) and washed with PBS. The number of colonies was counted under an inverted microscope (IX83; Olympus, Tokyo, Japan).

An Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Biolegend, Inc., San Diego, CA, USA) was used to measure the apoptosis rate of breast cancer cells. Cells were collected at 48 h after transfection, washed three times with cold PBS and resuspended in 100 µl binding buffer. Subsequently, cells were double-stained at 37°C with 5 µl Annexin V-FITC and 5 µl propidium iodide for 30 min in the dark. The percentage of apoptotic cells was measured using a flow cytometer (FACScan™; BD Biosciences, Franklin Lakes, NJ, USA). The data were processed using CellQuest™ version 5.1 software (BD Biosciences).

The migratory and invasive abilities of breast cancer cells were investigated using 24-well Transwell^® inserts (pore size, 8 µm) coated without or with Matrigel^® (both from BD Biosciences). In brief, a single-cell suspensions of the transfected cells was prepared and 1×10⁵ cells in 200 µl FBS-free DMEM were added to the upper compartment of Transwell^® inserts. DMEM supplemented with 10% FBS was added into the lower compartment to serve as a chemo-attractant. After 24 h of incubation at 37°C with 5% CO₂, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with 0.05% crystal violet (Beyotime Institute of Biotechnology) at room temperature for 20 min. The non-migrated or non-invading cells were carefully removed using a cotton swab, while the number of migrated or invaded cells was counted in five randomly chosen visual fields under an inverted microscope.

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Ethics Review Committee of The Fourth Affiliated Hospital of Harbin Medical University. Cells were transfected with miR-511 mimics or miR-NC and incubated at 37°C under 5% CO₂ for 24 h. Transfected cells were harvested after 24 h of incubation and then subcutaneously administered into the hind flanks of 4-week-old BALB/c nude mice (1×10⁶ cells per mouse). Tumor width and length were detected every 4 days for 4 weeks. All nude mice were sacrificed 4 weeks after tumor implantation and the tumor xenografts were excised and weighed. The tumor volumes were calculated using the equation: Tumor volume = 0.5 × tumor length × tumor width.

TargetScan 7.1 (http://www.targetscan.org/) and MiRanda (http://www.microrna.org/) were used to predict the potential targets of miR-511. SOX9 was identified as a candidate target gene of miR-511.

The 3′-UTR of SOX9 containing the wild-type or mutant miR-511 binding site was amplified by Shanghai GenePharma and inserted into the pMIR-REPORT plasmid (Promega Corporation, Madison, WI, USA). Cells were plated into 24-well plates with a density of 1×10⁵ cells/well and were co-transfected with the luciferase reporter plasmid (0.2 µg) together with miR-511 mimics (50 pmol) or miR-NC (50 pmol) using Lipofectamine^® 2000. Luciferase activity was determined 48 h after transfection using the dual-luciferase reporter assay system (Promega Corporation). Renilla luciferase activity served as an internal reference for Firefly luciferase activity.

Cells or tissues were lysed in ice-cold radioimmunoprecipitation buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) to extract total protein. The concentration of the isolated total protein was detected using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Equal amounts (30 µg) of protein were separated by SDS-PAGE on a 10% polyacrylamide gel. The separated proteins were then electronically transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking at room temperature in 5% fat-free milk powder for 2 h, the membranes were incubated overnight at 4°C with primary antibodies against SOX9 (cat. no. ab185966; 1:1,000 dilution), phosphor (p)-phosphoinositide 3-kinase (PI3K; cat. no. ab182651; 1:1,000 dilution), PI3K (cat. no. ab191606; 1:1,000 dilution) (all from Abcam, Cambridge, UK), p-Akt (cat. no. sc-81433; 1:1,000 dilution), Akt (cat. no. sc-56878; 1:1,000 dilution) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or GAPDH (cat. no. ab181603; 1:1,000 dilution; Abcam). Following three washes with Tris-buffered saline containing 0.1% Tween^®-20, the membranes were further incubated with the goat anti-rabbit (cat. no. ab6721; Abcam; used for SOX9, p-PI3K, PI3K and GAPDH primary antibodies) or goat anti-mouse (cat. no. ab205719; Abcam; used for p-Akt and Akt primary antibodies) corresponding horseradish peroxidase-conjugated secondary antibody (both 1:5,000 dilution) at room temperature for 2 h and visualized using an enhanced chemiluminescence kit (EMD Millipore). GAPDH was used as a loading control. Protein expression was quantified using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

SPSS software (version 19.0; IBM Corp., Armonk, NY, USA) was used to perform all statistical analyses. Data are presented as the mean ± standard deviation from three independent experiments. The statistical differences between groups were analyzed using Student’s t-test or one-way analysis of variance combined with multiple comparisons using the Student-Newman-Keuls post-hoc test. The χ² test was used to assess the association between miR-511 expression and the clinicopathological features of patients with breast cancer. Spearman’s correlation analysis was employed to identify the association between the expression levels of miR-511 and SOX9 mRNA in breast cancer tissues. P<0.05 was considered to indicate a statistically significant difference.

To determine the expression of miR-511 in breast cancer, miR-511 expression was detected using RT-qPCR in 51 pairs of breast cancer tissue and adjacent normal breast tissue. miR-511 was significantly reduced in breast cancer tissues compared with adjacent normal breast tissues (P<0.05; Fig. 1A). To further confirm this observation, miR-511 expression was analyzed in four human breast cancer cell lines (MCF-7, BT-474, MDA-MB-231 and SKBR3) and a human breast epithelial cell line (MCF-10A). The expression of miR-511 was significantly lower in all four breast cancer cell lines compared with MCF-10A cells (P<0.05; Fig. 1B).

To characterize the clinical characteristics of miR-511, all patients with breast cancer were divided into either the miR-511 low expression group (n=26) or miR-511 high expression group (n=25) based on the median value of miR-511. The results of the statistical analysis indicated that decreased miR-511 expression was associated with lymph node metastasis (P=0.001) and tumor stage (P=0.017; Table I). However, no significant associations were observed between miR-511 expression and other clinicopathological characteristics, including age, tumor diameter and histology grade (all P>0.05). The results suggest that the downregulation of miR-511 may be involved in breast cancer initiation and progression.

To gain insight into the detailed roles of miR-511 in breast cancer, miR-511 mimics were transfected into MCF-7 and MDA-MB-231 cells to increase endogenous miR-511 expression. The data obtained by RT-qPCR analysis revealed that miR-511 was overexpressed in miR-511 mimics-transfected MCF-7 and MDA-MB-231 cells relative to that in cells transfected with miR-NC (P<0.05; Fig. 2A). The CCK-8 assay was performed to evaluate the effect of miR-511 upregulation on the proliferative ability of breast cancer cells. Ectopic miR-511 expression caused an obvious reduction in proliferation in MCF-7 and MDA-MB-231 cells (P<0.05; Fig. 2B). Colony formation assay was also used to confirm the regulatory effect of miR-511 on breast cancer cell proliferation. The results indicated that miR-511 overexpression significantly decreased the colony formation abilities of MCF-7 and MDA-MB-231 cells (P<0.05; Fig. 2C). Apoptosis is associated with the alterations in cell proliferation, suggesting that a high apoptosis rate mat cause suppression of cell proliferation. Thus, the apoptosis rate of MCF-7 and MDA-MB-231 cells transfected with miR-511 mimics or miR-NC was determined. As displayed in Fig. 2D, enforced miR-511 expression increased the rate of apoptosis in MCF-7 and MDA-MB-231 cells in comparison with the miR-NC groups (P<0.05). Furthermore, migration and invasion assays were performed to determine whether miR-511 was involved in the regulation of breast cancer cell metastasis. miR-511-overexpressing MCF-7 and MDA-MB-231 cells exhibited decreased migratory (P<0.05; Fig. 2E) and invasive (P<0.05: Fig. 2F) capacities as compared with the cells transfected with miR-NC. The findings suggest that miR-511 upregulation may inhibit the aggressiveness of breast cancer cells.

To elucidate the mechanism by which miR-511 may act as a tumor suppressor in breast cancer, bioinformatics analysis was performed to search for putative targets of miR-511. SOX9 was predicted to be a major target of miR-511, as two complementary binding sites of miR-511 were identified in the 3′-UTR of the SOX9 gene (Fig. 3A). SOX9 was chosen for further experimental confirmation as this gene was previously reported to be involved in the pathogenesis of breast cancer. A luciferase reporter assay was performed to determine whether miR-511 directly recognized and bound to the 3′-UTR of SOX9. The data revealed that miR-511 upregulation decreased the luciferase activity of the plasmid harboring the wild-type (1 and 2) 3′-UTR of SOX9 in MCF-7 and MDA-MB-231 cells (P<0.05; Fig. 3B), but did not affect the activity of the mutant (1 and 2) 3′-UTR plasmid. To further confirm that SOX9 is a target of miR-511, RT-qPCR and western blot analysis were performed to detect SOX9 mRNA and protein expression in MCF-7 and MDA-MB-231 cells with overexpressed miR-511. The mRNA (P<0.05; Fig. 3C) and protein (P<0.05; Fig. 3D) levels of SOX9 were lowered in MCF-7 and MDA-MB-231 cells transfected with miR-511 mimics. Collectively, these findings suggest that SOX9 is a direct target gene of miR-511 in breast cancer cells.

To further illustrate the relationship between miR-511 and SOX9 in breast cancer, RT-qPCR was performed to measure SOX9 expression in breast cancer tissues and adjacent normal breast tissues. Compared with adjacent normal breast tissues, the level of SOX9 mRNA (P<0.05; Fig. 4A) was strikingly upregulated in breast cancer tissues. Then, Spearman’s correlation analysis was performed to analyze the association between miR-511 and SOX9 expression in breast cancer tissues, which revealed a statistically significant negative correlation between the miR-511 and SOX9 mRNA levels in these breast cancer tissues (r=−0.5683, P<0.0001; Fig. 4B). These results further supported the conclusion that SOX9 is a downstream target of miR-511 in breast cancer.

SOX9 was validated as a direct target gene of miR-511 in breast cancer cells; thus, it was hypothesized that SOX9 knockdown may mimic the tumor-suppressor role of miR-511 overexpression in breast cancer cells. To test this hypothesis, SOX9 siRNA was applied to knock down endogenous SOX9 expression in MCF-7 and MDA-MB-231 cells (P<0.05; Fig. 5A). Subsequent functional assays demonstrated that SOX9 inhibition impeded proliferation (P<0.05; Fig. 5B) and colony formation (P<0.05; Fig. 5C), increased apoptosis (P<0.05; Fig. 5D) and prohibited migration (P<0.05; Fig. 5E) and invasion (P<0.05; Fig. 5F) of MCF-7 and MDA-MB-231 cells. Thus, the downregulation of SOX9 had functional effects that were similar to those of miR-511 upregulation in breast cancer cells, suggesting that SOX9 is a functional target of miR-511 in breast cancer cells.

To further demonstrate whether SOX9 inhibition was essential for the tumor-suppressor activity of miR-511 in breast cancer, a SOX9 overexpression plasmid lacking the 3′-UTR (pcDNA3.1-SOX9) was transfected into miR-511-overexpressing MCF-7 and MDA-MB-231 cells. Western blot analysis indicated that the downregulation of SOX9 protein caused by miR-511 upregulation was re-established in MCF-7 and MDA-MB-231 cells following co-transfection with pcDNA3.1-SOX9 (P<0.05; Fig. 6A). CCK-8 and colony formation assays revealed that SOX9 reintroduction re-established the miR-511-induced inhibition of MCF-7 and MDA-MB-231 cell proliferation (P<0.05; Fig. 6B) and colony formation (P<0.05; Fig. 6C). Additionally, the miR-511 mimics-mediated promotion of cell apoptosis was partially reversed by SOX9 restoration in MCF-7 and MDA-MB-231 cells (P<0.05; Fig. 6D). Furthermore, the restoration of SOX9 overexpression blunted the inhibitory effects of miR-511 overexpression on the migratory (P<0.05; Fig. 6E) and invasive (P<0.05; Fig. 6F) abilities of MCF-7 and MDA-MB-231 cells. Together, these results provided sufficient evidence to demonstrate that miR-511 may inhibit the development of breast cancer cells, at least partially by suppressing SOX9 expression.

SOX9 participates in the regulation of the PI3K/Akt signaling pathway (30,31); thus, whether miR-511 inactivates PI3K/Akt signaling in breast cancer cells was examined. To test this hypothesis, western blot analysis was performed to detect the molecules associated with the PI3K/Akt pathway in MCF-7 and MDA-MB-231 cells following co-transfection with miR-511 mimics and pcDNA3.1 or pcDNA3.1-SOX9. It was demonstrated that enforced miR-511 expression suppressed the levels of the p-PI3K and p-Akt phosphoproteins in MCF-7 and MDA-MB-231 cells; however, miR-511 overexpression did not affect the total levels of the PI3K and Akt proteins. Additionally, co-transfection with pcDNA3.1-SOX9 restored the miR-511 overexpression-inhibited levels of p-PI3K and p-Akt in these cell lines (Fig. 7). These findings suggest that miR-511 targets SOX9 to inactivate the PI3K/Akt pathway in breast cancer cells.

A xenograft tumor experiment was performed to assess whether miR-511 contributes to the tumorigenesis of breast cancer cells in vivo. MDA-MB-231 cells were transfected with miR-511 mimics or miR-NC and were subcutaneously administered into the hind flanks of nude mice. In order to meet ethical requirements, all the nude mice were sacrificed before the tumor xenografts reached 2 cm. The maximum tumor burden was 1.8 cm. Consistent with the in vitro results, miR-511-overexpressing xenografts exhibited a lower tumor volume (P<0.05; Fig. 8A and B) and tumor weight (P<0.05; Fig. 8C) compared with miR-NC expressing tumors. In addition, the data obtained by RT-qPCR analysis revealed that miR-511 was specifically upregulated in the xenografts of the miR-511 mimics-overexpressing group compared the miR-NC-transfected group (P<0.05; Fig. 8D). Western blot analysis demonstrated that the xenografts of the miR-511-overexpressing group expressed lower levels of SOX9, p-PI3K, and p-Akt than the xenografts of the miR-NC-transfected group (Fig. 8E). Together, these results suggest that miR-511 targets SOX9 to inhibit breast cancer tumor growth in vivo via deactivation of the PI3K/Akt pathway.