Human breast cancer cell lines (MCF-7, BT474, MDA-MB-231, and MDA-MB-468), normal breast epithelial cell line MCF-10A and 293T cells were purchased from the American Type Culture Collection (Manassas, VA, USA). BT474 and MDA-MB-231 cells were cultured in Hyclone RPMI-1640 medium (GE Healthcare Life Sciences, Logan, UT, USA) while MCF-7, MDA-MB-468, MCF-10A and 293T cells were cultured in Hyclone Dulbecco's modified Eagle's medium (GE Healthcare Life Sciences). All cells were grown in medium containing 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) supplemented with 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) in a humidified atmosphere of 5% CO₂ at 37°C.

Total RNAs were extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). To detect miR-3666 expression, cDNA was synthesized by Moloney murine leukemia virus reverse transcriptase (Takara Biotechnology, Co., Ltd., Dalian, China). To detect SIRT7 expression levels, cDNA was synthesized using a miScript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany). The RT-qPCR was conducted using a SYBR-Green master mix kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with an Applied Biosystems AB7500 Real Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the following procedure: 94°C for 5 min; 30 cycles of 94°C for 20 sec, 55°C for 25 sec, and 72°C for 35 sec; and 72°C for 10 min. Small nuclear RNA U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal controls. The relative gene expression was calculated using the 2^−ΔΔCq method as compared with U6 or GAPDH (40). The fold-change of gene expression was obtained by normalization against the control group. The primers used were as follows: Forward, 5′-ACACTCCAGCTGGGCAGTCAAGTGTAGA-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′ for miR-3666; forward, 5′-CGCTTCGGCAGCACATATACTAA-3′ and reverse, 5′-TATGGAACGCTTCACGAATTTGC-3′ for U6; forward, 5′-GTGGACACTGCTTCAGAAAG-3′ and reverse, 5′-CACAGTTCTGAGACACCACA-3′ for SIRT7; and forward, 5′-CCATGTTCGTCATGGGTGTG-3′ and reverse, 5′-GGTGCTAAGCAGTTGGTGGTG-3′ GAPDH.

The miR-3666 mimics and negative control (miR-NC) were obtained from Origene Technologies, Inc. (Beijing, China) and transfected into cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 50 nM. SIRT7 small interfering RNA (siRNA) and negative control (NC siRNA) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and transfected into cells according to the manufacturer's instructions. SIRT7-overexpressing vector was generated by cloning SIRT7 cDNA without a 3′-UTR into a pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). The pcDNA3.1-SIRT7 vector was transfected into cells using Lipofectamine 2000.

Cells were seeded into 96-well plates at a density of 1×10⁴ cells/well and cultured overnight. Cells were then transfected with miR-3666 mimics and cultivated for 48 h, and 20 µl of MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well and incubated at 37°C for 4 h. Subsequently, the culture media were discarded and 200 µl dimethyl sulfoxide was added to each well. Optical density values at a wavelength of 490 nm were detected using an ELISA reader (Bio-Rad Laboratories, Inc.).

The BrdU assay was performed using a BrdU cell proliferation assay kit (Cell Signaling Technology, Inc., Danvers, MA, USA) in accordance with the manufacturer's instructions. Briefly, cells were seeded into 96-well plates (1×10⁴ cells/well) and transfected with miR-3666 mimics for 48 h. Thereafter, 10 µl BrdU solution was added to each well and cultured for 2 h. Following removal of the culture media, 150 µl denaturing solution was added to each well and incubated at room temperature for 1 h. Subsequently, peroxidase conjugated anti-BrdU was added and incubated for 1 h at room temperature. The optical density values at a wavelength of 450 nm were measured by an ELISA reader (Bio-Rad Laboratories, Inc.).

Caspase-3 activity assay was performed using a commercial kit (Roche Applied Science, Madison, WI, USA) according to the manufacturer's instructions. Briefly, following treatment, cells were lysed and the supernatant was harvested followed by incubation with DEVD-pNA substrate (Roche Applied Science) at 37°C for 2 h. Optical density values at a wavelength of 405 nm were determined using an ELISA reader (Bio-Rad Laboratories, Inc.).

miRNA targets were predicted using the algorithms of TargetSan (https://www.targetscan.org) (41). The 3′-UTR of SIRT7 containing the wild-type or mutant binding sites of miR-3666 were cloned into pmirGLO vector (Promega Corporation, Madison, WI, USA) followed by transfection into 293T cells with miR-3666 mimics using Lipofectamine 2000. After 48 h of incubation, cells were harvested and detected using a Dual-Luciferase assay kit (Promega Corporation). The relative luciferase activity was calculated according to the following formula: Firefly luciferase/Renilla luciferase.

Total protein was extracted using RIPA buffer (Sigma-Aldrich; Merck KGaA). Equal quantities (40 µg) of proteins were loaded onto 10% sodium dodecyl sulfate polyacrylamide gels (Sangon Biotech Co., Ltd., Shanghai, China) for separation. The separated proteins were then transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) followed by incubation with 3% nonfat milk for 1 h. The membrane was incubated with primary antibodies at 4°C overnight. Anti-SIRT7 (cat. no. sc-135055; dilution, 1:500) and anti GAPDH (cat. no. sc-367714; dilution, 1:800) primary antibodies were both purchased from Santa Cruz Biotechnology, Inc. Subsequently, the membrane was washed with Tris-buffered saline containing 0.1% Tween-20 three times and then blotted with horseradish peroxidase conjugated secondary antibodies (cat. no. A0208; dilution, 1:1,000, Beyotime Institute of Biotechnology, Haimen, China) for 1 h at 37°C. The protein bands were visualized using enhanced chemiluminescence (EMD Millipore). The intensity of the bands on the membrane was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Relative protein expression was calculated by normalization against GAPDH. The fold-change of protein expression was obtained by normalization with the control group.

All values are presented as means ± standard deviation and the statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). Differences were analyzed by one-way analysis of variance with a Bonferroni correction. P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential relevance of miR-3666 in breast cancer, its expression was examined in breast cancer cell lines using RT-qPCR. The results demonstrated that the expression level of miR-3666 was significantly downregulated in breast cancer cell lines (MCF-7, MDA-MB-468, BT474 and MDA-MB-231) compared with the normal breast epithelial cell line, MCF-10A (Fig. 1), indicating a tumor suppressive role of miR-3666 in breast cancer.

As miR-3666 demonstrated a lower expression level in BT474 and MDA-MB-231 cells, these two cell lines were selected for subsequent experiments. To investigate the potential biological effect of miR-3666 in breast cancer, gain-of-function experiments were performed by transiently transfecting miR-3666 mimics into BT474 and MDA-MB-231 cells. The expression level of miR-3666 was markedly upregulated by miR-3666 transfection, as detected by RT-qPCR (Fig. 2A). The effect of miR-3666 overexpression on cell viability and growth was then examined by MTT assay. It was observed that miR-3666 overexpression significantly suppressed breast cancer cell growth (Fig. 2B). In addition, BrdU assay indicated that proliferation of BT474 and MDA-MB-231 cells was markedly inhibited by miR-3666 overexpression (Fig. 3A). Furthermore, overexpression of miR-3666 significantly promoted apoptosis of BT474 and MDA-MB-231 cells (Fig. 3B). These results indicate that miR-3666 functions as a tumor suppressor.

To elucidate the molecular mechanism by which miR-3666 regulates breast cancer cell proliferation, bioinformatic analyses were performed using TargetScan to predict potential target genes. Among these target genes, SIRT7, which is a novel oncogene, was notable. The putative binding sites of miR-3666 within the 3′-UTR of SIRT7 are presented in Fig. 4A. To verify whether SIRT is a direct target of miR-3666, a Dual-Luciferase reporter system containing either wild-type or mutant 3′-UTR of SIRT7 was used. Co-transfection with miR-3666 mimics markedly inhibited the luciferase activity of the reporter containing the wild-type 3′-UTR (Fig. 4B). However, miR-3666 overexpression demonstrated no significant effect on mutant 3′-UTR of SIRT7 (Fig. 4B). Subsequent RT-qPCR and western blot analysis indicated that SIRT7 expression levels were significantly suppressed in BT474 and MDA-MB-231 cells following transfection with the miR-3666 mimics (Fig. 5A and B). Taken together, these results indicate that SIRT7 is a direct target of miR-3666.

To investigate whether SIRT7 is involved in regulating breast cancer, SIRT7 was silenced by transfecting SIRT7 siRNA (Fig. 6A), and its effect on cell proliferation and apoptosis was detected. It was found that silencing SIRT7 using siRNA significantly inhibited proliferation (Fig. 6B) and promoted apoptosis (Fig. 6C) of breast cancer cells. The results indicate that SIRT7 is involved in regulating breast cancer cell proliferation and apoptosis.

To investigate whether miR-3666 induced its anti-tumor effect via SIRT7, a rescue assay was performed using MDA-MB-231 cells. Recombinant SIRT7 lacking the 3′-UTR sequence (pcDNA3.1/SIRT7) was exogenously expressed in MDA-MB-231 cells. Western blotting demonstrated that co-transfection of pcDNA3.1/SIRT7 and miR-3666 mimics restored the decreased protein expression level induced by miR-3666 overexpression (Fig. 7A). Overexpression of SIRT7 significantly reversed the inhibitory effect of miR-3666 overexpression on cell proliferation (Fig. 7B and C). Furthermore, the increased apoptosis induced by miR-3666 overexpression was markedly reversed by SIRT7 overexpression (Fig. 7D). Thus, these results indicate that miR-3666 exerts its tumor suppressive role via SIRT7.