Nine paraffin-embedded breast cancer and corresponding adjacent tissue samples were collected from Zhejiang Cancer Hospital (Hangzhou, China). This study was approved by the ethics committee of the Zhejiang Sci-Tech University (Hangzhou, China) and Zhejiang Cancer Hospital. All the samples were stored at 4°C until RNA extraction.

Total RNA was extracted from paraffin-embedded tissues using Recover All™ Total Nucleic Acid Isolation (Ambion, Austin, TX, USA) following the manufacturer's instructions. RNA concentrations were measured by the NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA reverse transcription was performed with 100 ng of total RNA. Expression of let-7c-5p, ERCC6 and internal control GAPDH were detected using SYBR Premix Ex Taq™ II (Takara, Dalian, China). qRT-PCR was performed on the Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Relative expression was calculated using the 2^−∆∆Ct method, the expression of let-7c-5p and ERCC6 was normalized with GAPDH. Primers for qRT-PCR are listed in Table I.

The MCF-7 human breast cancer cell line was obtained from Zhejiang Sci-Tech University, cultured in DMEM-high glucose medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), at 37°C in a 5% CO₂ humidified incubator (HF90, Heal Force, Hong Kong). Mimics for miR-NC (5′-CAGUACUUUUGUGUAGUACAA-3′) and let-7c-5p (5′-UGAGGUAGUAGGUUGUAUGGUU-3′) were purchased from GenePharma (Shanghai, China). Reverse transfection was performed in this study using GeneTran™ III High Efficiency Transfection Reagent (Biomiga, Inc., San Diego, CA, USA) according to the manufacturer's recommendations.

MCF-7 cells were seeded in 96-well plates at approximately 4000 cells per well and transfected with miR-NC or let-7c-5p mimics. MTT (Sigma, St. Louis, MO, USA) was used to examine the effects of let-7c-5p on cell proliferation at 24, 48 and 72 h post-transfection. At each time, 20 µl MTT solution was added to each well, and incubated at 37°C for 4 h, the formazan produced by viable cells was dissolved in 150 µl dimethylsulfoxide (DMSO). The cell numbers were evaluated by reading the absorbance at 490 nm (15).

Cells were seeded in 6-well plates at approximately 3×10⁵ cells per well and transfected with miR-NC or let-7c-5p mimics. All cells were harvested at 24 h post-transfection, washed with cold PBS for twice, stained with FITC and PI using BD Pharmingen FITC Annexin V™ apoptosis detection kit I (BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Flow cytometry analysis was finished on BD Accuri C6 flow cytometer with C6 software.

The potential target genes of let-7c-5p were predicted using the algorithms of TargetScan (http://www.targetscan.org/vert_71/), PicTar (http://pictar.mdc-berlin.de/) and starBase (http://starbase.sysu.edu.cn/). ERCC6 was taken as a potential target gene of let-7c-5p. There was only one potential complementary site for let-7c-5p in the 3′UTR of ERCC6 mRNA, and the wild-type 3′UTR fragment containing putative binding site was amplified by PCR and cloned into the XbaI and NotI sites downstream of Renilla luciferase vector pRL-TK (Promega, Beijing, China), the recombinant plasmid was named pRL-ERCC6-wt. The putative let-7c-5p binding site was deleted by overlap-extension PCR, and the recombinant plasmid was named pRL-ERCC6-mut. Primers are listed in Table II, constructs were verified by sequencing.

For dual luciferase reporter assay, cells were seeded in 24-well plates in triplicate and cotransfected with the firefly luciferase vector pGL3 and recombinant plasmid pRL-ERCC6-wt or pRL-ERCC6-mut, together with mimics for either miR-NC or let-7c-5p at a final concentration of 20 nM. After 48 h, luciferase activities were measured using the dual luciferase reporter assay system (Promega). Renilla luciferase activity was normalized with firefly luciferase activity.

Cells were harvested at 48 h post-transfection and lysed in RIPA buffer on ice. Total proteins were separated by 10% SDS-PAGE, electroblotted onto PVDF membranes. Membranes were blocked with 5% non-fat milk powder for 2 h at room temperature and incubated overnight at 4°C with a polyclonal antibody: anti-ERCC6 antibody (1:2000 dilution; Abcam, Cambridge, MA, USA) or anti-GAPDH antibody (1:2000 dilution; HuaBio, China). After washing three times with TBS-T, membranes were incubated with a secondary antibody anti-rabbit HRP-conjugate (1:2000 dilution; HuaBio) for 2 h at room temperature (16). Antibody detection was performed with ECL Western Blotting Substrate (Solarbio, Beijing, China), and photos were taken using the Tanon 5500 imaging system (Tanon, Shanghai, China).

Statistical analysis was performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). All data were presented as mean ± standard deviation (SD). Statistical significance was tested by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, expression of let-7c-5p was significantly decreased in the nine breast cancer tissues, indicating that let-7c-5p could potentially serve as a biomarker for breast cancer.

To determine the transfection efficiency, cells were harvested after transfection with 40 or 80 nM let-7c-5p mimics for 24 h. Total RNA was isolated from cells using the traditional TRIzol method. qRT-PCR was performed to measure the expression of let-7c-5p. As shown in Fig. 2A, let-7c-5p expression was enhanced significantly by let-7c-5p mimics.

To examine the effects of let-7c-5p on MCF-7 cell proliferation in vitro, MTT assay was performed at 24, 48 and 72 h post-transfection, the cell growth curve showed that the proliferation ability of transfected cells was reduced remarkably compared to control group cells (Fig. 2B).

Additionally, we explored whether cell apoptosis could be induced by let-7c-5p. After transfection for 24 h, cells were harvested, and cell apoptosis was detected by flow cytometer. As shown in Fig. 2C, early stage apoptotic cells are presented in the lower right quadrant, a higher rate of apoptosis was observed in transfected cells, especially in those transfected with 80 nM let-7c-5p mimics (Fig. 2D).

TargetScan, PicTar and starBase were used to predict the target genes of let-7c-5p, and the intersection of the prediction results from the three algorithms showed that there were 41 candidate genes with at least one binding site of let-7c-5p (Table III). Of these genes, ITGB3 and MAP4K3 have been verified as targets of let-7c-5p in human non-small cell lung cancer. In this experiment, we aimed to find a new target of let-7c-5p in breast cancer. ERCC6, which has been reported to be associated with cancer risk, was selected as a putative target gene.

The let-7c-5p binding sites in 3′UTR of ERCC6 mRNA were predicted by TargetScan, as a result, there was only one potential complementary sequence for let-7c-5p at nt 125–132 (Fig. 3A). To validate whether let-7c-5p targets the 3′UTR of ERCC6 mRNA, we used the firefly luciferase vector pGL3 as an internal control plasmid, and co-transfected pGL3 and recombinant plasmid pRL-ERCC6-wt or pRL-ERCC6-mut along with miR-NC or let-7c-5p mimics into MCF-7 cells. Luciferase activities were measured after co-transfection for 48 h, as shown in Fig. 3B, dual luciferase reporter assay demonstrated that the Renilla luciferase activity of pRL-ERCC6-wt, which contained the wild-type 3′UTR fragment of ERCC6, was decreased by approximately 50% by let-7c-5p, compared to the miR-NC group. In addition, the luciferase activity of pRL-ERCC6-mut, in which the predicted binding site has been deleted, was not suppressed significantly. These results showed that ERCC6 was a direct target gene for let-7c-5p in breast cancer.

To better understand the effects of let-7c-5p on ERCC6, we evaluated the ERCC6 mRNA and protein expression level in MCF-7 cells after treatment with let-7c-5p mimics for 48 h. As shown in Fig. 3C and D, protein expression of ERCC6 was clearly inhibited by let-7c-5p, but the mRNA expression of ERCC6 did not change significantly. These findings suggested that let-7c-5p regulated ERCC6 mainly through suppression of ERCC6 protein accumulation, but not affecting the mRNA level.