The normal mammary epithelial cell line MCF-10A and the breast cancer cell lines MCF7, SK-BR-3 and MDA-MB-231 were obtained from the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences (Beijing, China). MCF-10A cells and MDA–MB–231 cells were cultured in Dulbecco's minimum essential medium (DMEM; Hyclone, Logan, Utah, USA) with 10% fetal bovine serum (FBS; Lanzhou National Hyclone Bio-engineering, Lanzhou, China). MCF7 cells were cultured in RPMI 1640 (Hyclone) with 10% FBS and SK-BR-3 cells were maintained in RPMI 1640+HEPES with 10% FBS. All of the above cells were cultured at 37°C in a 5% CO₂ atmosphere incubator.

Total RNA samples were isolated using Invitrogen TRIzol reagent (Thermofisher Scientific, Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription was then conducted using All-in-One miRNA qRT-PCR Detection kit (GeneCopoeia, Rockville, MD, USA) to obtain cDNA. The total reaction system was incubated at 42°C for 60 min, then 70°C for 10 min and cooled on ice. The PCR reaction was performed using All-in-One miRNA qRT-PCR Detection kit (GeneCopoeia) with a Bio-Rad iCycler single-color real-time detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RNU6 was used as the internal control of miRNA. The primers for miR-147 and RNU6 were designed and sythesized by GeneCopoeia. The relative expression values of miRNA were calculated by the 2^−ΔΔCq method. Cq is the intensity value of fluorescent signal detected by the thermal cycler. ΔΔCq=(CqmiRNA - CqU6). Using 2^−ΔΔCq as the expression of miR in each experimental group. The average expression level of MCF7 cells was set to 1. The expression levels of the other cancerous groups were normalized to that of the MCF7 cell group, as the MCF7 cell group exibited the least intra-group difference with regard to miR-147 expression.

miR-147 mimic transfection was performed using mimics of miR147 or C. elegans miRNA (Guangzhou RiboBio Co., Ltd., Guangzhou, China) following the manufacturer's protocol. The final reagent concentration of the mimics was modulated to 50 nM. The si-Akt expressing plasmid vector was designed, synthesized and packaged by Shanghai GenePharma Co., Ltd (Shanghai, China). The quantity of si-Akt expressing plasmid vector used was 2 µg/well (6 well plate). A total of 1.25 µl/well Invitrogen Lipo2000 (Thermofisher Scientific, Inc.) was used to transfect the miR mimic or siRNA into MDA-MB-231 cells at a density of 2.5×10⁵ cells/well, according to the manufacturer's protocol. Cell proliferation assays were performed every 24 h for seven days after transfection. Total RNA and total protein were extracted two days after transfection, and the cells were dissociated two days after transfection for the Transwell assays.

A total of 2.5×10³ cells/well were seeded into 96-well plates with 5 repeated wells. The absorbance values were detected every 24 h for 7 days. Before the detection, 20 µl MTT (Sigma-Aldrich, St. Louis, MO, USA) was added in each well and the cells were incubated for 4 h. After incubation, the upper liquid was removed and 200 µl DMSO (Chengdu Kelong Chemical Co., Ltd., Chengdu, China) was added to each well. The absorbance value was determined at 490 nm using a Multiskan Spectrum microplate spectrophotometer (Thermofisher Scientific, Inc.).

Boyden Well (BD Biosciences, Franklin Lakes, NJ, USA) was used for the assessment of the migration and invasion of transfected cells. Polycarbonate Microporous Membrane was placed between the upper and lower chambers. For the invasion assay, the membrane of the upper chambers were coated with 6 mg/l Matrigel (BD Biosciences) and incubated at 37°C for 30 min. For the migration assay, the wells were not coated with Matrigel. MDA-MB-231 cells were seeded at a density of 1×10⁵ cells in each upper chamber and DMEM+10% FBS was placed in the lower chambers. The plates were then incubated at 37°C, 5% CO₂. After 24 h, the membranes were removed and fixed in 4% paraformaldehyde (Chengdu Kelong Chemical Co., Ltd.), stained with Giemsa (Sigma-Aldrich) and washed with PBS buffer. The migration and invasion of each group were quantified as the mean number of cells found in ten microscope fields [magnification, ×400; Eclipse E200; Nikon Instruments (Shanghai) Co., Ltd., Shanghai, China].

Cells were dissolved using RIPA buffer to obtain the cell lysates. Next, 5X Loading Buffer (Chengdu Cetme Science and Technology Co., Ltd., Chengdu, China) was added to the cell lysates, and incubated at 70°C for 10 min. Total proteins were separated on 8–12% gradient SDS-polyacrylamide gels and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). β-actin was used as loading control. The PVDF membranes were then blocked in TBST with 5% nonfat milk for 1 h, and incubated with the following antibodies: monoclonal rabbit anti-human anti-Akt (1:1,000), anti-p-Akt (1:1,000), anti-P70S6K (1:1,000), anti-p-P70S6K (1:1,000), anti-4E-BP-1 (1:1,000), anti-p-4E-BP-1 (1:1,000) (CST, Boston, Massachusetts, USA) and monoclonal mouse anti-human anti-β-actin (1:1,000) (Santa Cruz Biotechnology, Dallas, Texas, USA). Following washing with TBST the membranes were incubated with polyclonal goat anti-rabbit or rabbit anti-mouse IgG secondary antibodies (1:10,000) (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 1 h then washed with TBST. The blots were developed using ECL reagents (EMD Millipore). The exposure and image capture was performed with a Gel Dox XR+ imaging system (Bio-Rad Laboratories, Inc.).

All quantitative data are expressed as the mean ± standard error. The results were analyzed by Prism 6 software, version 6.01 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical analyses were performed using Student's t-test for comparisons between each case group and the control group. Linear regression was used for cell growth curve comparisons. P<0.05 was considered to indicate a statistically significant difference.

To determine the function and mechanism of miR-147 in breast cancer, miR-147 expression was detected in breast cancer cell lines. Normal mammary epithelial cell line (MCF-10A), two less aggressive breast cancer cell lines (MCF7 and SK-BR-3), and a highly aggressive breast cancer cell line (MDA-MB-231) were cultured in vitro. The endogenous levels of miR-147 in the above cell lines were examined by RT-PCR. The level of miR-147 expression in MCF-10A, MCF7 and SK-BR-3 cells was relatively high, while the expression of miR-147 in MDA-MB-231 was too low to be detected. The difference in miR-147 expression level between MDA-MB-231 and the other 3 cell lines was statistically significant (Fig. 1; P<0.001), indicating that the expression of miR-147 may be negatively correlated with the aggressiveness of breast cancer. The MDA-MB-231 cell line exhibits high aggressiveness and low miR-147 expression, and was chosen to be used in further experiments.

Proliferation, invasion and migration are examples of the characteristic aggressive properties of cancer cells. A 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to investigate the effect of miR-147 on cell proliferation. MiR-147 was transiently transfected into the cell line MDA-MB-231 to form the ‘miR-147’ group. In the ‘si-Akt’ group, siRNA was transfected to knockdown the expression of Akt. Fig. 2 shows that there was a significant decrease in the MTT value in miR-147 transfected MDA-MB-231 cells on day 7, in comparison to the blank control cells [MDA-MB-231 cells cultured in DMEM±10% FBS; blank control, (BC)] and negative control miR transfected cells (P<0.001). In addition, in comparison with the BC cells, the knockdown of Akt in MDA-MB-231 cells also resulted in a reduced MTT value (P<0.001). Although miR-147 transfected cells had a higher MTT value compared with knockdown of Akt cells on day 4 (P=0.007), a statistically significant difference was not observed between the MTT value of the miR-147 group and si-Akt group on overall trend (P=0.086).

Transwell assays were used to measure the cell invasive and migrating capability. The number of cells that invaded the Matrigel or traversed the microporous membrane are presented in Fig. 3. The number of invasive and migratory miR-147 transfected cells was significantly less than the BC and miR transfected negative control cells both in the invasion assay (P<0.01; Fig. 3A and B) and migration assay (P<0.001); Fig. 3C and D). Similarly, the number of invasive and migratory cells following the knockdown of Akt was less than the BC cells both in invasion assay (P<0.001; Fig. 3A and B) and migration assay (P<0.001; Fig. 3C and D). The number of invasive cells following knockdown of Akt was less than the number of invasive miR-147 transfected cells in the invasion assay (P=0.047), however no significant difference between the two in the number of migrating cells was observed in the migration assay (P=0.769).

Previous studies have reported that Akt may be a potential target of miR-147 (13,15). To investigate how miR-147 affects AKT, and the role of Akt in miR-147 induced cell proliferation, invasion and migration inhibition, western blotting was performed to assess the expression and phosphorylation of the Akt protein and the downstream key effectors in the Akt/mTOR pathway. Fig. 4 demonstrates that miR-147 expression repressed the phosphorylation of Akt, P70S6k and 4E-BP-1 in MDA-MB-231 cell, but appeared to have no effect on the total level of these proteins. Knockdown of Akt restrained both Akt phosphorylation and total Akt level, and the downstream phosphorylation of P70S6k and 4E-BP-1. The total P70S6K level was slightly upregulated in the ‘si-Akt’ group. Generally, the effect of miR-147 on Akt/mTOR pathway was analogous to that of siRNA directed at Akt.