Bioinformatics prediction
TargetScan, miRanda, and miRDB were used to predict putative miRNA that potentially targets NRARP. As shown in Fig. 2(a), with a perfect seed match model (7mer-m8), TargetScan indicated that miR-130a-3p potentially block NRARP. Other algorithms also predicted miR-130a-3p as a potential regulator of NRARP.
Cell Lines and Culture
The MCF-7 and SKBR3 human breast cancer cell lines, as well as MCF-10A as a non-cancerous breast epithelial cell line, were obtained from the National Cell Bank of Iran (NCBI, Tehran, Iran). To verify that the cells are free of mycoplasma, a PCR test was performed. In order to culture BC cell lines, complete cell culture medium containing high-glucose Dulbecco's modified eagle medium (DMEM), 10% fetal bovine serum (FBS-Gibco, USA), 100 units/ml penicillin, and 100 g/ml streptomycin was used. A medium containing 10% horse serum (HBS-Gibco, USA) was utilized for the growth of MCF-10A cell line. The cells were incubated at 37 °C with 90% humidity and 5% CO2. Every 24 hours, the cells were observed under an inverted microscope for cellular density, morphology, and contamination.
RNA Extraction
To assess the mRNA expression of miR-130a-3p, NRARP, and Wnt/β-catenin pathway genes in BC cells or normal breast epithelial cells, the cellular RNA was isolated using hybrid-RTM RNA extraction kit (GeneAll, Korea) according to the manufacturer's protocol. Briefly, the cells were lysed with 1ml of RiboExTM lysis buffer for 5 minutes. Next, the cells were transferred to a microtube and each tube was mixed intensely with 0.2 ml of chloroform for 15 seconds and then centrifuged for 15 minutes at 12000 × g for phase separation. Afterward, the upper phase was moved to a small spin column. The column was centrifuged for 30 seconds (~ 10000 x g), and then passed-through material was removed. The spin column was washed twice using wash solutions, and then RNase-free water was used to elute the purified RNA. The cellular RNA isolated from MCF-10A was considered as control sample. NanoDrop (Aosheng, China) and denaturing agarose gel electrophoresis were used to determine the concentration and integrity of isolated RNAs, respectively. Extracted RNAs were stored at -80 °C for future experiments.
Synthesis of cDNA and qRT-PCR
In a reaction catalyzed by the RevertAid enzyme (Thermo Scientific, USA), cDNA was synthesized from isolated RNA in the presence of stem-loop primers for miR-130a-3p and SNORD47, and random hexamer primers for genes. The StepOne plusTM thermal cycler (ABI, USA) was used to perform qRT-PCR using SYBR Green fluorescent dye and primers listed in Table1. The PCR was carried out for 10 minutes at 95°C for initial denaturation. After that, 40 cycles of 95°C for 15 seconds, 60°C for 40 seconds, and 72°C for 50 seconds were performed. At the end of the experiment, the qRT-PCR amplification efficiency and cycle threshold (Ct) was analyzed using LinRegPCR program (J.M. Ruijter, Version 1.2). Expression values were normalized using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference gene for the NRARP and Wnt/β-catenin pathway genes, and SNORD47 (U47) for the miR-130a-3p, and presented as a relative expression using the 2 –ΔΔCt method.
Transient transfection
The miR-130a-3p miRNA mimics or scramble miRNA as negative control were purchased from Exiqon-Qiagen (Germany). Transfection of these sequences (5nM) into BC cell lines was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. In brief, MCF-7 or SKBR3 cells from low passage were seeded in plates (24-well) and incubated at 37 °C until 90% confluence was achieved. The complete medium in each well was then changed with FBS-free medium. Following that, the cells were exposed to respective sequences. Cells were processed two days after transfection for further testing. Non-transfected MCF-7 or SKBR3 cells (untreated cells) were investigated as control.
qRT-PCR after transfection
Total RNA was extracted, cDNA was synthesized, and qRT-PCR was performed on either transfected or untreated cells, as described above. For normalization of qRT-PCR data, SNORD47 and GAPDH were used as reference genes.
Proliferation assay
The proliferation of BC cells after miR-130a-3p restoration was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In brief, BC cells were dissociated and suspension of 3 × 103 cells for each well were transferred into a 96-well plate. The cells were transfected as described above or left untreated, and then were further grown for 24, 48, and 72 hours. Following that, the cells were incubated in the MTT reagent (0.05 g/ml in PBS) for 3 hours at 37 °C in order to develop formazan crystals. In the next step, the formazan crystals were dissolved using dimethyl sulfoxide (DMSO), and color intensity was quantified spectrophotometrically at 570 nm after 5–10 minutes shaking. Non-transfected MCF-7 or SKBR3 cells were investigated as control.
Soft agar colony formation assay
The ability of BC cells to grow anchorage-independently in response to miR-130a-3p restoration was tested using a soft agar colony formation assay, which evaluates cellular transformation in vitro [16]. First, the cells were exposed to respective treatments as described above or left untreated. Next, the experiment was carried out in a 6-well plate with two layers of agar; the bottom layer containing 0.8% agar and 2X cell culture medium in a 1:1 ratio, and the upper layer containing 0.5% agar and the suspension of cells (5 ×103 cells for each well) in a 1:1 ratio. After chilling and forming agar layers, plates were transferred to 37 °C temperature and 5% CO2 incubator. After 3 weeks, each well was stained with 0.2 ml of nitroblue tetrazolium chloride solution. The plates were then incubated at 37 °C for 24 hours, and the colonies were counted.
Scratch test
Wound closure or scratch test is a type of whole-cell mass migration that is commonly used by researchers to determine the migratory potential of cancer cells [17]. For performing the scratch test, the cells were seeded at a density of 1 × 105 cells per well in a 24-well plate and grown to form a cell monolayer. The scratch was created on the cell layer using a pipette tip. Following that, the cells were washed thoroughly twice with PBS, and a low-FBS medium (0.5 %) was added to the wells. The first image of the scratch area was captured after it was formed and viewed under an inverted microscope, and subsequent images in the same area were captured at regular time intervals for up to 48 hours. ImageJ software (v1.8.0, NIH, USA) was used to quantify snapshots, and the following formula was used to determine the migration area:
Migration Area = (the scratch area measured after the scratch has formed) – (the scratch area measured at regular time intervals)
In vitro cell migration assay
The ability of BC cells to migrate in response to attractant was appraised using an 8 μm pore transwell plate (SPL, Life Bioscience, Korea). To do so, at first. the cells were exposed to respective treatments as described above or left untreated. Next, the upper compartment of the insert was filled with 0.2 ml of FBS-free cell solution (5 × 104 cells per well), and to the well of the plate (bottom of the lower chamber), 0.7 ml DMEM supplemented with 10% FBS as a chemoattractant was added. The cells were incubated for 24 hours at 37 ºC, and then the cells that migrated from the upper to lower surface of the membrane were fixed with 70% ethanol (Sigma, Germany), exposed to the 0.05% crystal violet (Merck-Darmstadt, Germany), randomly different views were chosen, and the average of the cells per views was computed. Non-transfected MCF-7 or SKBR3 cells were investigated as control.
Statistical analysis
GraphPad Prism version 8.0.1 (GraphPad, La Jolla, CA) was used for statistical analysis. For multiple comparisons, analysis of variance (ANOVA) test was employed followed by Tukey post hoc tests. The results are presented as the mean ± SD of three independent experiments, and the P-value of less than 0.05 was considered statistically significant.