Cell culture
LNCaP, DU145, PC-3 and RWPE-1 (American Type Culture Collection, Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and penicillin (100 U/ml). The cells were cultured at 37°C with 5% CO2.
Plasmid construction and transfection
A miR-30c expression vector was constructed by cloning the fragment, amplified from human genomic DNA by polymerase chain reaction (PCR) with the primers, hmiR30c-EcoRI (forward, 5′-CCGGAATTCAACATAGTGTGGGGATGGGGT-3′) and hmiR30c-BamHI (reverse, 5′-CGCGGATCCAGGTTAATGGGAAACAGGGCT-3′) into the EcoRI and BamHI sites of the pLV-mCherry(2A)puro vector. The pLV-mCherry(2A)puro vector and packaging vector were co-transfected into HEK293T cells (American Type Culture Collection) to obtain high titer virus particles with replication defects. For lentiviral infection, cells were plated at a concentration of 1×105 cells/well onto 6-well culture plates and then infected with indicated lentiviruses in the presence of 8 µg/ml polybrene. Following infection for 72 h, puromycin was added to the medium at a concentration of 0.5 µg/ml for PC-3 cells and cell populations were selected for 2 weeks for following experiments.
Isolation of total RNA and quantitative PCR (qPCR)
Total RNA was extracted from collected cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and mRNA was then reverse transcribed to cDNA (Promega Corporation, Madison, WI, USA). U6 small nuclear RNA was used for normalization. The PCR reactions were performed with the following primers: hsa-miR-30c forward, 5′-CCCGCTGTAAACATCCTACACTC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; and U6 forward, 5′-CGCTTCGGCAGCACATATAC-3′ and reverse, 5′-CAGGGGCCATGCTAATCTT-3′. qPCR was performed using the ABI 7900 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.).
Cell cycle analysis
The cell cycle was assessed at 24 h following transfection by propidium iodide staining, and measured with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). MTT PC-3 cells were trypsinized and seeded onto 96-well plates at a density of 3×103 cells/well for culture. Cell proliferation was measured using the MTT assay 24, 48 and 72 h after seeding. Briefly, 10 µl of MTT solution (5 mg/ml) was added to each well, and the cells were incubated for 3 h at 37°C. The medium was then aspirated, and 150 µl of dimethyl sulfoxide was added. The optical density was measured at 570 nm with a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Three independent experiments were performed in quadruplicate. The cell growth inhibition rate (%)=(1-value of experimental group/negative control group) ×100.
Colony formation assay
The two cells types were harvested and 1,000 cells were inoculated in each pore of a 6-well plate. The medium was changed every 2 days. After 14 days, cells were rinsed with 1X PBS and then fixed with methanol for 10 min. Subsequently, cells were stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for >1 h.
Wound healing assay
The cells were seeded onto 6-well plates and cultured with RPMI-1640 medium. Following 24 h, the cells were wounded with a 200 µl pipette tip. Serum-free RPMI-1640 medium was added and wound closure was observed for 30 h using an XSP-4C microscope at a magnification of ×200 (Shanghai Changfang Optical Instrument Co. Ltd., Shanghai, China).
Transwell assay
Cell motility was measured using an 8 µm pore polycarbonate membrane Boyden chamber insert in a Transwell apparatus (EMD Millipore, Billerica, MA, USA). The transfected cells were treated with 0.2% trypsin/EDTA solution and washed once with serum-containing RPMI-1640 medium. A total of 1.5×105 cells in 0.2 ml serum-free RPMI-1640 medium were seeded onto a Transwell apparatus. RPMI-1640 medium containing 10% FBS (600 µl) was added to the lower chamber. An invasion assay was conducted following the aforementioned procedure, with the exception that the filters of the Transwell chambers were coated with 45 µg Matrigel (BD Biosciences). Following incubation of the cells for 24, 48 and 72 h at 37°C in a 5% CO2 incubator, the cells on the top surface of the insert were removed by wiping with a cotton swab. The cells that invaded to the bottom surface of the insert were fixed in the 100% precooling methanol for 10 min, stained with 0.1% crystal violet for 30 min, rinsed in PBS and then subjected to microscopic inspection (XSP-4C microscope; magnification, ×200). The values for invasion were obtained by counting three fields per membrane and were represented as the average of the three independent experiments.
Western blot analysis
The total proteins were prepared from the established cells. The proteins were fractionated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane (EMD Millipore), blocked in 5% dry milk at room temperature for 1 h and immunostained with antibodies at 4°C overnight using anti-KRAS (dilution, 1:1,000; #sc-30; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-GAPDH (dilution, 1:5,000; #sc-47724; Santa Cruz Biotechnology, Inc.). All results were visualized through an electrochemiluminescence substrate western blotting detection system (Pierce; Thermo Fisher Scientific, Inc.) and then exposed by the Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Inc.). The integrated density of the band was quantified by ImageJ software v1.37 (Bio-Rad Laboratories, Inc.).
Statistical analysis
The 2−∆∆Cq method was used to analyze the results of qPCR in all the experiments performed in the present study (21). Statistical analysis was performed using SPSS 10.0 (SPSS, Inc., Chicago, IL, USA) and presented with GraphPad prism software v6.01 (GraphPad Software, Inc., La Jolla, CA, USA). The results obtained from experiment in vitro assays are presented as the mean ± standard deviation from at least 3 independent experiments, and the data were analyzed by the t-test. P<0.05 was considered to indicate a statistically significant difference.