Clinical specimens.
In total 30 samples from patients diagnosed with CRC and 30 samples from healthy individuals, who were identified via health checks and did not exhibit any health concerns during their physical examinations, were collected from Beijing Huairou Hospital (Beijing, China) from January 2018 to December 2020. Samples were snap-frozen and stored at -80˚C until further use. Informed consent was obtained from each patient and ethics approval was granted by the Ethics Committee of Beijing Huairou Hospital. The present study was performed in accordance with 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.
Cell culture and transfection.
The human colon cancer HCT116 and SW480 cell lines were purchased from the American Type Culture Collection and were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), HEPES (20 mM), streptomycin (100 µg/ml) and penicillin (100 U/ml). Cells were incubated in a humidified atmosphere at 37˚C with 5% CO2. All transfections were performed using lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol.
Plasmid construction.
The miR-561-3p mimic, the miR control, miR-561-3p inhibitor [miR-561-3p-antisense oligonucleotide (ASO)] and the normal scrambled negative control (NC; ASO-NC) were purchased from Shanghai GenePharma Co., Ltd. The uPAR overexpression vector was constructed using the coding sequence region of the uPAR gene, which was cloned into the pcDNA3.1 vector.
The small interfering RNA-uPAR was annealed and inserted into the plasmid of the pSilencer2.1-U6 neo vector. The uPAR 3’UTR containing the miR-561-3p wild-type (WT) or mutant (MUT) binding sites was annealed and cloned into the pmirGLO luciferase vector (Promega Corporation). All the constructed plasmids were confirmed via DNA sequencing.
Reverse transcription-quantitative PCR (RT-qPCR).
Total RNA was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The experimental procedure of RNA extraction and RT-qPCR used in the present study was as previously described (Wan et al., 2014; Zhao et al., 2015). All primers are presented in Table I.
Dual-luciferase reporter assay. HCT116 cells were seeded into 48-well plates prior to transfection. Subsequently, the cells were co-transfected with the WT or MUT uPAR-3’UTR luciferase reporter and the miR-561-3p mimic, miR-561-3p-ASO or the corresponding control respectively. Luciferase activity was determined using the Dual-Glo Luciferase Assay System (Promega Corporation) according to the manufacturer’s protocol.
MTT and colony formation assays.
The MTT and colony formation assays were performed according to the manufacturer’s protocols, which were described previously (Wan et al., 2014; Zhao et al., 2015).
Migration and invasion assays.
Transfected HCT116 and SW480 cells (1x105 cells) in serum-free medium were seeded into the upper chamber, either precoated with Matrigel for 2 h at 37˚C or uncoated. Medium with 20% FBS was added to the lower chamber. Following 24 h incubation, cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Subsequently, cells were quantified in five random visual fields using a light microscope.
Cell apoptosis assay.
Transfected HCT116 and SW480 cells were seeded into six-well plates and treated for 24 h before being serum-starved for a further 24 h. The cells were then harvested. Apoptosis analysis was performed using the Annexin V-FITC Apoptosis Detection Kit II (BD Biosciences) according to manufacturer’s protocol.
Bioinformatics.
miR-561-3p and target uPAR were identified using miRecords online software (http://miRecords.umn.edu/miRecords.)(Xiao et al., 2009).Among the potential miRNAs, miR-561-3p was chosen as the miRNA that markedly affected the growth phenotype of CRC cells based on previous data.
Western blotting.
Western blotting was performed to detect protein expression levels using previously described methods. The antibodies used were as follows: anti-GAPDH (Catalog No: SRP13406, 1:3,000 dilution), uPAR (Catalog No: SRP10752, 1:200 dilution), phosphorylated (p)-AKT (Catalog No: ab278559, Abcam, UK, 1:300 dilution), AKT (Catalog No: SRP00437, 1:300 dilution), p-PI3K (Catalog No: ab182651, Abcam, UK, 1:300 dilution) and PI3K (Catalog No: SRP01115, 1:300 dilution). The antibodies were used to incubate at 4°C overnight. Anti-GAPDH/uPAR/AKT were purchased from Tianjin Saierbio Technology, Inc.
Statistical analysis.
Statistical analysis was performed using Prism 6 (GraphPad Software, Inc.). The two-tailed unpaired Student’s t-test or Mann Whitney U test were performed to make statistical comparisons for continuous variables and the χ2-squared test was used for categorical variables. All data are presented as the mean ± SD of at least three experimental repeats. P ≤ 0.05 was considered to indicate a statistically significant difference.