Patient tissue specimens
Forty paired CRC tissues and matched adjacent nontumour tissues were obtained from patients after receiving surgical resection at The First Affiliated Hospital of Zhengzhou University. None of the patients received any preoperative chemotherapy or radiotherapy. Survival was calculated by months. Overall survival (OS) was defined as the time from tumour excision to death by any cause. Pathological diagnoses of colorectal cancer were determined by three pathologists. The tumour stage was determined according to the eighth edition of the International Union Against Cancer (UICC)/American Joint Committee on Cancer (AJCC) TNM classification . All patients signed informed consent forms, and this protocol was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University.
Cell Culture, Transfection And Stable Cell Line Construction
HCT116 cells were obtained from iCell Bioscience Inc. (Shanghai, China) and authenticated by STR before use. SW480 cells were obtained from the Biotherapy Centre of The First Affiliated Hospital of Zhengzhou University. All cells were cultured in DMEM (high glucose) (HyClone, Logan, Australia) with 10% foetal bovine serum (BI, Israel), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C and 5% CO2. A plasmid containing the precursor sequence of miR-103a-3p (pre-miR-103a-3p) was obtained from Vigene (Rockville, MD). The pMIF-cGFP-ZEO/miR-103a-3p plasmid, pSilencer-1-cGFP/miR-103a-3p shRNA plasmid, and siRNAs were obtained from RiboBio (Guangzhou, China). The pLVX-TRE3G-ZsGreen1/YAP1, pLVX-TRE3G-ZsGreen1/HIF1A and pLVX-TRE3G-ZsGreen1/TEAD1 plasmids were purchased from Vigene, and the corresponding lentiviruses/shRNAs were purchased from Vigene. The psiCHECK-2-LATS2-3′-UTR and psiCHECK-2-SAV1-3′-UTR WT plasmids and the corresponding mutants were purchased from Vigene. According to the manufacturer’s instructions, Lipofectamine 3000 (Invitrogen, ThermoFisher Scientific, Carlsbad, CA, USA) was used for transient siRNA and plasmid transfection. MiR-103a-3p, HIF1A, YAP1, and TEAD1 siRNAs were purchased from RiboBio. The siRNA sequences are shown in Table S1. MiR-103a-3p mimics and inhibitors were stably transfected into SW480 cells and HCT116 cells, respectively. Then, the cells were cultured with puromycin to obtain stable cell lines.
RNA Extraction And Quantitative Real-time PCR
Total RNA was extracted from cells and tissues with RNAiso Plus reagent (Takara, Dalian, China) according to the manufacturer’s instructions. The concentration and purity of RNA were detected using a NanoDrop 2000 (Thermo Scientific, USA). First-strand cDNA was synthesized from 1 µg of total RNA using the Prime Script RT Master Mix Kit (Takara), and real-time PCR was performed using GoTaq qPCR Master Mix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The primers are listed in Table S1. GAPDH or U6 was used as an endogenous control for normalization.
Total proteins were extracted by RIPA buffer supplemented with PMSF (Solarbio, Beijing, China) and quantified by a BCA kit. Then, the proteins were separated in SDS-PAGE gels and transferred into PVDF membranes (Millipore, Massachusetts, USA). Membranes were blocked with TBST with 5% skim milk powder and incubated overnight at 4 °C with primary anti-P-YAP, VEGFA, HK2, LDHA, and HIF1A antibodies (Proteintech, Wuhan, China). The next day, blots were washed with PBS and then incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Secondary antibodies were incubated with the membrane at room temperature for 1 h. The membrane was visualized using a chemiluminescence kit (Absin, Shanghai, China) and quantified by densitometry analysis using ImageJ software. GAPDH and tubulin were used as loading controls.
The transfected HCT116 and SW480 cells were seeded in 12-well plates. After the cells were grown to 80–90% confluence, scratch wounds were generated by a 10 µl plastic pipette tip, which was recorded as 0 h. Cell migration was assessed by measuring the movement of cells into the scratch wounds. Then, the scratch was imaged at 24 h, 48 h, 72 h, and 96 h. Wound width was measured with an ocular ruler to ensure that all wounds were the same width at the beginning of each experiment.
To assess the migration and invasiveness of HCT116 and SW480 cells, we used Transwell chambers (Corning, NY, USA). Briefly, approximately 3 × 105 cells in serum-free medium were seeded in the upper chambers with 8 µm pore size membranes to perform the migration assay in the absence of Matrigel (Corning, NY, USA) and the invasion assay with Matrigel. Dulbecco’s modified Eagle’s medium (500 µl) supplemented with 10% foetal bovine serum was added to the lower chamber. After incubation in a humidified atmosphere containing 5% CO2 at 37 °C for 72 h, the migrated cells were fixed, and the other cells were wiped off. Then, the migrated cells were stained by Giemsa (Solarbio, Beijing, China). Stained cells were imaged under an IX53 inverted microscope (Nikon, Tokyo, Japan), and the Image-Pro Plus software programme (Media Cybernetics, Rockville, MD) was used to count the cells.
Cell Proliferation Assay
Cells were seeded in 96-well plates at 0.8 ~ 1 × 103 per well. Cell proliferation was evaluated using Cell Counting Kit-8 (Dojin Laboratories, Tokyo, Japan) according to the manufacturer's instructions. We collected cell samples at 24 h, 48 h, 72 h, 96 h, and 120 h. Then, 10 µl of CCK-8 solution was added to the culture medium and incubated for 2 h at 37 °C. Viable cells were evaluated by measuring the absorbance at 450 nm with a reference wavelength of 570 nm.
5-Ethynyl-2'-deoxyuridine Assay (EdU)
A total of 4 × 103 cells per well were seeded into 96-well plates, cultured overnight, washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and incubated with 2 mg/ml glycine for 5 min. Based on the kFluor488-EdU (5-ethynyl-2′-deoxyuridine) manufacturer’s instructions (RiboBio), 200 µl of 1 × Apollo dyeing solution was added to each well, followed by incubation at room temperature for 30 min. Next, 100 µl of 0.5% Triton X-100 was used to wash the cells two to three times (10 min per wash). Following staining with Hoechst 33342 at room temperature for 30 min in darkness and one or two washes with PBS, the cells were observed using a Micro imaging system (ImageXpress, Downingtown, PA, USA). Five fields were randomly selected and imaged, and the number of EdU-positive cells was calculated.
Tube Formation Assay
Twenty-four-well plates were coated with 60 ml Matrigel (BD Biosciences, USA) at 37 °C for 1 h for gel formation. A total of 1 × 105 stably transfected cells in medium containing 10% FBS were plated into the pre-solidified Matrigel and allowed to start the process of forming capillary tubes and networks once seeded on Matrigel. Six hours after incubation, the plates were observed under a microscope and imaged (Nikon, Japan). The numbers of branching points generating at least three tubules were counted.
Dual-luciferase Reporter Assay
Luciferase activity assays were performed with the Dual-Luciferase Reporter Assay System (Promega, Beijing, China). Validation of miRNA targets was performed by cloning partial LATS2 (SAV1) 3′-UTRs containing the sequence recognized by the miR-103a-3p seed sequence. HCT116 cells and HEK293 T cells were cotransfected with miR-103a-3p mimics, the Renilla luciferase reporter vector (Promega), and either wild-type (WT) or mutant LATS2 (SAV1) reporter constructs using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s instructions. After 48 h of transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Correction for differences in transfection efficiency was performed by normalizing firefly luciferase activity to total Renilla luciferase activity.
HCT116 cells transfected with miR-103a-3p inhibitors were fixed by 4% paraformaldehyde and permeabilized by 0.1% Triton X-100 in PBS for 10 min. The cells were blocked with 5% BSA for 30 min at 37 °C and incubated with primary antibody (YAP1) overnight at 4 °C. The next day, the cells were washed with PBS and then incubated with the corresponding secondary antibody for 30 min at 37 °C, followed by nuclear staining with DAPI. Fluorescent images were acquired using an OLYMPUS FV1000 confocal microscope. The relative fluorescence densities were analysed by ImageJ and plotted using GraphPad Prism 6 software.
Chromatin Immunoprecipitation (ChIP)
ChIP assays were performed as described previously . Formaldehyde-fixed cells were immunoprecipitated overnight with anti-YAP1 and anti-TEAD1 antibodies (Santa Cruz, CA, USA) or rabbit IgG, and the associated genomic DNA was assessed by PCR and agarose gel electrophoresis. PCR was performed with HIF1A promoter-specific primers that amplified the YAP1/TEAD1 binding regions. The primers were HIF1A Forward 5′-TACTCAGCACTTTTAGATGCTGTT-3′ and Reverse: 5′-ACGTTCAGAACTTATCCTACCAT-3′.
Subcutaneous Xenotransplantation Model
All mouse procedures were approved by the Institutional Animal Care and Use Committee of Zhengzhou University. All BALB/c nude mice, 6 weeks old, were acquired from Vital River Laboratory (Beijing, China). Logarithmic phase HCT116 cells (1 × 106/100 µl) were inoculated subcutaneously into the dorsal flank. After 12 days, according to the completely randomized design using a random comparison table, the mice with xenograft tumours were randomly divided into two groups, intratumoural injection of miR-103a-3p antagomir group and negative control group, to examine tumourigenicity. These mice were treated with miR-103a-3p antagomir every 3 days. The tumour size was measured by a slide calliper, and tumour volume was evaluated by the following formula: volume =(D × d2)/2, where D was the longest diameter and d was the shortest diameter. All animals were sacrificed 32 days after inoculation, and the tumours were excised, weighed, fixed, and paraffin embedded for haematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining detected under a microscope. All specimens were examined under a light microscope (Nikon, Japan).
H&E and IHC staining of formalin-fixed paraffin-embedded xenograft tumour sections was performed with antibodies against the following antigens: P-YAP (1:1000), HK2 (1:5000), PKM1 (1:2000), PCNA (1:1000), and KI-67 (1:500) obtained from Cell Signalling Technology. The anti-goat IgG-HRP (Santa Cruz Biotechnology) secondary antibody was also used.
All statistical analyses were carried out with SPSS version 18.0 (MT, USA) and GraphPad Prism 5.0 software (CA, USA). Data are expressed as the mean ± SEM. All differences between two independent groups were evaluated by a two-tailed Student’s t-test. Survival curves were generated using the Kaplan–Meier method and compared using the log-rank test. The MedCalc software was used to generate the ROC curve, and the data were analyzed by two-tailed t test. Pearson’s coefficient was used to assess the correlation between two independent groups. The associations of miR-103a-3p expression and clinicopathologic variables were assessed by the Chi-square test or Fisher’s exact test. The indicated P values (*P < 0.05 and **P < 0.01) were considered statistically significant.