Colorectal cancer tissues and adjacent normal tissues from the same patients were collected from the Endoscopy Centre, Tianjin Nankai Hospital. This study was approved by the Clinical Trial Ethics Committee of Tianjin Nankai Hospital (Approval No: NKYY_YX_IRB_2018_039_01), and informed consent was obtained from all patients before their enrollment. All tissues (17 cases) were subjected to histology and pathology, and all samples were immediately frozen in liquid nitrogen at the time of surgery and stored at − 80 °C before use.
The human CRC cell lines HCT116 and RKO and HEK293T were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), streptomycin (100 μg/mL) and penicillin (100 μg/mL). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
Vector construction and establishment of stable cell lines
Human colorectal cancer HCT116 and RKO cell lines stably expressing miR-3065-3p (HCT116-miR-3065-3p and RKO-miR-3065-3p) or negative control (HCT116-NC and RKO-NC), HCT116 cell lines stably expressing CRLF1 (HCT116-CRLF1) or vector control (HCT116-MCS) were established by using a lentivirus transfection system according to the manufacture’s instruction (Biosettia, San Diego, CA, USA).
HCT116 and RKO human colorectal cancer cells were transfected with miR-3065-3p mimics or inhibitor using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. The cells were collected for 48 h after transfection. miR-3065-3p mimics or inhibitor, negative control (NC) miRNA and inhibitor negative control (I.NC) were synthesized by RiboBio (Guangzhou, China).
Real-time quantitative PCR (RT-qPCR)
Total RNA was extracted from the cultured cells and tissues using TRIzol reagent (Invitrogen), and reverse transcription was performed using the TransScript First-Strand cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China) according to the manufacturer’s recommendations. RT-qPCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems Thermo Fisher). U6 small nuclear RNA (snRNA) was used as an internal control for miR-3065-3p, and the mRNA levels were normalized to those of GAPDH. The relative gene levels normalized to the control were calculated using the equation 2−ΔΔCT, ΔCT = CTgene − CTU6/GAPDH, ΔΔCT = ΔCTtest − CTctrl. Primers were designed and synthesized, and the sequences are as follow: hsa-miR-3065-3p, forward: 5ʹCGTCAGCACCAGGATATTG3ʹ and reverse: 5ʹGTGCAGGGTCCGAGGT3ʹ; hsa-U6, forward: 5ʹCTCGCTTCGGCAGCACATATACT3ʹ and reverse: 5ʹACGCTTCACGAATTTGCGTGTC3ʹ; homo-NANOG, forward: 5ʹTCTGGACACTGGCTGAATCCT3ʹ and reverse: 5ʹCGCTGATTAGGCTCCAACCAT3ʹ; homo-OCT4, forward: 5ʹGCTCGAGAAGGATGTGGTCC3ʹ and reverse: 5ʹCGTTGTGCATAGTCGCTGCT3ʹ; homo-SOX2, forward: 5ʹGCCTGGGCGCCGAGTGGA3ʹ and reverse: 5ʹGGGCGAGCCGTTCATGTAGGTCTG3ʹ; homo-TMEM47, forward: 5ʹTTGGACATCTGGCACTGCGAGT3ʹ and reverse: 5ʹCCTTCGAGATCCCACGCAGATA3ʹ; homo-CRLF1, forward: 5ʹCCCAGAGAAACCCGTCAACAT3ʹ and reverse: 5ʹACTGTGTGGTACTCCTCACAT3ʹ; homo-CLDN11, forward: 5ʹGGCTGGTGTTTTGCTCATTCTGC3ʹ and reverse: 5ʹAGCACCAATCCAGCCTGCATAC3ʹ; homo-GAPDH, forward: 5ʹCTCTGATTTGGTCGTATTGGG3ʹ and reverse: 5ʹTGGAAGATGGTGATGGGATT3ʹ.
RIPA lysis buffer (Sigma, St. Louis, MO, USA) including protease inhibitor cocktail (Roche, Mannheim, Germany) was used to lyse cells and tissues for total protein extraction. The concentrations of the extract proteins were measured by the BCA kit (SolarBio, Beijing, China). Protein lysates were electrophoresed on 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The immunoblots were blocked with 5% fat-free dried milk in TBST at room temperature for 1 h and incubated at 4 °C overnight with primary antibodies against NANOG (ab80892, 1:1000, Abcam, Cambridge, MA, USA), OCT4 (ab19857, 1:1000), SOX2 (ab97959, abcam, 1:1000), CRLF1(ab211438, abcam, 1:1000), and β-Actin (ab8226, abcam, 1:5000). The membranes were subsequently incubated with HRP-linked secondary antibodies (ZB-2301 or ZE2305, 1:5000; ZSGB-BIO, Beijing, China). Bound antibodies were detected with enhanced chemiluminescence reagent (Millipore).
Transwell migration assay
A total of 1 × 105 cells were suspended in serum-free medium and seeded into upper Transwell chambers (8 μm pore size, 24-well plate). The bottom chamber was filled with 500 μL of medium containing 10% FBS. After incubation for 24 h, the inserts were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and stained with 0.1% crystal violet staining solution. Nonmigratory cells were removed from the upper chamber with cotton swabs, and migrated cells were visualized using an Olympus microscope (Olympus Co., Tokyo, Japan) and quantified in five fields per chamber under blinded conditions [21,22,23].
Wound healing assay
A total of 1 × 106 cells in 2 mL DMEM containing 10% FBS were seeded in each well of a 6-well plate. After the cells were incubated for 24 h, a “wound” was generated by scratching the cells vertically with a 10 μL pipette tip. The floating cells were gently removed with PBS and then cultured with DMEM containing 2% FBS. The wound healing process was assessed at 0 h, 24 h and 48 h using an Olympus microscope (Olympus Co., Tokyo, Japan). The wound healing area was measured in ImageJ software.
Sphere formation assay
A total of 1 × 103 cells were seeded in ultralow attachment 6-well plates in 3 mL serum-free DMEM supplemented with 1 × B27 (1:50, Invitrogen), 20 ng/mL human epidermal growth factor (Invitrogen), and 20 ng/mL basic fibroblast growth factor (Invitrogen). Spheres larger than 50 μm in diameter were counted after 12 days [12, 24].
An ALDEFLUOR™ Kit was purchased from Stemcell Technologies. A total of 5 × 105 cells were seeded in 6-well plates and then transiently transfected with miR-3065-3p inhibitor or CRLF1 plasmid. The cells were collected after transfection for 48 h, and the Aldefluor assay was performed according to the manufacturer’s instructions. Stained cells were analyzed on a FACS flow cytometer (Beckman Coulter, Inc.). DEAB, a specific ALDH inhibitor, served as a negative control.
Dual-luciferase reporter assay
The CRLF1 3ʹUTR containing a predicted miR-3065-3p-targeting seed region or corresponding mutant seed region was inserted into the pmirGLO reporter vector (Promega, Madison, WI, USA) to obtain the pmirGLO-CRLF1 3ʹUTR-WT and the pmirGLO-CRLF1 3ʹUTR-Mut plasmid. To determine the direct targeting relationship between miR-3065-3p and the CRLF1 3ʹUTR, HEK293T cells were cotransfected with miR-3065-3p mimics or scramble negative control (NC) and the wild-type, mutant or empty reporter plasmid [12, 25]. Luciferase activity was measured 40 h later and normalized to Renilla luciferase activity using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol.
To establish a tumor xenograft mouse model, twenty in total 6-week-old female BALB/c nude mice were randomly divided into four groups with five in each group. HCT116 cells (1.5 × 107) expressing negative control (NC), miR-3065-3p, vector control (MCS) or CRLF1 were subcutaneously injected into the dorsal flanks of mice. Tumor volume was measured twice a week and calculated using the standard formula: length × width2/2. The mice were sacrificed one month after injection, and tumor tissues were obtained for further analysis. To construct a mouse model of liver metastasis, cells (3 × 106) were injected into the spleens of mice (five in each group). Liver tissues were obtained 4 weeks after injection, fixed with 4% PFA and stained with hematoxylin and eosin. All animal experiments were performed strictly according to the guidelines for laboratory animals of Tianjin Nankai Hospital and approved by the Institutional Ethics Committees of Tianjin Nankai Hospital (Approval No: NKYY-DWLL-2020-001).
Hematoxylin–eosin (HE) and immunohistochemical (IHC) staining
Tumors and livers tissues were fixed in 4% PFA, embedded in paraffin, sectioned, and then stained. H&E-stained sections were independently evaluated at 40× and 100× magnification by two pathologists who were blinded to the experimental conditions. IHC-stained sections were processed with an SP Kit (Ovitalin-Biotin Detection System for Streptomyces Rabbits) purchased from ZSGB-BIO according to the manufacturer’s protocol. Positive signals were detected by using DAB color developing agent. Images were captured using a Leica microscope. The protein expression level was determined by measuring the mean densitometry and assessed with ImageJ software. By measuring the integrated optical density (IOD) and area of each image, the average optical density (mean density = IOD/area) was calculated, which reflects the per unit area concentration of the target protein. The signal density of the tissue areas was measured in at least three sections.
RNA-seq data of colorectal cancer patients from TCGA database using the UALCAN (http://ualcan.path.uab.edu/) were used for miR-3065-3p or CRLF1 expression analysis . Kaplan–Meier analysis was performed using the OncomiR tool (http://www.oncomir.org/) . miRNA target prediction and analysis were performed using algorithms from TargetScan (http://www.targetscan.org/) , miRDB (http://www.mirdb.org/) , and GEPIA (http://gepia.cancer-pku.cn/) .
All data shown are representative of at least three independent experiments and are presented as the mean ± SEM. Student’s t-test or nonparametric test was used to compare two independent groups and paired groups. One-way ANOVA followed by the Tukey–Kramer multiple comparisons test was performed to compare three or more groups within the same experiment. Pearson correlation coefficients were used to assess the degree of association between biomarkers. All statistical analyses were computed and are presented via GraphPad Prism 8 software. The results were considered statistically significant when *P < 0.05, **P < 0.01, and ***P < 0.001.