Patients and specimens
Human colorectal mucosal biopsies were obtained from healthy controls (n=29), active ulcerative colitis (n=49) and inactive ulcerative colitis (n=47) patients who underwent endoscopy examination at the Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). A total of 82 CRC tissues and their paired non-cancerous tissues were collected from The First Affiliated Hospital of Zhengzhou University. This research was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University and all patients have given written informed consent.
A panel of human colon cancer cell lines including SW620, LoVo, HT-29, Caco-2, HCT116 and SW480, as well as normal human colonic epithelial cell line CCD841 were purchased from the American Type Culture Collection (ATCC). HCT116 and Caco-2 cells were grown in DMEM medium replenished with 10% FBS (Fisher Scientific, USA) and 1% penicillin/ streptomycin. Cells were maintained under the standard condition (37°C, 90% humidity and 5% CO2) and have been confirmed without mycoplasma contamination. Cell culture dishes and plates were purchased from Hangzhou Xinyou Biotechnology Co., Ltd. (China). The inhibitors of JAK2/STAT3 signaling BP-1-102 and FLLL32 were purchased from Selleckchem (shanghai, China).
Plasmids construction and cell transfection
pLKO.1-SLC6A14 vector, pCDH-CMV-MCS-EF1α-SLC6A14 vector or their corresponding control vectors combined with packaging plasmids (ps-PAX2 and pMD2.G) were transfected into 293T cells using Lipofectamine 3000 (Invitrogen, CA, USA). After 48h, lentivirus-containing supernatants were collected, filtered and added into Caco-2 and HCT-116 cells accompanied with Polybrene (10 μg/mL). Thereafter, puromycin (5 μg/mL) was utilized to select stable SLC6A14-knockdown and SLC6A14-overexpression CRC cells for 7 days.
RNA extraction and qRT-PCR
Total RNA from tissues and cells were isolated by using RNAiso Plus reagent and quantified with a NanoDrop spectrophotometer. Then 1000 ng total RNA was reverse-transcribed to generate first-strand cDNA by using the PrimeScript™ RT Master Mix (TaKaRa, Japan) following the protocols of the manufacturer. Gene expression was analyzed by quantitative real-time PCR with SYBR Green Master Mix using a QuantStudio 7 Flex system. GAPDH and
β-actin were used as reference genes and all experiments were conducted in triplicate. The relative mRNA expression was calculated using the 2-ΔΔCt method [19, 20].
Immunohistochemistry, Ki-67 and TUNEL staining
Immunohistochemistry staining was carried out on paraffin-embedded colorectal tissue sections, which were deparaffinized in xylene, rehydrated in alcohol, and immersed in 3% hydrogen peroxide to prevent endogenous peroxidase activity. Afterwards, the slides were separately immunostained with the corresponding primary antibody at 4°C overnight. After rinsing, the sections were then incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. The slides were stained with DAB solution and analyzed with a light microscope (Olympus, Japan). For the expression and localization of Ki-67, the slides were deparaffinized, rehydrated and treated according to a standard protocol. After incubating with primary antibody against Ki-67 overnight at 4°C and corresponding secondary antibody, the sections were incubated with DAPI for 10 min to exhibit the location of the nucleus. Images of Ki-67 were captured by using an inverted fluorescence microscope. For TdT-mediated dUTP nick end labelling (TUNEL) staining, paraffin sections of the biopsies were immersed in xylene to deparaffinize and a series of decreasing concentrations of alcohol to rehydrate. Then the slides were treated with 3% hydrogen peroxide to block the activity of endogenous peroxidase, followed by the incubation with proteinase K. DNA fragmentation was analyzed by an In Situ Cell Death Detection Kit (Roche, Germany) according to the manufacturer’s instruction. TUNEL-positive cells were observed and captured with an Olympus microscope.
Cells and colorectal mucosal tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors (Beyotime, China). Total protein concentrations were measured by using a BCA protein assay kit (Thermo Scientific, USA). Equal amounts of cell protein samples (20 μg) and human tissue samples (50 μg) were loaded onto an SDS-PAGE gel and electrophoresed. Then the separated protein samples were transferred onto PVDF membranes (Millipore, USA). Then the membranes were blocked with 5% BSA in TBST buffer at room temperature for 1 hour and incubated with appropriate primary antibodies at 4°C overnight. After washing the membranes with TBST for three times, the membranes were incubated with corresponding HRP-conjugated secondary antibodies at room temperature for 1 hour. Finally, ECL chemiluminescence reagents were added to the membranes to magnify immunoreactive signals, which were subsequently detected and visualized by a Bio-Rad detection system.
Cell proliferation and colony formation assay
Cell growth rate was detected by using commercial Cell Counting Kit-8 reagent as previously described . CRC cells at a density of 1×103 per well were seeded into a 96-well plate with five repeats. After 24 hours’ incubation, cells in each well were added with 10 μl CCK-8 solution and 90 μl cell culture medium and incubated for another 2 hours at 37°C. Finally, the absorbance at 450 nm was detected with a multifunctional microplate reader for consecutive five days. For colony-formation experiments, about 500 cells resuspended in the complete medium were seeded into six-well plates and cultured in the incubator for consecutive 14 days. The medium change was conducted at three-day intervals. Finally, cells were fixed with 4% paraformaldehyde, washed with PBS and stained with crystal violet. Images of colonies were captured for further analysis.
Cell apoptosis analysis by flow cytometry
Annexin-V/propidium iodide (PI) staining is a typical flow cytometric method for detecting cell apoptosis . Cells at a density of 1 × 106 were collected and resuspended in 500 μl binding buffer containing 5 μl Annexin V and 10 μl PI. After incubation in the dark for 15 minutes, samples were analyzed immediately with a FACSCanto flow cytometer (BD Bioscience, USA).
Cell migration and invasion assay
Matrigel for invasion assay was thawed slowly on the ice at 4°C overnight. Chambers inserts for invasion assay were coated with a layer of diluted matrigel and dried at 37°C. For migration and invasion assays, CRC cells were grown and treated in the same manner as previously described. In brief, 1×105 CRC cells per well resuspended in serum-free media were plated into the upper chamber and 20% FBS-containing complete medium was added into the bottom room as a chemoattractant. After incubation at 37°C for 24 hours, the migrated cells were fixed with 4% paraformaldehyde and non-migratory cells in the upper chamber were removed by a sterile cotton swab. Then the attached migratory cells were stained with 0.3% crystal violet and quantified with a Nikon inverted microscope.
Animal studies and AOM/DSS-induced colorectal carcinogenesis model
All animal experimentation was conducted under the approval of the Laboratory Animal Care and Welfare Committee of Zhengzhou University. For the induction of Azoxymethane/ Dextran sulfate sodium (AOM/DSS) model, wild-type C57BL/6 male mice (6-8 weeks old) were administrated with a single dose of 12.5 mg/kg AOM (Sigma-Aldrich, USA) via intraperitoneal injection on the first day and recovered for next 5 days. Then the mice were treated with two cycles of 2.5% dextran sulfate sodium (DSS, MP Biochemicals, USA) for 5 days and regular drinking water for 14 days. After the final cycle of 2% DSS for 5 days and recovered for a month, the mice were euthanized. The colons were dissected longitudinally, polyps and tumors were blindly assessed and counted. During the whole period, mice were weighed and monitored every three days for bodyweight changes and other symptoms to evaluate CRC progression. For in vivo tumor xenograft model, female Balb/c nude mice (6-week-old) were purchased from Shanghai SLAC Laboratory Animal and maintained with free access to diet or water. The mice were divided into two groups at random and injected subcutaneously with equal OE-SLC6A14 and pCDH cells (106 cells). Tumor size was checked with a caliper once a week and recorded. After a month, the mice were euthanized, and tumors were isolated.
Experimental data were analyzed by GraphPad Prism 8.0 software and presented as mean ± standard deviation (SD). Differences between groups were analyzed by paired Student's t-test, unpaired Student's t-test. Chi-square test was used to analyze the relationship between SLC6A14 expression and clinicopathological parameters. Clinical correlations in human colorectal tissues were performed using Pearson's correlation coefficient. Overall survival curves were analyzed by using Kaplan-Meier method. Data for statistical analysis were from at least three representative independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered as statistical significance.