Cell culture and transfection
Human colon cancer cell lines HCT116, HT29, SW620 and SW480 were purchased from ATCC (USA). HCT116 and SW620 cells were cultured in RPMI 1640 medium (Gibco, USA), HT29 cells and SW480 cells were cultured in DMEM/F12 (Gibco, USA). All cells were cultured in medium supplemented with 10% FBS (Gibco, USA) in humidified air at 37 °C with 5% CO2. The comparison of Cldn7 knockdown efficiency between three lentiviral shRNA vectors (sequence #1:5′-ATGGGTGGAGGCATAATTT-3′; sequence #2: 5′-CTAAGTCCAACTCTTCCAA-3′; and sequence #3: 5′-GCTCCTATGCGGGT GACAA-3′) was documented in our previous studies . We selected lentiviral shRNA2 with the highest transfection efficiency and the control vector (sequence: 5′-TTCTCCGAACG TGTCACGT-3′) to transfect HCT116CD133+CD44+ cells. The cells were collected after transfection for qRT-PCR and Western blot analysis.
Cell suspensions of HCT116 cells in logarithmic growth phase were collected, washed and centrifuged. PE-labelled anti-CD133 (eBioscience, USA) and FITC-labelled anti-CD44 (Biotech, China) antibodies were then added and incubated for 20 min at room temperature. Cell suspensions without added antibodies were used as a control. The cells were resuspended and subjected to flow cytometry analysis or flow sorting. CD133- and CD44-positive cells were sorted and cultured in complete medium containing penicillin and streptomycin (Gibco, USA).
Cell proliferation assay
Cell proliferation assays were performed using a cell counting kit (CCK-8) assay (Dojindo Laboratories, Japan). Cldn7 knockdown HCT116CD133+CD44+ cells and control cells were seeded in 96-well plates at a density of 2 × 103 cells per well. After 24, 48, 72 and 96 h, CCK-8 reagent (10 μL/well) was added to each well, and the cells were incubated with the reagent for 2 h at 37 °C. The absorbance of the cells at 450 nm was then measured using a microplate reader.
Cell migration assay
Cldn7 knockdown HCT116CD133+CD44+ cells and control cells were seeded evenly into 6-well plates. After the bottom of the well was covered with cells, the cell monolayer was lightly scraped using a 10-μL pipette tip to form a scratch. After washing away the floating cells with PBS, the cells were further cultured in serum-free medium at 37 °C. At 0, 6, 12, and 24 h, scratch wound healing was observed with an inverted microscope and photographed.
Cell apoptosis assay
Apoptosis assays were performed using a Caspase-Glo® 3/7 kit (Promega, USA). Cldn7 knockdown HCT116CD133+CD44+ cells and control cells were seeded in 96-well plates at a density of 104 cells per well. After 24 h of cell culture, Caspase-Glo® Buffer and Caspase-Glo® Substrate were thoroughly mixed at room temperature and then added to each well (100 μL/well). The mixture was shaken gently for 30 s and incubated for 1 h at room temperature. Then, the fluorescence values of the cells were measured with a microplate reader.
Tumour sphere formation assay
Cldn7 knockdown HCT116CD133+CD44+ cells and control cells were seeded in 6-well plates at a density of 104 cells per well. The cells were cultured in DMEM/F12 containing 20 ng/mL EGF (Peprotech, USA), 20 ng/mL basic FGF (Peprotech, USA), 2% B27 (Gibco, USA) and 1% double antibiotics (penicillin and streptomycin, Gibco, USA). The medium was changed every three days. Tumour sphere growth was observed and photographed. Some tumour spheres were inoculated in complete medium containing serum, and the growth of the cells was continuously observed.
In vivo subcutaneous xenograft model
Six- to eight-week-old male and female BALB/c nude mice weighing 17–20 g were used for this experiment. Cldn7 knockdown HCT116CD133+CD44+ cells and control cells were diluted to 1 × 106 cells/ml, mixed evenly and injected into the back of each nude mouse (each injection was 100 μL). Then, the growth of the subcutaneously implanted tumours was recorded. The long and short diameters of the tumours were recorded every 3 days. The tumour volume was calculated as V = 1/2 the long diameter × the short diameter2. After 30 days, the nude mice were sacrificed by cervical dislocation, and the tumours were removed and photographed. After the tumour volumes were recorded, the tumour formation rate was calculated, and growth curves for subcutaneous tumour formation were drawn.
Total protein was extracted from cells and tissues, and the protein concentrations were measured using a BCA kit (Thermo Fisher Scientific, USA). The proteins were then separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose (NC) membrane. The membranes were blocked with Tris-buffered saline-Tween (TBST) solution containing 5% skim milk. Next, the membranes were incubated with anti-Cldn7 antibody (ab27487, 1:1000; Abcam), anti-Sox9 antibody (ab185230, Abcam), anti-Olfm4 antibody (ab105861, Abcam), anti-Ki67 antibody (ab16667, Abcam), anti-β-catenin antibody (ab32572, Abcam), anti-cyclin D1 antibody (ab134175, Abcam), anti-C-myc antibody (ab32072, Abcam), anti-E-cadherin antibody (ab40772, Abcam), anti-Snail-1 antibody (ab180714, Abcam) or anti-vimentin antibody (ab8978, Abcam) at 4 °C overnight, followed by an incubation with a donkey anti-rabbit IgG antibody (ab175780; Abcam) or anti-goat IgG antibody (ab175780; Abcam). Finally, a Western blot scanner was used to visualize the blots. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the cytoplasmic internal reference, and lamin B1 was used as the nuclear internal reference.
RNA extraction and quantitative reverse-transcription polymerase chain reaction (qPCR)
Total RNA was extracted from Cldn7 knockdown HCT116CD133+CD44+ cells and control cells using TRIzol reagent (Thermo Fisher Scientific, USA). Reverse transcription was performed using SYBR Green PCR Master Mix (TOYOBO, Japan) and an ABI7500 system (Thermo Fisher Scientific, USA). Primers were designed by Sangon Biotech (Shanghai, China). The sequences of the Cldn7 primers were 5′-AAAGTGAAGAAGGCCCGTATA-3′ (forward primer) and 5′-TAATGTTGGTAGGGATCAAAGG-3′ (reverse primer). The Sox9 forward primer was 5′-GCACATCAAGACGGAGCAG-3′, and the reverse primer was 5′-GTAGGTGAAGGTGGAGTAGAGG-3′. The Olfm4 forward primer was 5′-GTAGGTGAAGGTGGAGTAGAGG-3′, and the reverse primer was 5′-GGACGACAGGGGTGTTTTGAT-3′. The Ki67 forward primer was 5′-CACTCCACCTGTCCTGAA-3′, and the reverse primer was 5′-TGTTGACTTCGGCTGATAG-3′. The β-catenin forward primer was 5’-ACACCAAGAAGCAGAGATG-3′, and the reverse primer was 5′-ACGAACAAGCAACTGAACT-3′. The c-Myc forward primer was 5′-ACCGAGGAGAATGTCAAGA-3′, and the reverse primer was 5′-CGCACAAGAGTTCCGTAG-3′. The cyclin D1 forward primer was 5′-TGAACAAGCTCAAGTGGAA-3′, and the reverse primer was 5’-GCGGTAGTAGGACAGGAA-3′. The E-cadherin forward primer was 5′-TACACTGCCCAGGAGCCAGA-3′, and the reverse primer was 5′-TGGCACCAGTGTCCGGATTA-3′. The Snail-1 forward primer was 5′-CCACAAGCACCAAGAGTC-3′, and the reverse primer was 5′-AGAGGACACAGAACCAGAA-3′. The vimentin forward primer was 5′-CTTTGCCGTTGAAGCTGCTA-3′, and the reverse primer was 5′-GAAGGTGACGAGCCATTTCC-3′. The GAPDH forward primer was 5′-TGACTTCAACAGCGACACCCA-3′, and the reverse primer was 5′-CACCCTGTTGCTGTAGCCAAA-3′. After normalization to GAPDH gene expression, the gene expression levels were analysed using the comparative threshold cycle (2−ΔΔCt) method.
Immunofluorescence (IF) staining
Cldn7 knockdown HCT116CD133+CD44+cells and control cells were cultured in a 24-well confocal chamber (Nest, China), fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.1% Triton X-100 at room temperature for 3 min. The cells were then blocked with 1% BSA for 60 min and incubated with primary antibodies against β-catenin overnight at 4 °C. After washing, the cells were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:400, ZSGB-BIO, China) for 2 h at room temperature in the dark. Finally, DAPI was added to each chamber to stain the nuclei, and a fluorescent anti-quenching agent was added. Cellular fluorescence staining was observed using a laser scanning confocal microscope (Nikon, Japan).
Cldn7 inducible conditional gene knockout mice
We constructed Cldn7-floxed mice and then crossed them with villin-CreERT2 mice to obtain Cldn7 inducible intestinal conditional gene knockout mice (Cldn7 ICKO mice, genotype: Cldn7fl/fl; villin-CreERT2). The construction method and identification method were the same as our previous study . Six- to eight-week-old Cldn7 ICKO mice were selected, and each mouse was injected intraperitoneally with 100 μL of tamoxifen (10 mg/mL) every 5 days to activate Cre recombinase expression and achieve Cldn7 gene knockout . Cldn7fl/fl; villin-CreW mice, which were the control group, were administered the same dose. The dying mice and the control mice were sacrificed by cervical dislocation. The large intestine and small intestine were harvested, and the intestinal tract was observed using hematoxylin–eosin (HE) and immunohistochemical (IHC) staining.
All tumour, paracancerous, and metastatic tissues were embedded into wax blocks and cut into paraffin sections. Xylene was used to dewax the paraffin slices. Different concentration gradients of alcohol were used for hydration. Tissue sections were incubated for 10 min in 3% H2O2 and washed with 0.01 mol/L PBS. The sections were then incubated with anti-Cldn7 antibody, anti-Sox9 antibody, anti-β-catenin antibody, anti-cyclin D1 antibody or anti-c-Myc antibody, followed by incubation with the corresponding secondary antibody (ab175780, Abcam). Afterwards, the proteins were developed in diaminobenzidine (DAB) for colouration. Haematoxylin was used to stain the nuclei, and different concentration gradients of alcohol were used for dehydration. Neutral gum was used to seal the slides.
A ChIP assay was performed according to the manufacturer’s instructions (SimpleChIP Plus Sonication Chromatin IP Kit, CST).
An HCT116CD133+CD44+ cell pellet was subjected to cross-linking, cross-linking suspension, cell and nuclear lysis, ultrasonic disruption, and chromatin dilution. The lysate was immunoprecipitated with Sox9 antibody (Abcam, USA). Then, the purified DNA was quantitatively pulled down using qPCR. The sequences of the Cldn7 primers were 5’-TGTTGGGAAGAAAGGAAGG-3’ (forward primer) and 5’-CCAGGTGAGGAGGAAGAA-3’ (reverse primer).
Total protein was extracted from Cldn7 knockdown HCT116CD133+CD44+ cells and control cells. Then, a BCA Protein Assay Kit (Solarbio, Beijing) was used to determine the protein concentrations, and the sample protein concentrations were adjusted to a consistent level. Then, 20 μL of Protein A/G beads were added to 500 μL of protein sample and incubated at 4 °C for 30 min. The supernatants were incubated with anti-Cldn7, anti-Sox9 or anti-IgG antibodies for 1 h at 4 °C. Then, 20 μL of Protein A/G beads were added and incubated overnight at 4 °C. The beads were then washed four times with RIPA buffer, and 50 μL of 1 × loading buffer were added to the final pellet and incubated at 100 °C for 10 min. Bound proteins were eluted from the beads in SDS sample buffer and analysed using Western blotting.
CRC, paracancerous, metastatic cancer tissue samples
CRC tissue chips HColA150CS02 (Shanghai Outdo Biotech Co.,Ltd., China) and HLinAde075Met01 (Shanghai Outdo Biotech Co.,Ltd., China) were used in this study to analyze the expression of Sox9 in adjacent normal tissues, CRC and metastatic cancer tissues.
The demographic characteristics of these patients are shown in http://www.superchip.com.cn/biology/category_309/1272.html and http://www.superchip.com.cn/biology/category_309/1255.html. CRC tissues were collected from 75 patients with CRC who did not undergo preoperative radiotherapy or chemotherapy. For each patient, adjacent tissues located more than 5 cm away from the tumour were also collected. All of these adjacent samples were confirmed to be normal colorectal tissues by pathology. Remote metastatic CRC tissues were also collected, such as CRC liver metastasis tissues and lung metastasis tissues. IHC staining was used to detect the expression of Sox9 in all samples. Oral informed consent was obtained from all patients. This research was reviewed and approved by the Medical Ethics Committee of the Beijing Shijitan Hospital Affiliated Capital Medical University Institutional Review Board.
Statistical analyses were performed using IBM SPSS version 17.0 and GraphPad Prism version 6.0. All data are presented as the means ± standard deviations. The differences between Cldn7 knockdown HCT116CD133+CD44+ cells and control cells were analysed using Student’s t-test. The Sox9 expression positive rate in different tissues was analysed using the X2 test. Differences were considered significant when P < 0.05.