Human tissue specimens
The 80 pairs of CRC tissue samples and adjacent normal tissue samples were obtained from Department of General Surgery, the First Affiliated Hospital of Nanjing Medical University. The patients recruited to our study did not receive preoperative chemotherapy and radiotherapy. All the patients signed written informed consents before the study. The research was approved by the Institutional Ethical Board of the First Affiliated Hospital of Nanjing Medical University. The clinical stages of the CRC patients were determined according to the International Union Against Cancer (UICC) on Tumor-Node-Metastasis (TNM) staging system (7th edition). The collected tissues samples were stored in liquid nitrogen before use.
Cell lines and cell culture
The five CRC cell lines, including HCT116, SW480, HT-29, LoVo and DLD-1 were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Human normal colon epithelial cell line NCM460 was obtained from American Type Culture Collection (ATCC, USA). All of the cell lines were cultured in DMEM medium (Wisent, Canada), containing 10% fetal bovine serum (FBS, Wisent, Canada), 100 U/ml penicillin and 100 ug/ml streptomycin (Invitrogen, USA) in a moist incubator with 5% CO2 at 37 ℃.
Cell transfection
MiR-3622a-3p mimics lentivirus, miR-3622a-3p inhibitor lentivirus, the lentiviral vector containing SALL4 DNA sequencing (LV-SALL4) and the lentiviral vector containing SALL4 shRNA sequence (sh-SALL4) were purchased from GenePharma (Shanghai, China). We used puromycin (Sigma, Aldrich) to screen the transfected CRC cells to establish stable transfected cell lines according to the manufacturer’s protocol.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNAs were extracted with Trizol Reagent (Invitrogen, USA) from CRC tissues and cell lines. PrimeScript RT Reagent (Takara, Japan) and New Poly(A) Tailing Kit (ThermoFisher, China) were used for mRNA and miRNA reverse transcription respectively into cDNA following the manufacturer’s instructions. We carried out qRT-PCR with a 7500 Realtime PCR System (Applied Biosystems, USA) and SYBR
Green Master Mix (Roche, USA). Relative expression of miR-3622a-3p was normalized to snRNA U6. Beta-actin was taken as internal control for SALL4, Sox2, Nanog, Oct4, CD44 and CD133 detection. The 2-ΔΔCT analysis method was used to calculate the relative expression of miR-3622a-3p and SALL4. The primers for qRT-PCR were listed below: has-miR-3622a-3p forward, 5’-TCACCTGACCTCCCATGCCTGT-3’; Universal, 5’-GCGAGCACA GAATTAATACGAC-3’; U6 forward, 5’-CTCGCTTCGGCAGCACA-3’; U6 reverse, 5’-AACGCTTCACGAATTTGCGT-3’; SALL4 forward, 5’-TCGATGGCCAACTTCCTTC-3’; SALL4 reverse, 5’-GAGCGGACTCACACTGGAGA-3’; beta-actin forward, 5’-GCATCGTCACCAAC TGGGAC-3’; beta-actin reverse, 5’-ACCTGGCCGTCAGGCAGCTC-3’; Sox2 forward, 5’-ACACCAATCCCATCCACACT-3’; Sox2 reverse, 5’- GCAAACTTCCTGCAAAGCTC-3’; Nanog forward, 5’- CCTGATTCTTCCACCAGTCC-3’; Nanog reverse, 5’- TGCTATTCTTCGGCCAGTTG-3’; Oct4 forward, 5’- TTGAGGCTCTGCAGCTTAG-3’; Oct4 reverse, 5’- GCCGGTTACAGAACCACAC-3’; CD44 forward, 5’- TCACAGGTGGAAGAAGAGAC-3’; CD44 reverse, 5’- CATTGCCACTGTTGATCACT-3’; CD133 forward, 5’- CTGGGGCTGCTGTTTATTATTCTG-3’; CD133 reverse, 5’- ACGCCTTGTCCTTGGTAGTGTTG-3’.
Cell proliferation assay
To assess proliferation of stable transfected cells, we used a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). 2×103 cells were seeded into each well of a 96-well plate. The cells in each well were incubated with 10 ul CCK8 reagent for 2h at 37 ℃. Absorbance at 450 nm was measured by a microplate reader at the same time point for 5 days.
5-Ethynyl-2’-deoxyuridine (EDU) assay
DNA synthesis of stable transfected CRC cells was measured with an EDU assay kit (RiboBio, China). The cells were seeded at a density of 2×104 cells per well into a 24-well plate and cultured in DMEM containing 10% FBS for 24 h. After incubation with 50 uM EDU reagent at 37 ℃ for 2 h, the cells were fixed and permeabilized with 4% formaldehyde and 0.5% TritonX-100 respectively at room temperature (RT). Then we added 1× Apollo R reaction cocktail (400 ul) to each well. After 30 min, 400 ul Hoechest33342 was added to stain the nuclei of stable transfected cells. Red and blue signals were observed and taken by a Nikon microscope (Nikon, Japan).
Colony formation assay
The stable transfected cells were seeded into a 6-well plate (500 cells/well) and cultured in DMEM (10% FBS) for 15 days. After being fixed with 75% ethyl alcohol, the colonies were stained with crystal violet (Beyotime, China). We washed the colonies with phosphate buffered solution (PBS) for three times and counted the number of colonies.
Flow cytometric analysis
The stable transfected cells were digested with trypsin and then collected. After being washed with PBS twice and fixed with 75% ethyl alcohol, the cells were stored at -20 ℃ overnight. The cells were then washed with PBS, incubated with RNase and stained with a Cell Cycle Staining Kit (Multi Sciences, China) for 15 min in the dark. The cell-cycle was analyzed with a FACScan flow cytometer (BD, USA). To evaluate apoptosis of stable transfected cells, the cells were collected and stained with an Annexin V‐FITC/PI Apoptosis Kit (Multi Sciences, China) based on the manufacturer’s protocol. The ratio of apoptotic cells was determined by a FACScan flow cytometer (BD, USA). The collected stable transfected cells were incubated with PE-conjugated CD133 antibody (MiltenyiBiotec, Germany) at RT for 90 min. Then the cells were placed on ice for 10 min and washed with precooled PBS before flow cytometric analysis for CD133(+) cells detection.
Transwell migration and invasion assays
To assess the migration ability of stable transfected cells, we used a Transwell plate (Corning, USA). 2×104 stable transfected cells were seeded into the upper chamber and cultured in 200 ul DMEM without FBS. 500 ul DMEM medium containing 10% FBS which acted as chemoattractant was added to the lower chamber. The plate was incubated at 37 ℃ for 24 h. Part of the cell migrated to the underside of the membrane. The cells which did not penetrate the membrane were removed and the cells on the lower surface of the membrane were fixed and stained with 75% alcohol and crystal violet respectively. The stained cells were counted with a microscope. To perform invasion assay, 100 ul Matrigel (BD, USA) was coated on the upper side of the membrane before cell plating. The remaining steps were similar to those in migration assay.
Dual-luciferase reporter assay
The 3’-UTR sequences of SALL4 containing wild-type (WT) or mutated (MUT) miR-3622a-3p binding site were designed and synthesized by GeneScript (Nanjing, China). The sequences were cloned into a a pGL-3 luciferase reporter vector (Promega, USA). DLD-1 was co-transfected with miR-3622a-3p mimics or miR-NC and pGL3-WT-SALL4 or pGL3-MUT-SALL4. HCT116 was co-transfected with miR-3622a-3p inhibitor or miR-NC and pGL3-WT-SALL4 or pGL3-MUT-SALL4. The luciferase activity was determined with a Dual Luciferase Reporter Assay System (Promega, USA). The ratio of firefly luciferase to renilla luciferase was defined as the relative luciferase activity.
Western blot analysis
RIPA lysis buffer (Beyotime, China) was used to extract total proteins from paired CRC tumors and adjacent normal tissues and CRC cells. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After being blocked in 5% non-fat milk at RT for 2 h, the membranes were incubated with primary antibodies at 4 ℃ all through the night. The membranes were washed with TBST buffer for three times and incubated with secondary antibodies at RT for 2 h the next day. Finally, we washed the membranes for three times using TBST buffer and the proteins on the membranes were visualized by an enhanced chemiluminescence (ECL) detection system (Millipore, USA). Primary antibodies, including anti-SALL4, anti-GAPDH, anti-Sox2, anti-Nanog, anti-Oct4, anti-CD44, anti-CD133, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Snail, anti-TWIST, anti-beta-catenin, anti-Cyclin D1, anti-TCF1 and anti-c-myc, were purchased from Abcam (Cambridge, UK). The secondary antibodies used in our study, including anti-rabbit IgG-HRP and anti-mouse IgG-HPR antibodies, were purchased from Santa Cruz (Dallas, USA).
Sphere formation assay
Stable transfected cells were seeded into a 6-well ultra‐low attachment surface plate (Corning, USA) with 5×103 cells per well. The cells were cultured in serum-free DMEM/F12 (Gibco, Australia). The culture medium was supplemented with B27 (Invitrogen, USA), N2 (Invitrogen, USA), EGF (Invitrogen, USA) and basic FGF (Invitrogen, USA). The spheres were observed and photographed 10 days later.
TOPflash/FOPflash luciferase reporter assay
The Topflash/FOPflash reporter plasmids were obtained from Upstate Biotechnology (NY, USA). The cells were transfected with the plasmids using Lipofectamine 3000 (Invitrogen, USA). The luciferase activity was detected by a Dual Luciferase Reporter Assay System (Promega, USA). The results were shown as normalized TOPFlash/FOPFlash values.
Animal experiment
The BALB/c nude mice (aged 5 weeks) used in our study to build tumor xenograft model and tumor metastasis model were purchased from Animal Center of Nanjing Medical University (NJMU). The animal experiments were approved by NJMU Animal Ethics Committee. 1×106 CRC cells stably transfected with miR-3622a-3p mimics or inhibitor were injected into the flanks of nude mice (6 mice/group). The volume of the tumors was measured every 4 days with a vernier caliper following calculation formula: volume= (width2×length)/2. The nude mice were sacrificed on day 24. For in vivo metastasis assay, 1×106 stable transfected CRC cells suspended in 100 ul PBS were injected into lateral tail veins of nude mice. After 4 weeks, the distant metastases of 8 nude mice in each group were visualized with an IVIS Imaging system (Caliper life Sciences, USA). The remaining of the nude mice were kept to analyze the effect of miR-3622a-3p on survival of nude mice with 12 weeks as cutoff.
Immunochemical staining
The subcutaneous tumors of nude mice were obtained and fixed in 4% formaldehyde. Then they were embedded in paraffin and cut into 4 um thick sections. The sections were incubated with primary antibodies, such as anti-ki67 and anti-SALL4 (Abcam, UK) overnight at 4 ℃. After being washed with PBS for three times, the sections were incubated with HRP-polymer-conjugated secondary antibody at RT for 1 h. We used 3,3’-Diaminobenzidine (DAB) solution to stain the sections for 3 min and hematoxylin to counterstain nuclei. The percentage of positive cells was determined based on three random fields of the sections.
TUNEL assay
We used a TUNEL apoptosis detection kit (Beyotime, China) for this assay according to manufacturer’s protocol. The subcutaneous tumor sections were rehydrated in the ethanol and fixed in 4% formaldehyde. The sections were then incubated with proteinase K at RT for 20 min and 3% hydrogen peroxide was used to inactivate endogenous peroxidases. Working solution and chromogenic agent were prepared following manufacturer’s instructions. Hematoxylin was used to stain nuclei of the cells.
The percentage of apoptotic cells in randomly selected fields was determined with a microscope (Nikon, Japan).
Hematoxylin and eosin staining
The lungs of the nude mice were fixed in 4% formaldehyde and embedded in paraffin. The sections prepared from paraffin mass was incubated with hematoxylin for 3 min and washed with deionized water. Then we used eosin Y solution to dye the sections and 95% alcohol followed by absolute ethanol to dehydrate the specimens. Eventually, xylene was adopted for alcohol extraction and neutral balsam was used to seal the sections.
Establishment of CRC organoid model
The CRC organoid model was constructed based on the protocols published previously [31]. The organoids were transfected with miR-3622a-3p mimics or inhibitor lentivirus. After 12 days of culture, the diameters of the organoids were measured. Then the organoids were collected from Matrigel for miR-3622a-3p and SALL4 detection with qRT-PCR.
Statistical analysis
All the statistical analyses adopted in the study were performed with Statistical Product and Service Solutions (SPSS) 20.0 software. The data was shown as mean ± standard deviation (SD). All the experiments were carried out at least three times. Linear correlation analysis was performed to analyze the correlation between miR-3622a-3p expression and SALL4 expression. χ2 test was used to determine the relationship between miR-3622a-3p expression level and the CRC patients’ clinicopathological features. The Caplan-Meier method was adopted in survival analysis. Student’s t-test and analysis of variance (ANOVA) were performed to analyze the data obtained from experiments. P<0.05 (*) and P<0.01 (**) were considered statistically significant.