CRC patients and clinical tumor tissues
Eighty pairs of CRC tissues and the adjacent non-tumor tissues were collected in the Second Affiliated Hospital of Fujian Medical University during Jan. 2019 to Dec. 2019. The collection was approved by the Ethical Committee of the Second Affiliated Hospital of Fujian Medical University and all patients were informed of details before admission. No additional treatment was conducted in the collected patients before surgery. The tissues were snap-frozen in liquid nitrogen and stored at -80°C.
Cell culture and transfection
Human colorectal cancer cells HCT116, SW620, HT29, and SW480, normal colonic epithelial cell line NM460 and HEK293T were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). All the cells were cultured in DMEM-High glucose (Gibco) containing 10% fetal bovine serum (PAN biotech) and 100 u/mL penicillin/streptomycin at 37 °C under 5% CO2 in a humidified chamber.
The miR-338-3p mimics, miR-negative control (miR-NC), siRNAs for circRAE1(si-circRAE1#1,2 and 3) and TYRO3 (si-TYRO3), and siRNA-negative control (si-NC) were from Shanghai Gene-Pharma. circRAE1-overexpression plasmid was constructed using the Pcd-ciR vector. All the recombinant plasmids were sequenced confirmed by Fuzhou Biosune. circRAE1-overexpression plasmid was then co-transfected with RRE, REV, and VSV/G into HEK293T cells to produce lentivirus-circRAE1 (lv-circRAE1). si-circRAE1 (100 nM), si-TYRO3 (100 nM) and miR-338 mimics (50 nM) were transfected through Lipofectamine 2000 reagent (Life) according to product specification. Lentivirus-circRAE1 infected CRC cells with 10 μg/mL polybrene. Cell, RNA and protein samples were collected 24 h or 48 h after transfection.
Quantitative RT-PCR assays (qRT-PCR)
Total RNA was isolated with the TRIzol reagent as per the product specification. cDNA was synthesized using reverse transcription kit (Sangon, China) for circRNA and mRNA analysis, while RiboBio microRNA reverse transcription kit (Guangzhou, China) was used for miRNA. Quantitative PCR was conducted using GoTaq® qPCR Master Mix (Promega, USA). Relative expression levels of circRNAs, mRNAs and miRNAs were calculated as the method of 2−ΔΔCt, and normalized to GAPDH for circRNAs and mRNAs or U6 for miRNAs. Each experiment was in triplicate. Primer sequences are showed in Table 1.
Western blot assay
Western blot assays were conducted as previously described [13]. Briefly, equal amount of proteins were separated on 10-12% SDS-PAGE, transferred onto PVDF membranes (0.45 μM), blocked with 5% skim milk. The membranes were then incubated with various primary antibodies at 4 °C for more than 10 h. Afterwards, the membranes were blotted with HRP-conjugated secondary antibodies. The signal was visualized with ECL (Beyotime Biotechnology, Jiangsu, China) using the LiCor C-DiGit Blot scanner with LiCor Biosciences Image Studio Software. Anti-E Cadherin antibody [HECD-1], Anti-Vimentin antibody [RV202], and Anti-TYRO3 antibody [EPR4308] were from Abcam.
Stability analysis of circRAE1
SW620 and HT29 were treated by Actinomycin D (2 μg/mL) and then total RNA was extracted after 0, 4, 8, 12 and 24 h. Levels of circRAE1 and its linear subtype were detected by qRT-PCR. As for RNase digestion assay, total RNA was co-incubated with 3 units of RNase R per 1μg RNA for 30 min at 37 °C. Then re-purified RNA was subjected to qRT-PCR for circRAE1 and its linear subtype.
Fluorescence in situ hybridization
In situ hybridization was employed to explore the intracellular location of circRAE1 and miR-338-3p in HT29 and SW620. Cells were grown on cover slips and fixed with 4% paraformaldehyde. The procedure was carried out as previously reported [14]. Briefly, the slides were treated with CSK Buffer (0.5% TritonX-100, 10 mM VRC) for 10-12 min and then treated with 70% alcohol for 10 min at 4 °C, , followed by incubating with series alcohol to dehydration. After air drying, the slides were prehybridized for 1 h at 55 °C in prehybridization solution. Cy3-circRAE1 and/or Digoxin-labeled miR-338-3p (DIG-miR338-3p) probe in hybridization buffer were denatured at 76°C for 10 min, and then added to each slide and hybridized overnight at 37 °C in dark humidified chamber. After washing, the anti-DIG [21H8]-FITC (Abcam, USA) was added to the slides and the slides were incubated at 37 °C for 1 h in the humidified chamber. The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) after washing.
Luciferase reporter assay
PCR method was used to amplificate the wild-type (WT) and mutant (MUT) 3'-UTR of human TYRO3, and the WT and MUT of circRAE1 (shown as follow), which were then cloned into the psiCHECKTM-2-luciferase reporter plasmid. HEK293T cells were co-transfected with WT or MUT psiCHECKTM-2-circRAE1 plasmids or psiCHECKTM-2-TYRO3 (3'-UTR), and miR-338-3p mimics or miR-NC using Lipofectamine 2000 reagent. 48 h later, cells were collected and subjected to the commercial Dual-Luciferase reporter assay system (Promega) following the manufacturer's instructions for measuring both firefly and Renilla luciferase activity.
Wound-healing assay
A wound migration model for in vitro assay was used as previously described [15]. Culture-Inserts (Ibidi) were used in the wound-healing assay. Briefly, HT29 and SW620 cells was seeded to each well of the Culture-Inserts. Cells were incubated at 37˚C for 24 h, a cell-free gap of 500 μm was created after removal of the Culture-Insert. An inverted phase-contrast microscope was employed to capture images at 0, 24 and 48 h. Five randomly chosen fields were used to calculate the percent of wound closure using the ImageJ software.
Transwell migration assay
Transwell chambers (Corning, USA) was used in the transwell migration assay as previously described[9]. SW620 and HT29 cells were seeded into the upper chambers with serum-free DMEM, and the lower wells were filled with DMEM containing 20% FBS. 24 h later, cells in the top chamber were removed with cotton swabs and those on the lower surface were fixed using 4% paraformaldehyde (PFA) and stained with 0.1% crystal violet for 15 min. After washing twice with PBS, cell numbers in five randomly chosen fields were calculated under the microscope (Olympus, Japan).
Transwell invasion assay
Transwell invasion assay was detected as previously described [16]. Briefly, the upper chambers (Corning, USA) were coated with diluted Martrigel (BD, USA). Afterwards, SW620 and HT29 cells were seeded into the upper chambers with serum-free DMEM, and then the transwell chambers were incubated in wells filled with DMEM containing 10% FBS. Following 24 h incubation at 37 ˚C, cells inner the chambers and the remaining Matrigel were removed with cotton swabs and the cells on the outside surface of the lower chambers were fixed using 4% PFA, stained with 0.1% crystal violet for 15 min. After washing twice with PBS, cell numbers in five randomly chosen fields were calculated under the microscope.
Statistical analysis
SPSS 22.0 statistical software was employed for the statistical analysis. Data was shown as mean ± the standard deviation (SD). The significant differences between groups were estimated by One-Way ANOVA. P < 0.05 was considered as statistically significant.