In this study, tissue samples of 52 patients with CRC from the Third Xiangya Hospital of Central South University, China between May 2018 and December 2018 were collected. No patient received radiotherapy or chemotherapy before surgery, and the postoperative pathological diagnosis was adenocarcinoma (high, medium, and low differentiation). For each specimen, two copies of cancer tissues and adjacent normal tissues were collected. The surgical specimens were cryopreserved in liquid nitrogen immediately after excision. This study was approved by the Ethics Committee of the hospital, and all patients gave informed consent.
Cell culture: Human CRC cell lines (SW480, SW620, HCT116, HT29) and normal colonic epithelial cell lines (NCM460) were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). SW480 and SW620 cells were cultured in L15 medium (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) containing 10% fetal bovine serum (FBS: Biological Industries Israel Beit-Haemek, Beit-Haemek, Israel). HCT116 and HT29 were cultured with McCoy's 5A medium (Nanjing KeyGen Biotech Co., Ltd.) containing 10% FBS. NCM460 was cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific ™, Beijing China) containing 10% FBS. All cells were incubated at 37 °C in a 5% CO2 incubator.
siRNA-1, siRNA-2, siRNA-3, shRNA-3, and negative-control siRNA were all designed by Suzhou Genepharma Co., Ltd., China. Cell transfection was done according to the manual of Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).
Tablet cloning assay
The tumor cell lines that were successfully transfected with shRNA-3 were harvested, counted, and adjusted to a cell concentration of 500 cells/ml, then were seeded in 6-well plates (1 × 103/well), and 3 duplicate wells were set for each cell line. The 6-well plates were incubated at 37 °C in a 5% CO 2 incubator for 2 weeks. The cell culture medium was changed every 2 to 3 days. The 6-well plates were taken out after 2 weeks, and the medium was removed. Cells were then washed with phosphate-buffered saline (PBS) 2 times, dried naturally, and then fixed by adding 1 ml of 4% paraformaldehyde in each well. After cells were fixed at room temperature for 30 min, the paraformaldehyde solution was aspirated. Cells were dried and stained with 0.1% crystal violet for 30 min. After washing with PBS twice, the cells were photographed and counted.
Cell Counting Kit-8 (CCK-8) assay
SW480 and HCT116 (5 × 103/well) were mixed well in 100 µl normal medium containing 10% FBS, seeded into 96-well plates, and incubated at 37 °C in a 5% CO2 incubator. Each well received 10 µL of CCK-8 reagent at the specified time point (0, 24, 48, and 72 hours). After incubation for another 3 hours, the absorbance at 450 nm of each well was measured by an EnVision microplate reader (PerkinElmer, Inc., Waltham, MA, USA).
A marker was used to draw horizontal lines with the help of a ruler on the back of a 6-well plate. The lines were evenly drawn to 1 cm long, and there were at least 5 lines for each well. A total of 5 × 105 cells were inoculated into each well of a 6-well plate. Cells were transfected when they reached a density of approximately 70% confluence. When the cells grew to just cover the entire well, a 10 µl pipette tip was used to make a scratch, with the help of a ruler, perpendicular to the horizontal lines on the back of the plate. The detached cells were washed with PBS, and then serum-free medium was added to continue the culture for 72 hours. Samples were taken at 0, 24, 48, and 72 hours, the cells were photographed, and the scratch width was measured.
Migration and invasion assays
Migration and invasion assays were performed using a Transwell chamber (8 µm, 24-well insert; Corning Incorporated, Corning, NY, USA). After 3 × 105 cells were mixed in 200 µl of serum-free medium, they were seeded in the upper chamber, 500 µl normal medium containing 20% FBS was added into the lower chamber, and then the plates were incubated for 48 hours at 37 °C in a 5% CO2 incubator. The upper chamber and the lower chamber were washed twice with PBS. The cells in the upper chamber were cleaned with a cotton swab to remove cell debris. The chamber membrane was fixed with 4% paraformaldehyde for 30 min. The paraformaldehyde solution was aspirated and cells were dried, followed by 0.1% crystal violet stain for 30 min and two washes with PBS. Cells on the lower side of the chamber membrane were observed under an inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan). The cells were photographed and cell numbers were calculated.
Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNAs in tissues and cells were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA). RT-qPCR was performed according to the manual of the Toyobo RT kit (Toyobo Life Science, Osaka, Japan) and Hieff® qPCR SYBR® Green Master Mix (Yason Biotech Co., Ltd.). CircRNA primers were designed by Beijing Tsingke Biological Technology Co., Ltd. (Hunan, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control, and the relative expression levels were calculated by the 2−ΔΔCt method.
CircRNA-miRNA-mRNA coexpression network construction
circRNA-miRNA interactions were predicted using Arraystar’s homemade miRNA target prediction software (Rockville, MD, USA) based on TargetScan (15) and miRanda (16). The miRNAs were scored and sequenced using the miRNA support vector regression (mirSVR) algorithm to focus on target miRNAs (17). Therefore, for each circRNA, we identified the top 5 miRNAs in the miRVR score system to establish a top-5 circRNA-miRNA network (1 circRNA connecting to 5 miRNAs). To further predict the interactions between miRNA and mRNAs, the miRNA-mRNA overlapping set predicted by databases of miRDB (18), miRTarBase (19), and TargetScan were used to plot circRNA-miRNA-mRNA interaction networks in Cytoscape (version 3.4.0) (20).
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of circRNAs: The Database for Annotation, Visualization and Integrated Discovery (DAVID; http://david.ncifcrf.gov (Version6.7)) (21) is an online bioinformatics database that integrates biological data and analysis tools. It provides a complete set of gene and protein functional annotation information for users to extract biological information. KEGG is a database resource used to categorize high-level functions and biological systems from large-scale molecular datasets generated by high-throughput experimental techniques (22). GO is an important bioinformatics tool for annotating genes and analyzing their biological processes (23). To analyze the functions of circRNAs, the online database of DAVID was used for biological analysis. P < 0.05 indicated statistical significance.
Data analysis: SPSS 19.0 software was used for statistical analysis (IBM Corp., Armonk, NY, USA): The data were imaged using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA), and the data are expressed as the mean ± standard deviation. The differences between two groups were analyzed using the independent-sample t test, and intragroup differences were analyzed using one-way analysis of variance (ANOVA). P < 0.05 indicated that a difference was statistically significant.