Tissues collection
50 CRC tissues and adjacent normal tissues were collected from patients during operation at Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. The patients have not received any treatment prior to the study. The specimens were preserved at -80°C before use.
Cell culture
Two CRC cell lines (SW480 and SW620) were bought from the American Type Culture Collection (ATCC, Manassas, VA, USA) and one normal human colon mucosal epithelial cell line (NCM460) was bought from Beina Biotechnology Co., Ltd. (Beijing, China). These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) at 37°C in a humidified atmosphere consisting of 5% CO2.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The extraction of total RNA in tissues and cells was performed with RNAiso Plus kit (Takara, Dalian, China). After total RNA was examined on a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), M-MLV Reverse Transcriptase Kit (Promega, Madison, WI, USA) or miRNA 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) was adopted for reverse transcription experiment. Next, qRT-PCR was conducted on an ABI 7900 PCR system (Applied Biosystems, Foster City, CA, USA) using AceQ Universal SYBR qPCR Master Mix (Vazyme). The relative expression was examined by the 2-ΔΔCt method. U6 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was chosen as the internal control. The primers sequences were: circSEC24A: (F: 5’-GCTCTCCTTAAACAGGATATACACA-3’ and R: 5’-TGTCCACTGAGAAGGAATAAGTCA-3’); SEC24A: (F: 5’-GGCCACAAGCCTTTACTCAG-3’ and R: 5’-CTAGTCAGCGTGCATCGAAA-3’); miR-488-3p: (F: 5’-CGGGGCAGCUCAGUACAG-3’ and R: 5’-CAGTGCGTGTCGTGGAGT-3’); TMEM106B: (F: 5’-GGAGGAACCGGCAATTGATGGGAAAGTCTCTTTCTCACTTACC-3’ and R: 5’-GGCGGGCCTCCTCGAGTTATTGTTGTGGCTGAAGGACATT-3’); GAPDH: (F: 5’-TGTTGCCATCAATGACCCCTT-3’ and R: 5’-CTCCACGACGTACTCAGCG-3’); U6: (F: 5’-CTCGCTTCGGCAGCACATATACTA-3’ and R: 5’-ACGAATTTGCGTGTCATCCTTGCG-3’).
Actinomycin D assay
To block transcription, 2 μg/mL Actinomycin D (Abcam, Cambridge, MA, USA) was added into the cell culture medium. After being treated for 0 h, 4 h, 8 h, 12 h and 24 h, total RNA in SW480 and SW620 cells was isolated and the abundance of circSEC24A and linear SEC24A was measured by qRT-PCR assay.
RNase R digestion assay
10 μg total RNA extracted from SW480 and SW620 cells was digested with or without RNase R (20 mg/mL; Solarbio, Beijing, China) at 37°C for 20 min. Thereafter, qRT-PCR was adopted to test the levels of circSEC24A and linear SEC24A.
Cell transfection
Small interfering RNA against circSEC24A (si-circSEC24A), miR-488-3p mimics (miR-488-3p), miR-488-3p inhibitors (anti-miR-488-3p), the overexpression vector of TMEM106B (TMEM106B), short hairpin RNA targeting circSEC24A (sh-circSEC24A) and their corresponding controls (si-NC, miR-NC, anti-miR-NC, vector and sh-NC) were synthesized by GeneCopoeia (Guangzhou, China). The transfection of SW480 and SW620 cells was conducted with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay
MTT assay was employed to assess the proliferation of SW480 and SW620 cells. In brief, transfected cells were plated into 96-well plates (1×103 cells/well). Next, 20 μL MTT (5 mg/mL; Solarbio) was added into each well after 0 h, 24 h, 48 h and 72 h of incubation. After being kept for another 4 h, 150 µL dimethyl sulfoxide (DMSO; Solarbio) was put into the well for the dissolution of formazan crystals. Finally, the optical density at 490 nm was monitored by a microplate reader (BioTek, Winooski, VT, USA).
Western blot assay
Total RNA was obtained from tissues and cells by using RIPA buffer (Beyotime, Shanghai, China) and measured using a BCA protein assay kit (Tiangen, Beijing, China). Then 20 μg proteins were resolved by 10% sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) and then transferred onto polyvinylidene difluoride membranes (Pall Corporation, New York, NYC, USA). Next, the samples were immersed with 5% skim milk for 1 h and incubated with primary antibodies: cyclin D1 (ab16663; Abcam), proliferating cell nuclear antigen (PCNA; ab92552; Abcam), TMEM106B (ab264323; Abcam) or GAPDH (ab181602; Abcam) at 4°C overnight followed by incubation with relevant secondary antibody (ab205719; Abcam) for 2 h at room temperature. Finally, an enhanced chemiluminescence kit (Vazyme) was utilized to visualize the blots.
Flow cytometry analysis
For the analysis of cell apoptosis, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (Beyotime) was used. After 48 h of transfection, SW480 and SW620 cells were harvested and stained with 5 μL AnnexinV-FITC (Beyotime) and 10 μL PI (Beyotime) for 15 min in the dark. Next, a flow cytometer (BD Biosciences, San Jose, CA, USA) was adopted to examine the percentage of apoptotic cells.
For the analysis of cell cycle, transfected SW480 and SW620 cells were collected, washed in PBS (Solarbio) and fixed in 70% ethanol overnight at 4°C. Then the ethanol was removed, cells were washed again and resuspended in PI (Beyotime) containing RNase (Solarbio) for 30 min at room temperature followed by detection with a flow cytometer (BD Biosciences).
Transwell assay
The transwell chambers (Corning, Corning, NY, USA) pre-coated with Matrigel (Corning) were used to determine cell invasion, while the chambers without Matrigel (Corning) were used to determine cell migration. Briefly, transfected SW480 and SW620 cells were harvested and fixed in serum-free medium and then seeded into the upper compartments. The bottom compartments were filled with 500 μL DMEM (Gibco) including 10% FBS (Gibco). After 48 h, migrated or invaded cells were fixed with 4% formaldehyde (Sangon, Shanghai, China), dyed with 1% crystal violet (Sangon) and then counted under a microscope (Olympus, Tokyo, Japan).
Dual-luciferase reporter assay
The circSEC24A or TMEM106B 3’UTR sequences containing the wild-type or mutant binding sequences of miR-488-3p were cloned and inserted into pmirGLO vector (Promega) to create luciferase reporter vectors: circSEC24A WT, circSEC24A MUT, TMEM106B 3’UTR WT and TMEM106B 3’UTR MUT. Then miR-488-3p or miR-NC was transfected into SW480 and SW620 cells together with corresponding luciferase reporter vectors. 48 h later, the luciferase activity was measured by Dual-Luciferase Reporter Assay Kit (Promega).
Murine xenograft model
4-6 weeks old BALB/c nude mice were bought from Shanghai SLAC Laboratory Animal Co., Ltd, (Shanghai, China) and assigned to 3 groups (6 mice/group). Lentiviral vector for the silencing of circSEC24A (sh-circSEC24A) or its control sh-NC was transfected into SW480 and SW620 cells. Then transfected cells were subcutaneously injected into the right flank of the mice. Tumor volume was monitored every 5 days and calculated with the equation: (Length×Width2)/2. After 30 days of injection, the mice the euthanized and tumors were weighed and saved at -80°C for further experiments.
Statistical analysis
The data were collected from three independent experiments and analyzed using GraphPad Prism 7 (GraphPad Inc., La Jolla, CA, USA). The data were displayed as mean±standard deviation. Student’s t-test or one-way analysis of variance (ANOVA) was utilized to estimate the difference. If P<0.05, the differences were defined as significant.