Human samples

A total of 45 CRC patients were recruited from Zhengzhou Central Hospital Affiliated to Zhengzhou University from August 2021 to May 2022, including 26 males and 19 females. CRC tissues and matched adjacent normal tissue were stored at -80 °C. All patients signed a written informed consent and were diagnosed for the first time. They had completed clinical information and did not suffer from other malignancies. This study was approved by the Medical Ethics Committee of Zhengzhou Central Hospital Affiliated to Zhengzhou University.

Cultivation and treatment of cells

Human CRC cell lines DLD1 (YS100C) and SW480 (YS284C), and fetal human colon (FHC, YS1779C) were acquired from YaJi Biological (Shanghai, China). Cells were cultured in RPMI 1640 (11,875,119, Gibco, Carlsbad, CA, USA) or DMEM (11,965,092, Gibco) plus 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C in humidified air with 5% CO2. Lentiviral vectors harboring short hairpin RNA (shRNA) against circ_0008345 (sh-circ_0008345), METTL3 (sh-METTL3), CYP1A2 (sh-CYP1A2) and their scramble control (sh-NC), miR-182-5p inhibitor and negative control (inhibitor-NC), miR-182-5p mimic and its control (miR-control), circ_0008345 overexpression vector (OE-circ_0008345) and control vector (OE-NC) were all designed and purchased from VectorBuilder (Guangzhou, Guangdong, China). After viral infection or transfection of plasmids, the cells were treated with puromycin (3 μg/μL) for 1 week, and surviving cells were selected as stable cells.

RNA extraction, reverse transcription, and RT–qPCR

TRIzol reagent (15,596,026, Gibco) was applied to extract RNA from tissues and cells, and PrimeScript RT kit (RR014B, Takara Holdings Inc., Kyoto, Japan) was used for the reverse transcription of cDNAs other than miR-182. The reverse transcription of miR-182-5p or miR-151-3p was performed using TaqMan™ MicroRNA Reverse Transcription Kit (4,366,596, Gibco). Finally, cDNA and specific primers (Table 1) were mixed with ChamQ Universal SYBR qPCR Master Mix (Q711-02, Nanjing Vazyme Biotech Co., Ltd., Nanjing, China), and PCR reactions were performed in the PCR system. The expression of mRNAs and circ_0008345 or the miR-182 was calculated relative to the expression of GAPDH or U6 small nuclear RNA, respectively. The comparative cycle threshold values (2−ΔΔCt) were calculated to analyze the expression.

Table 1 The oligonucleotides used in this studyActinomycin D assay

The DLD1 or SW480 cells were treated with 2 mg/L actinomycin D (A4262, Sigma-Aldrich Chemical Company, St Louis, MO, USA) and incubated at 37 °C. The treated cells were collected at 15 min, and RNA was extracted to remove the genomic DNA. The cDNA was synthesized using reverse transcription, and qPCR was performed to detect the level of circ_0008345. Finally, the data were analyzed, and the half-life of the target RNA was calculated.

Luciferase reporter assay

Based on the binding site between miR-182-5p and circ_0008345 or CYP1A2 3’UTR, wild-type (WT) and mutant (MUT) fragments of circ_0008345 or CYP1A2 3’UTR were ligated to the pmirGLO reporter vector (E1330, Promega) to construct circ_0008345-WT/MUT or CYP1A2 3’UTR-WT/MUT vectors. The reporter vector (circ_0008345-WT/MUT or CYP1A2 3’UTR-WT/MUT) and miR-182-5p mimic or miR-NC were co-transfected into CRC cells for 48 h. Cellular luciferase activity was detected using a dual luciferase reporter gene assay kit (E1910, Promega).

Pull-down assay using biotinylated miRNA

The biotinylated miR-182-5p, miR-151-3p, or control RNA (Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China) was transfected into DLD1 and SW480 cells at a final concentration of 100 nM. Two days after transfection, the whole cell lysates were harvested. Biotinylated RNA complexes were pulled down by incubating cell lysates with streptavidin-coated magnetic beads on a spinner at 4 °C overnight. TRIzol LS Reagent (10,296,010, Gibco) was used to extract RNA from input beads and pull-down beads. The abundance of circ_0008345 in the binding fraction was assessed by RT-qPCR analysis.

Nuclear and cytoplasmic RNA extraction

Nuclear and cytoplasmic RNA of DLD1 and SW480 cells were extracted according to the instructions of PARISTM Kit (AM1921, Gibco), respectively, followed by reverse transcription and fluorescence real-time quantitative PCR to detect circ_0008345 subcellular localization. β-actin or U6 was used as cytoplasmic and nuclear controls.

Colony formation and cell proliferation assays

Stably transfected CRC cells were seeded into 6-well plates (200 cells/well). After 14 days of incubation at 37 °C, colonies were fixed using crystal violet (G1063, Beijing Solarbio Life Sciences Co., Ltd., Beijing, China) and counted under a microscope.

Following the instructions in the BeyoClick™ EdU-488 Cell Proliferation Assay Kit (C0071S, Beyotime Biotechnology Co., Ltd., Shanghai, China), transfected CRC cells in 96-well plates (1 × 104 cells/well) were treated successively with the prepared EdU solution and Click reaction solution. The signals were observed under a fluorescent microscope. EdU-positive (EdU+) cell rate was analyzed using ImageJ software.

Cell migration and invasion assays

In migration assays, transfected CRC cells (1 × 105 cells/well) floating in serum-free medium were seeded into the apical chamber of Transwell plates (CLS3513, Corning Glass Works, Corning, N.Y., USA), and the complete medium was added to the basolateral chamber. After 24 h of incubation, the cells were fixed in paraformaldehyde and stained with crystal violet, and then five fields of view were randomly selected under a microscope for observation and counting. In the invasion assay, CRC cells (4 × 105 cells/well) were loaded into the apical chamber pre-coated with Matrigel. Other steps were the same as for the detection of migration.

Immunoblotting

Protein extraction was performed using RIPA lysis buffer (P0013C, Beyotime) containing protease inhibitors. After quantification using the BCA kit (23,230, Thermo Fisher Scientific Inc., Waltham, MA, USA), proteins (30 μg) were separated using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, which were sealed with 5% skim milk powder for 2 h. The membranes were then probed with the primary antibodies against CYP1A2 (1:5000, ab151728, Abcam, Cambridge, UK) and β-actin (1:200, ab115777, Abcam) at 4 °C overnight. The secondary antibody (1:10,000, ab6721, Abcam) was incubated at room temperature for 60 min. Signals were visualized using ECL reagent (WBULS0100, Merck Millipore, Darmstadt, Germany). Relative protein expression was analyzed using ImageJ software.

Immunohistochemistry

Paraffin-embedded human CRC tissue or paracancerous tissue blocks were cut to 4 μm thickness and stained for immunohistochemistry using the 3,3-diaminobenzidine kit. To eliminate endogenous peroxidase activity, tissue sections were de-paraffinized, rehydrated, and incubated in methanol containing 3% H2O2 for 15 min at room temperature before performing antigen retrieval at 95 °C for 20 min. The sections were probed with the primary antibody to CYP1A2 (1:250, ab217377, Abcam) at 4 °C overnight. After incubation with secondary antibody (1:1000, ab6721, Abcam) for 0.5 h at room temperature, the slides were incubated with HRP-peroxidase complex for 30 min at room temperature. The reaction products were visualized, and hematoxylin solution (105,174, Merck) was used for counter-staining. Finally, the expression of CYP1A2 protein in the tissues was determined by observing the number of positive cells under the microscope.

m6A RNA immunoprecipitation (MeRIP)

The Magna MeRIP™ m6A Kit (17-10499, Merck) was used according to the manufacturer’s instructions. Briefly, purified circRNA from the cells was fragmented to ∼ 100 nt using RNA fragmentation reagent and incubated at 94 °C. After fragmentation, the termination buffer was added, followed by standard ethanol precipitation and collection. The m6A antibody (12 μg) was pre-incubated with 50 μL of beads in IP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH = 7.4) for 1 h at room temperature. Next, 6 μg of the RNA fragment was supplemented to the antibody-bead mixture and incubated on a spinner for 4 h at 4 °C. After sufficient washing, the immunoprecipitation mixture was detached using high concentrations of proteinase K. Bound RNA was extracted using the phenol-chloroform method and ethanol precipitation for qPCR analysis. The remaining steps and primer sequences were described in RT-qPCR above.

RNase R assay

The CRC cell lines were treated with RNase R and then incubated for 30 min at 37 °C. The treated RNA was then reverse transcribed with specific primers (see above) and detected by real-time quantitative PCR assay, as described previously.

Tumor xenografts in nude mice

All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Zhengzhou Central Hospital Affiliated to Zhengzhou University. The report of animal experiments is under the ARRIVE guidelines. BALB/c nude mice were acquired from Vital River (Beijing, China). Transfected DLD1 cell suspension (2 × 106 cells/0.2 mL PBS) was injected into the right abdomen of BALB/c nude mice. The tumor size was recorded every week after injection, and the length (L) and width (W) of the tumors were measured. The tumor volume was calculated using the formula V = (L × W2) × 0.5. Four weeks after injection, the mice were euthanized with an intraperitoneal injection of pentobarbital, and the tumors were resected and weighed.

Statistics

GraphPad Prism version 8.0 (La Jolla, CA, USA) was used for graph drawing and statistical analyses. Paired t-tests were conducted for comparisons between any two groups. One-way/two-way ANOVA with Tukey’s post hoc test was performed to compare more than two groups. If not otherwise stated, all values are mean ± SD. All experiments were repeated three times. Differences with p < 0.05 were considered significant.