Cell culture and tissues

HepG2, SK-Hep1, HEK293T cells and HUVECs were purchased from ATCC (Manassas, VA, USA). The cells were cultured in high-glucose Dulbecco’s modified Eagle’s Medium (DMEM, VivalCell, Armenia) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin solution (Gibco) at 37 ℃ in an incubator with 5% CO2. The HCC samples and adjacent tissues were collected from HCC patients who underwent surgical resection at the Department of Hepatobiliary Surgery, the First Affiliated Hospital of Jinan University (Guangzhou, China).

Design of sgRNAs targeting the OC2 gene and vector construction

Two sgRNA sequences targeting exon 1 of OC2 were designed via the sgRNA design website (http://crispr.mit.edu/). The sequences of the sgRNAs are shown in Supplementary Tables 1 and were synthesized by Tsingke Biotechnology Limited Company (Guangzhou, China). The sgRNA was ligated into the LentiCRISPRv2 plasmid via T4 ligase to construct the recombinant plasmid LentiCRISPR-sgRNA. The OC2 overexpression plasmid (pcDNA3.1(+)-OC2) was constructed by Wuhan Gene Create Biological Engineering Corporation (Wuhan, China). The OC2 CDS region in this plasmid has been codon optimized, and there is no sgRNA binding site.

Packaging and infection of lentivirus

HEK293T cells were cotransfected with LentiCRISPR-sgRNA, pSPAX2 and pMD2G plasmids using lip2000 (GK20005, GLPBIO). The lentivirus was produced in recombinant HEK293T cells. The lentivirus in the supernatant of the HEK293T cells was collected at 96 h and concentrated in PEG 8000 at 5000×g for 30 min at 4 ℃. SK-Hep1 cells were plated in 6-well plates and infected with 100 µL of lentivirus for 24 h. The cells were treated with 2 µg/mL puromycin (HY-K1057, MCE) for two weeks to attenuate noninfected cells. After selection with puromycin, the SK-Hep1 cells were cloned by limited dilution in 96-well plates. The monoclonal cells were harvested 14 days after transfer to 24-well plates and sequenced at Sangon Biotech (Guangzhou, China).

Western blot

The cells were lysed with RIPA lysis buffer containing PMSF, and the protein concentration was quantified via a BCA kit (E112-01, Vazyme). The proteins were separated by SDS‒PAGE and transferred onto PVDF membranes (IPVH00010, Millipore). The membranes were incubated with primary antibodies, followed by interaction with a secondary antibody (Cat: 7074; Cell Signaling Technology, USA). The primary antibodies used included an anti-OC2 antibody purchased from Proteintech (21916-1-AP). The anti-Ac-p53 (2525), anti-AKT (4685), anti-angiogenin (62224), anti-Bax (41162), anti-Bcl-2 (4223), anti-CD31 (77699), anti-FGF2 (46879), anti-GAPDH (5174), anti-MAPK (4695), anti-PARP (9532), anti-PDGF-B (3169), anti-p21 (2947), anti-p27 (3686), anti-p53 (2527), anti-p-AKT (4060), anti-p-MAPK (9101), anti-p-p53 (9284), anti-TGF-β1 (3709), and anti-VEGFA (50661) antibodies were purchased from Cell Signaling Technology.

Real-time quantitative PCR (qRT‒PCR)

Fresh HCC and adjacent tissues and mouse xenograft tumor tissues from each treatment group were ground in liquid nitrogen, and total RNA was extracted via the RNA-Easy Isolation Reagent (Vazyme, Nanjing, China). cDNA was prepared via reverse transcription using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). qRT‒PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme) to amplify the target sequence. The relative mRNA expression levels were determined by the 2–ΔΔCt method and normalized to those of the control group. The primers used for qRT‒PCR are listed in Supplementary Table 2.

Immunohistochemistry (IHC) assay

Sections of liver tissue and xenograft tumor tissues were prepared at Servicebio (Wuhan, China). The sections were incubated with primary antibodies against OC2 (Proteintech, 21916-1-AP, 1:250), SKP2 (Cell Signaling Technology, 2652, 1:200), KI67 (Cell Signaling Technology, 12202, 1:400) and CD31 (Cell Signaling Technology, 77699, 1:200) at 4 °C overnight. The next day, peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were added, and the sections were incubated for 30 min at room temperature. The signal was visualized after 2 min of treatment with diaminobenzidine. Images were captured using an optical microscope (Olympus, Japan).

Cell viability

Cell viability was measured using a Cell Counting Kit 8 (HY-K0301, MCE). The cells were grown in 96-well plates, and 10 µL of CCK-8 solution was added to each well at 0 h, 24 h, 48 h, 72 h, and 96 h. The absorbances of the 96-well plates at 450 nm were detected via a microplate absorbance reader (BioTek).

Colony formation assay

The cells were cultivated in 6-well plates at 2000 cells per well for approximately 7‒9 days. The positive clonal clusters grew to 50 cells or more. The cells were washed with PBS, fixed with 4% paraformaldehyde for 1 h, stained with 1% crystal violet, and finally, colonies with 50 or more cells were counted via ImageJ.

Wound-healing assay

The HCC cells in each treatment group were seeded in 6-well plates at 3 × 105 cells per well. When the cell density reached 70-80%, the cells were scratched with 200 µL pipette tips. Images were taken at 0 h, 24 h, and 48 h using an inverted microscope (Olympus, Tokyo, Japan). The wound area was measured via ImageJ to calculate the migration rate.

Tube formation assay

The 96-well plate was coated with Matrigel (50 µL) and incubated at 37 °C for 1 h. HUVECs adjusted to 1 × 105 cells/mL in FBS-free DMEM were added to each well of a 96-well plate, and the culture supernatant (100 µL) of HCC cells was added. The plate was placed in a 37 ℃ incubator for 4 h. The tube formation of HUVECs was photographed under a microscope, and the numbers were determined via ImageJ.

ELISA assay

An AuthentiKine™ Human VEGF/FGF2 ELISA Kit (KE00216/KE00129, Proteintech) was used to detect human VEGFA and FGF2 concentrations in the supernatant of HCC cells. The primary antibodies used were anti-VEGFA and anti-FGF2, and the secondary antibodies used were HRP-conjugated goat anti-human monoclonal antibodies. The optical density (OD value) was measured at a wavelength of 450 nm with a reference wavelength of 630 nm with a CLARIOstar Microplate Reader (BMG LABTECH, Germany).

Apoptosis assay

Apoptotic cells were identified via an Annexin V-APC/7-AAD Apoptosis Kit (Elabscience, Cat: E-CK-A218). The HCC cells in each treatment group were digested with EDTA-free trypsin and adjusted to 2 × 105 cells in DMEM supplemented with 10% FBS. The cells were washed with PBS and treated according to the manual of the Annexin V-APC/7-AAD Apoptosis Kit. The cells were assayed with a flow cytometer (Attune NxT).

Dual-luciferase reporter assays

The cells (2 × 105) were seeded in a 12-well plate for 12 h. The dual-luciferase reporter assay system and OC2 overexpression plasmid were cotransfected into the cells using Lipofectamine 2000 (Invitrogen, USA). After 48 h, the transfected cells with the luciferase reporter genes were lysed and then centrifuged for 3 min. Luciferase activity (firefly luciferase at a wavelength of 560 nm and Renilla luciferase at a wavelength of 465 nm in the supernatant) was measured with the substrates of the Dual Luciferase Reporter Assay Kit (Vazyme, Nanjing, China) with a CLARIOstar Microplate Reader (BMG LABTECH, Germany).

ChIP‒qPCR

The chromatin coimmunoprecipitation was performed via the BeyoChIP™ Enzymatic ChIP Assay Kit (Protein A/G Magnetic Beads) (Beyotime, Shanghai, China). Following the manual’s instructions, the HCC cells were fixed with 1% formaldehyde and lysed with cell lysis buffer and nuclear lysis buffer. The supernatant was subjected to ultrasonic treatment for 30 min at 4 °C at high power to obtain fragments of 200–500 bp in size, which were then immunoprecipitated from the protein‒DNA complex with anti-Flag antibodies. The protein‒DNA complex was combined with protein‒A‒agarose beads. The bound DNA fragments were isolated and purified via phenol‒chloroform-isoamyl alcohol (25:24:1) and precipitated with ethanol and acrylamide. The purified DNA fragments were used for ChIP‒qPCR. The primers used for ChIP‒qPCR were listed in Supplemental Table 3.

Mouse xenograft model

Four-week-old BALB/c nude mice were treated humanely according to the Guidelines for the Care and Use of Experimental Animals of Jinan University, and all animal assays were approved by the Experimental Animal Science Ethics and Welfare Committee of Jinan University. Monoclonal OC2 knockout cells (2 × 106 in 100 µL) with an equal volume of Matrigel were subcutaneously injected into the shoulders of the mice. The control group was injected with PBS and Matrigel. The volume of the tumors (length×width2)/2 was measured every 3 days. After 21 days, the mice were euthanized, and the xenograft tumors were harvested for IHC analysis and weight measurements.

TUNEL assay

The TUNEL assay was performed with a one-step TUNEL apoptosis assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The sections were fixed with a fixative and permeabilized with Triton X-100. The slides were incubated with the TUNEL reaction mixture at 37 °C for 1 h. The samples were re-stained with DAPI and analyzed via fluorescence microscopy.

Statistical analysis

All of the experiments were performed three times, and the data were analyzed with GraphPad Prism 8. The results are shown as the means ± SDs. Statistical differences between groups were analyzed via one-way analysis of variance or Student’s t test.