Tissue specimens and patients
HCC tumor tissue and para cancer tissue were taken from HCC patients who had hepatectomy at Huazhong University of Science and Technology’s (HUST) Tongji Hospital (Wuhan, China). The Ethics Committee of Tongji Hospital, HUST, examined and approved all procedures, which were carried out in accordance with the Declaration of Helsinki Principles. Each patient’s written consent was obtained before the collection of specimens.
After the mouse subcutaneous tumor formation experiment, the tumor xenografts were removed from the mice, soaked in 4% formaldehyde, fixed for 1 to 24 hours, and then embedded in paraffin and made into sections. Place the slices in a 65-degree oven and bake for 30 minutes to 1 hour, then soak in xylene for 3 minutes for dewaxing, and soak in ethanol of high to low concentration for 3 minutes for rehydration. Soak the slices in 0.01 mol/l citrate buffer at 95 degrees Celsius for 30 minutes for antigen retrieval. The endogenous peroxidase activity was soaked in hydrogen peroxide (3%) at room temperature for 10 minutes in the dark, and the sections were incubated with 5% bovine serum albumin for 1 hour at 37 °C to reduce non-specific binding. Add antibody Ki-67 for immunostaining and incubate at 4 °C for 16 hours, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature. DAB was added dropwise to develop color for 1 minute. After brown staining appeared, the section was immersed in distilled water to stop the reaction. The sections were then counter-stained with 1% hematoxylin for 10 minutes and then immersed in low to high concentrations of ethanol for 3 minutes for dehydration. Use a high-resolution microscope or slice scanner for image capture.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
The FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China,RC112–01) was used to extract total RNA from tissues and cells according to the manufacturer’s instructions for RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR). A reverse-transcription system kit (Vazyme, China, R223–01) was used to complete the reverse transcription of lincRNA and mRNA, which was then evaluated by qPCR using a Universal SYBR qPCR Master Mix kit (Vazyme, China, Q711–02) Use GAPDH as an internal control to determine the mRNA level, according to the operation handbook. For relative quantification, the 2Δ-CT approach was applied. Three independent duplicates of each qRT-PCR experiment were carried out.
Cell culture and transfection
HCC Cells 7702 (CCTCC, No.ZHYC-0297), 97H (ATCC, No. CRL-2117), HLF (ATCC, No. CRL-2105), Hep3B (ATCC, No. HB-8064), HepG2 (ATCC, No. HB-8065), PLC/PRF/5 (ATCC, No.CRL-8024) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, China) and incubated in 5% CO2 at 37 °C.
Ribobio Technology designed and purchased antisense oligonucleotides (Asos) to knock down the HOXC-AS3 gene, as well as negative control Asos (Guangzhou, China). Lipofectamine 3000 Reagent was used to transfect Asos according to the manufacturer’s instructions (Invitrogen, USA). A full-length HOXC-AS3 was generated on the plasmid and processed into lentivirus, which was then introduced into HLF and 97H cells for stable overexpression.
Western blot and antibodies
It was separated for 2 hours on a 10% SDS-PAGE gel before being transferred to a PVDF membrane (0.45 μm, Roche). The membrane was blocked for 1 hour at 37 °C with 5% TBST, then the primary antibody was added and incubated for 8 hours at 4 °C. The membrane was then rinsed three times with TBST before being incubated for 1 h at 37 °C with HRP-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G secondary antibody (Jackson ImmunoResearch Laboratories). TBST washed the membrane three times more. The target western blot was detected using the ECL method (Bio-Rad, USA). Proteintech provided the CDK2(22060–1-AP) and p21(10355–1-AP) antibodies used in Western blotting in this investigation, while Cell Signaling Technology provided the Rb(#9313) and p-Rb(#8516) antibodies.
Cell proliferation assay
HCC cell proliferation was determined using the CCK-8 Cell Counting Kit (Vazyme, China, A311–01). After transfection, HLF, Hep3B, and 97H cells were seeded into 96-well plates (1000 cells/well). Then, according to the manufacturer’s protocol, the cells Culture the cells and record the 450 nm absorbance at 24, 48, 72, 96, and 120 hours respectively . All experiments were performed 3 times, and the results are shown as the average value of + SD.
Colony formation assay
Add HLF, Hep3B, and 97H cells to each well of a 6-well plate (2000 cells/plate), and continue to culture for 14 days in DMEM containing 10% FBS. Change the medium every 7 days. After 14 days, the cells were washed twice with PBS, fixed with 4% formaldehyde for 15 minutes, and stained with crystal violet for 15 minutes. Count and analyze cell clones .
EdU incorporation assay
HCC cells (20,000 cells per well) are inoculated into a 24-well plate and cultured overnight. When the cell density reaches 40%, add 100 μl of 50 M EdU solution to the cells (BeyoClickTM EdU Cell Proliferation Kit with a Fluor 594, Beyotime, China,C0078S) and cultivate for another 2 hours. PBS was used to wash the cells, 4% paraformaldehyde was used to fix them, and 0.5% TritonX-100 was used to rupture them. After 30 minutes, stain the cell nucleus with 1X Hoechst 33342 solution and 100 μl 1X Apollo solution. Take images using a fluorescent microscope (EVOS FL auto imaging system, life technologies, USA) when the staining is finished to count the number of positive cells and analyze the data.
RNA Pulldown assay
The oe-HOXC-AS3 vector and the control vector were transfected into 293 T cells. Two groups of total RNA were extracted after 48 hours, 100 nmol RNA probes were added, and the mixture was incubated at 70 °C for 5 minutes. The RNA is then gradually cooled to room temperature, allowing HOXC-AS3 to hybridize with the probe. After that, add 50 μl streptavidin magnetic beads and incubate for 30 minutes at room temperature with stirring. Unbound RNA has washed away with 20 mM Tris, and a test tube containing streptavidin magnetic beads was filled with 100 μl RNA-protein binding buffer containing 100 μg total protein. Streptavidin Magnetic Beads were washed three times with washing buffer and then incubated with 50 μl elution buffer at 37 °C for 15 minutes with stirring after incubation for 1.5 hours with rotation at 4 °C. For Western blotting, collect the supernatant.
RNA immunoprecipitation (RIP)
RIP assay was performed to enrich Argonaute 2 (AGO2)-, Flag-P21, or Flag-CDK2-bound RNA using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Germany). The antibodies used for RIP assays included antibodies against AGO2 (Abcam, UK), Flag (Sigma), and mouse IgG (Millipore). qRT–PCR was subsequently performed to analyze the enriched RNA.
For the cell cycle investigation, HCC cells were isolated, fixed in 75% ethanol, and stored overnight at 4 °C. Following that, DNA Prep (Beckman Coulter, Brea, CA, USA) was used to stain the cells, and flow cytometry was utilized to quantify the percentage of cells at various stages based on DNA content, according to a report .
Fluorescence in situ hybridization analysis (FISH)
Fluorescence in situ hybridization (FISH) study was performed using fluorescence-conjugated HOXC-AS3 probes. The target RNA was created using known nucleotide probes and the HOXC-AS3 complementary bases pairing method. By using Zeiss fluorescent confocal microscopy of nucleic acid probe hybridization of cells and tissues under qualitative and quantitative or relatively localized target RNA, the location of the target RNA may be directly visualized.
The routine source of the experimental animals was performed as previously described .
Forty male BALB/c nude mice (4 weeks old) were separated into four groups (Vec, HOXC-AS3, shNC, and Aso-2) and injected with 1*106 HLF or 97H cells on the right side to create a subcutaneous xenograft model. The mice were euthanized after 28 days to perform cervical dislocation. The xenografts were then taken out, fixed, weighed, photographed, and stored. V (mm3) = length width2/2 was used to calculate the volume of the tumor. Total protein was collected from the tissues, and CDK2 expression was determined using Western blotting.
To create an orthotopic xenograft model in vivo, 28 male BALB/c nude mice (5 weeks old) were separated into four groups (Vec, HOXC-AS3, shNC, and Aso-2) and 1*106 cells were injected into their livers. The livers were then fixed, photographed, conserved, and H&E stained in preparation for analysis.
In HEK293T cells or HCC cells, expressed labeled cell protein extracts were incubated with antibodies (Flag, HA, CDK2, or IgG as control, Sigma), and binding protein A/G beads (Pierce) for 12 h at 4 °C. The materials were analyzed by western blotting after being washed three times with an IP buffer.
The mean ± standard error of the mean is used to express the findings (SEM). The data were analyzed using GraphPad Prism 8.3 and a two-tailed student’s t-test. P 0.05 was considered statistically significant.