TRIM36 inhibits tumorigenesis through the Wnt/β-catenin pathway and promotes caspase-dependent apoptosis in hepatocellular carcinoma

Subjects

Patients admitted to the Affiliated Cancer Hospital of Xinjiang Medical University between September 2014 and March 2021 who met the following criteria were enrolled: (1) complete medical records, (2) post-operative histological diagnosis of HCC, and (3) hepatectomy. The exclusion criteria were: (1) presence of other malignancies or AIDS and (2) fragmented clinical or pathological data. Ultimately, 92 patients with HCC were enrolled. All provided informed consent before inclusion. We collected clinical information: age, sex, HBV infection status, cirrhosis history, AFP level before surgery, Edmondson grade, and TNM tumor stage. We also collected imaging data: tumor size (cm, length × width), vascular invasion status, and tumor numbers. Patient follow-up data were recorded: recurrence-free survival (RFS) (months after surgery), survival status, and overall survival (OS) (months after surgery). Recurrence was primarily assessed by imaging. RFS was defined as the time between surgery and the date of recurrence or failure of follow-up. OS was defined as the time between surgery and death due to HCC or date without follow-up. All experiments followed the ethical principles stipulated in the 1964 Declaration of Helsinki and its subsequent revisions. The study was approved by the Institutional Research Ethics Committee of the Affiliated Tumor Hospital of Xinjiang Medical University(K-2021024). Patient characteristics are provided in Table 1.

Table 1 Correlation analysis between the clinical features and TRIM36 expression in HCCImmunohistochemistry (IHC)

Sections from paraffin blocks were first dewaxed and then boiled with citrate solution, incubated with H2O2 for 10 min, and primary antibodies against TRIM36 (1:200, Sigma, USA), cleaved caspase-3 (1:20, Sigma), Ki-67 (1:5000, Proteintech, China), and β-catenin (1:2000, Proteintech). After washing, secondary antibodies were added. After washing, sections were stained with diaminobenzidine solution and hematoxylin. Sections were washed, dehydrated, and sealed with neutral balsam. Images were taken using an AXIO Vert A1 (Carl Zeiss AG, Germany) and ZEN software at magnifications of 100× and 200× .

Immunostaining intensity was scored as 0 to 3 (0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining). Stained area scores ranged from 0 to 4 (0, no staining; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, more than 75% of tumor cells stained). The final IHC score was calculated as staining intensity × area score. Each slide was evaluated by two independent pathologists who were blinded to the sample origin.

RNA isolation and RT-qPCR

TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to tissues or cells. Chloroform was then added to the samples, followed by incubation for 5 min at room temperature. Then, centrifugation at 12,000 g × 15 min at 4 °C was performed followed by harvest of the aqueous phase. Isopropanol was added next and centrifuged at 12,000 g for 10 min at 4 °C. The precipitate was harvested, washed with 75% ethanol, air dried, and resuspended in RNase-free water. Complementary DNA was synthesized using the Evo M-MLV RT Kit with gDNA Clean for qPCR II, according to the manufacturer’s instructions (Accurate Biology, China). The cDNA was amplified using the SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biology, China), according to the instruction, using pre-denaturation at 95 ℃ for 30 s; amplification at 95 ℃ for 5 s and 60 ℃ for 34 s, 40 cycles; melting at 95 ℃ for 15 s, 60 ℃ for 60 s, and 95 ℃ for 15 s, 1 cycle. ACTB was used as an internal reference gene. Data were collected and analyzed using a Lightcycler96 instrument and the 2–∆∆Ct method was used for analysis. Primers are listed in Additional file 1: Table S1.

Protein extraction

For total protein, lysis solution with radioimmunoprecipitation assay (RIPA, Beyotime) lysis and 1% phenylmethanesulfonyl fluoride (PMSF, Beyotime) were added to the tissues or cells on ice, and 30 min later, the lysate was centrifuged at 15,000 rpm for 15 min, and the supernatant was harvested. A nuclear protein, nuclear protein, and cytoplasmic protein extraction kit (Beyotime, China) was used to isolate nuclear proteins according to the manufacturer’s instructions. BCA kits (Beyotime, China) were used to measure protein concentrations. Protein samples were boiled with 25% v/V loading buffer (Beyotime, China) at 100 °C for 5 min and stored at − 20 °C.

Western blotting

Proteins were separated by SDS-PAGE. After electrophoresis, gels were cut and transferred to PVDF membranes. Membranes were blocked with 5% w/V evaporated milk in PBST. Diluted primary antibodies were added and incubated overnight at 4 °C. Primary antibodies were against TRIM36 (1:1000, Sigma), BAX (1:10,000, Proteintech), and BCL2 (1:2000, Proteintech), caspase-3 (1:1000, Abclone), cleaved caspase-3 (1:200, Sigma), caspase-7 (1:5000, Proteintech), PARP1 (1:1000, Abclone), MMP-9 (1:500, Proteintech), cyclin D1 (1:1500, Proteintech), β-catenin (1:20,000, Proteintech), active β-catenin (1:1000, Cell signaling technology), c-JUN (1:2000, proteintech), Histone H3 (1:10,000, Proteintech), Actin (1:10,000, Proteintech). Thereafter, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) or HRP-goat anti-mouse IgG (H + L) secondary antibodies (Proteintech) for 50 min at room temperature. Chemiluminescence was measured using Super Signal West Atto (Thermo Fisher Scientific). Blots were imaged with Amersham Imager 600 software and analyzed using ImageJ software.

Cell culture and transfection

Human normal liver cell LO2, human hepatoma cell lines PLC/PRF/5, Huh7, Hep-G2, HCCLM3, and MHCC-97H were purchased from the Chinese Academy of Sciences. All were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml) (all from Gibco) at 37 °C and 5% CO2 in a cell incubator. The siRNA targeting TRIM36 and β-catenin, and the corresponding si-control were purchased from RIB-BIO (China) and transfected into Huh7 and HCCLM3 cell lines using Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. Transfection efficiency was determined using western blotting and qRT–PCR. The sequences are listed in Additional file 1: Table S1. Purified Wnt ligand WNT3a (98.68%) (Cat: HY-P70453) were purchased from MedChemExpress (NJ, USA), and treated cells at 100 ng/mL concentration.

Lentiviral transduction

To establish stable cell lines, lentiviral vectors for increasing TRIM36 expression levels and a vector-only control were purchased from GENECHEM (China). Lentiviral particles were transduced into HCCLM3 and Huh7 cells, followed by puromycin screening for 2 weeks. Elevated expression of TRIM36 was identified by western blotting and qRT–PCR; only cells with stably elevated TRIM36 expression were used for further examination.

Cell counting

Cells expressing the target gene were seeded in 96-well plates with 100 μL complete medium. Thereafter, 10 μL of CCK-8 reagent (Dojindo Laboratories, Japan) was added to each well at different time points (0, 1, 2, 3, 4, 5, and 6 d), according to the manufacturer’s instructions. After incubation at 37 °C for 1 h, optical density was measured at 450 nm.

Colony formation

HCCLM3 and Huh7 cells with desired expression patterns were harvested and resuspended. Cells were seeded at 1000/well into six-well plates for colony formation. After approximately 2 weeks of culture, cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min at room temperature in the dark. Visible colonies (diameter > 0.1 mm were photographed and counted.

Migration and invasion assays

For the invasion assay, Matrigel film (Corning, USA) was prepared before cell inoculation; a similar procedure was employed for the migration and invasion assays. Cells with targeted gene expression were collected and resuspended in serum-free medium to the same density. Cell suspensions (200 μL) were seeded in the upper chamber and 750 μL of medium with 10% FBS was added to the lower chamber. Cells were cultured for 48 h, fixed with formaldehyde, and stained with 0.1% crystal violet. Cells in the upper chamber were removed, and cells that crossed the membrane were retained. Images were taken with an EVOS XL Core Instrument (AMEX1000, Thermo Fisher Scientific, USA) at 100× and 200× magnifications. Cells were counted using ImageJ software for five fields at 200× magnification per chamber (n = 1) in triplicate. Images are shown at 100× magnification.

TUNEL apoptosis assay

Cells expressing TRIM36 were seeded into 96-well plates. Cells were fixed with 4% paraformaldehyde and incubated with 0.1% Triton X-100 for 2 min. After the addition of 50 μL TUNEL solution per well, cells were incubated in the dark at 37 °C for 60 min and washed twice with PBS. Fluorescence was captured using an AXIO Vert A1 fluorescence microscope (Carl Zeiss AG, Germany) and ZEN software at 200× magnification.

Annexin V/PI apoptosis assay

Apoptosis was measured by annexin V/PI staining (Thermo Fisher Scientific, USA). Cells with altered TRIM36 expression were harvested and resuspended in 200 μL of buffer containing annexin V and propidium iodide (PI) for 15 min at 37 °C in the dark. Staining intensities were measured by flow cytometry to distinguish between healthy, apoptotic, and dead cells. Data were analyzed using FlowJo version 10.8.0.

RNA-Seq

TRIM36 was knocked down in Huh7 cells using siRNA (RIB-BIO, China) in parallel with control cells with the control siRNA. TRIzol™ reagent (Thermo Fisher Scientific, USA) was used to purify total RNA from cells. An Illumina 6000-PE150 (Illumina, USA) was used for RNA sequencing. Data were annotated to the human genome and differentially expressed mRNAs were analyzed using R software.

Bioinformatics

Heatmap and volcano plots were drawn using R based on differential gene analysis. Gene Oncology (GO) was used to perform functional analyses of differentially expressed genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to identify enriched pathways. Gene set enrichment analysis (GSEA) was performed to identify significantly enriched genes using GSEA software (Broad Institute, Cambridge, MA).

Murine xenograft model

Five-week-old BALB/c male nude mice were purchased from the Chinese Academy of Sciences. Huh7 cells were transduced with either lenti-sh-TRIM36 (GENECHEM) or lenti-sh-NC (GENECHEM). Cells were suspended in serum-free DMEM and mixed with Matrigel (Corning) in a 1:1 ratio. Cells with lenti-sh-TRIM36 were injected subcutaneously into the right dorsum, and cells containing lenti-sh-NC were injected into the left dorsum as controls. Tumor width and length were measured every 3 days, and volumes were calculated according to the formula V = length × width2 × 0.52. Mice were euthanized with 5% Pentobarbital and necropsied after 3 weeks. Weights of mice and their subcutaneous masses were recorded. Excised tissues were stored separately for different measurements.

Statistical analysis

For HCC patients, clinical information was recorded and analyzed. A TRIM36 IHC score of 4 + was set as the cutoff, as the mean score in HCC tumor samples was 3.66. Thus, a score ≤ 4 represents a low expression level of TRIM36 and could be used to divide patients into comparable groups. Differences between groups were analyzed using the Kaplan–Meier method with the log-rank test. Cox univariate analysis was conducted for TRIM36 expression and other indicators commonly used for clinical prognosis. Significant variables (p < 0.05) were selected for Cox multivariate analysis, with a statistically significant p-value of < 0.05. At least three replicates were performed for both in vitro and in vivo assays. Data are presented as means ± standard deviations. Student’s t-test was used for comparisons between two groups. ANOVA was used to measure the differences between groups when they numbered three or more. All analyses were performed using SPSS software (Munich, Germany). Figures were generated using GraphPad Prism 8 (San Diego, CA, USA). Software used is shown in Additional file 2: Table S2.

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