GBAP1 functions as a tumor promotor in hepatocellular carcinoma via the PI3K/AKT pathway

Data of 374 liver hepatocellular carcinoma (LIHC) tissue samples and 50 normal liver tissues were downloaded from The Cancer Genome Atlas (TCGA, https://​cancergenome.​nih.​gov/​) [21]. The LICA-FR liver cancer data, including 150 liver cancer and 11 para-cancerous tissue samples from 150 patients with HCC, were acquired from the International Cancer Genome Consortium (ICGC; https://​icgc.​org) [22]. The GSE76427, GSE14520, GSE101685, GSE54236, and GSE64041 datasets were retrieved from Gene Expression Omnibus (GEO, https://​www.​ncbi.​nlm.​nih.​gov/​geo/​) [23]. The GSE76427 dataset included data for 115 cancer tissue samples and 52 paracancerous samples from 115 patients with HCC based on GPL10558 (Illumina HumanHT-12 V4.0 Expression BeadChip) [24]. The GSE14520 dataset was developed from 222 cancer tissue samples and 212 paracancerous samples detected by the Affymetrix HT Human Genome U133A Array [25, 26]. The GSE101685 dataset contained expression profiling data obtained by Affymetrix Human Genome U133 Plus 2.0 from 8 normal tissues and 24 HCC cases. The GSE54236 dataset included data for 80 normal liver samples and 81 HCC samples [27] and GSE64041 included data for 60 biopsy pairs from patients with HCC plus five normal liver biopsies [28]. The differential expression analysis and visualization of results were performed using R (https://​www.​r-project.​org/​) with the limma and VennDiagram packages. The criteria for DEG identification were |log2 fold change (log2 FC)| > 1 and adjusted p < 0.05. Expression of GBAP1 in multiple tumors was evaluated using the Tumor IMmune Estimation Resource (TIMER, cistrome.shinyapps.io/timer) database.

To annotate biological processes or pathways related to dysregulated genes in HCC, a Gene Ontology (GO) Biological Process enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and gene set enrichment analysis (GSEA) were performed using the clusterProfiler R package. For GSEA, “h.all.v7.1.symbols.gmt” from the Molecular Signatures Database (MsigDB; http://​software.​broadinstitute.​org/​gsea/​msigdb/​collections.​jsp#H) was applied using the expression matrix of HCC samples from TCGA. A gene set was considered enriched when p < 0.05. Then, the intersecting DEGs were evaluated using the Search Tool for the Retrieval of Interacting Genes (STRING) (http://​string-db.​org; version: 11.0) in Cytoscape to build a protein–protein interaction (PPI) network and hub genes were identified using the plug-in cytoHubba.

Hub genes strongly associated with prognosis and survival in HCC were screened out by a combination of LASSO Cox regression and univariate Cox regression analyses. Briefly, samples were classified into groups with low and high expression levels using the R package bestSeparation. A survival analysis was performed using the survival (https://​github.​com/​therneau/​surviva) and survminer packages (https://​github.​com/​kassambara/​survminer). Statistical significance was defined as log-rank p < 0.05. For GBAP1, univariate and multivariate Cox regression analyses were also performed.

The immortalized human hepatocyte (MIHA) cell line was purchased from Lonza (Basel, Switzerland), the human HCC cell lines Huh7, Hep3B, and HepG2 were obtained from ATCC (Rockville, MD, USA), and MHCC-97 was obtained from the Chinese Academy of Sciences Cell Bank. Cells were cultivated in Dulbecco’s Modified Eagle Medium Thermo Fisher Scientific, Carlsbad, CA, USA) under standard conditions at 37 °C with 5% CO₂. The 740Y-P reagent was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Cells were incubated with 740Y-P at a final concentration of 10 µM for 24 h for subsequent experimental detection.

To separate the nuclear and cytoplasmic fractions, RNAs were detached using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek Corp., Thorold, Canada) according to the manufacturer’s instructions. Approximately 1 × 10⁷ cells (HepG2) were lysed with lysis buffer on ice, and cytoplasmic or nuclear RNA was separated by centrifugation (14,000 × g) for 10 min, followed by q-PCR.

The coding sequence of GBAP1 was acquired from NCBI and used to design short hairpin RNAs (shRNAs) using RNAi Designer (Table 1). Next, shRNAs were cloned into the BamHI and EcoR I sites of the pLVX-puro vector. The lentiviral plasmid pLVX-shRNA2-puro and helper plasmids were co-transfected into 293T cells to generate lentiviral particles expressing the target fragment. The supernatant containing lentiviral particles was then harvested. Lentivirus infection was carried out using cells at 70% confluence with a multiplicity of infection (MOI) of 10 for HepG2 and Hep3B cells.

The CCK-8 assay (96992-100TESTS-F, Sigma, St. Louis, MO, USA) was performed to assess cell growth. Approximately 5 × 10³ cells per well were added to 96-well microplates. Then, 10 µl of CCK-8 was added to the cells for 1 h. Absorbance was evaluated at 450 nm for each well using a microplate reader (PerkinElmer, VICTOR NIVO, Waltham, MA, USA). All experiments were performed in triplicate.

Cells in the logarithmic growth phase were seeded in 96-well plates (4 × 10³~1 × 10⁵ cells/well) and 100 µL of 50 µM EdU medium was added to each well. Cells were fixed, washed with PBS, and 100 µL of 1×Apollo staining reaction solution was added to each well. The cells were then washed with PBS and 100 µL of 1×Hoechst33342 reaction solution was added to each well for DNA staining. The cells were observed under a fluorescence microscope and the EdU positive rate (number of EdU positive cells/total cells) was determined.

The Annexin V-FITC/PI Double Staining Kit (Jiangsu KeyGEN, KGA108-1, Nanjing, China) was used to detect apoptotic cells by flow cytometry, as described previously [29]. Briefly, positively stained cells (apoptotic cells) were quantified (ACEA Bio, 2040R; San Diego, CA, USA) and analyzed using FlowJo-V10.

Total RNA from tissue or cells was extracted with TRIzol reagent (Thermo Fisher Scientific, 15,596,026) following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized and PCR with cDNA templates was performed using a real-time detector (Analytik Jena AG, qTower 3.2G; Jena, Germany) using BeyoFast SYBR Green qPCR Mix (Bio-Rad, 1708882AP, Shanghai, China). The primer sequences were shown in Table 2. Transcript levels were normalized against GAPDH levels as an internal reference and were evaluated using the 2^−ΔΔCt method. All experiments were repeated three times.

The cells or tissues were lysed in 2× sodium dodecyl sulfate (SDS) lysis buffer. Protein concentrations were measured using the BCA Protein Concentration Assay Kit (Beyotime, Shanghai, China) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the cell lysates. The samples were moved to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against PCNA, MCM2, PI3K, p-PI3K, AKT, and p-AKT (1:1000; ABM, Vancouver, Canada) and GAPDH as a reference (1:6,000; Proteintech, Wuhan, China) overnight at 4 °C. The membrane was then incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibody (CST, Danvers, MA, USA) for 2 h at 25 °C. Signals were detected using the Enhanced Chemiluminescence Kit (NCM Biotech, Suzhou, China) and a chemiluminescence imaging system (Tanon, Shanghai, China). As there were many target proteins, the different incubation conditions of each antibody lead to different gel cutting and exposure time. In order to develop and expose in time, we cut it independently. As a result, different films were not exposed together.

Female BALB/c nude mice (5 to 6 weeks old) weighing approximately 18–20 g were acquired from the Shanghai Experimental Animal Center, Chinese Academy of Sciences (Shanghai, China). Mice were maintained in a specific pathogen-free (SPF) animal facility. Twelve mice were randomly classified into two groups: sh-lncRNA-GBAP1 and NC (n = 6 each). Approximately 5 × 10⁶ cells were subcutaneously injected into the oxter of each mouse. The subcutaneous tumor length and width were determined every 4 days. The tumor size was calculated as follows: bulk (m³) = 1/2 length × width². All mice were euthanized 30 days after inoculation.

Surgically resected tumor samples were fixed in formaldehyde and embedded in paraffin. The samples were washed with PBS three times, and 5% bovine serum albumin was used to block non-specific reactions. Antibodies against MCM2 (1:200 dilution) and PCNA (1:300 dilution) were added, followed by incubation at 4 °C overnight. Then, the slides were stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin according to the manufacturer’s protocol. Images were acquired using a Nikon Eclipse 80i microscope (Nikon Americas Inc., Melville, NY, USA).

In total, 21 pairs of fresh liver cancer tissues and adjacent normal tissues were acquired between January 2021 and April 2021 from patients with HCC who did not receive any preoperative anti-tumor treatment or surgery at Jiangsu Provincial People’s Hospital. All experiments were approved by the Ethics Committee (2021ZDSYLL245-P01).

Statistical analyses were performed using GraphPad Prism 8.0. The log-rank test was applied to assess differences between survival curves. For cell and animal experiments, Student’s t-tests were used for comparisons between two groups. Univariate comparisons among multiple groups were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s tests. Statistical significance was set at P < 0.05.