Search

Background: Hepatocellular carcinoma (HCC) accounts for a majority of primary liver cancer cases and related deaths. The purpose of this study was to assess the diagnostic value of splicing factor 3b subunit 4 (SF3B4) as a novel non-invasive biomarker for HCC and determine the association between SF3B4 expression and immune cell infiltration. Methods: An enzyme-linked immunosorbent assay (ELISA) was used to detect SF3B4 levels in plasma samples obtained from healthy controls (HCs) and patients with chronic hepatitis, liver cirrhosis, and HCC. The expression levels of autoantibodies that detect SF3B4 in the plasma samples of each group of patients were measured. Small extracellular vesicles (EVs) were isolated from patient sera, and the expression levels of EV-SF3B4 were measured using quantitative reverse transcription PCR. Results: ELISA results confirmed that the expression levels of SF3B4 proteins and autoantibodies in the plasma of patients with HCC were higher than those in HCs. However, their diagnostic performance was not better than that of alpha-fetoprotein (AFP). The mRNA expression of SF3B4 in serum EV increased but not in the buffy coat or serum of patients with HCC. Serum EV-SF3B4 displayed better diagnostic power than AFP for all stages of HCC (AUC = 0.968 vs. 0.816), including early-stage HCC (AUC = 0.960 vs. 0.842), and this was consistent in the external cohort. Single-cell RNA sequencing indicated that SF3B4 expression was correlated with myeloid-derived suppressor cells. The Tumor Immune Estimation Resource database reconfirmed the correlation between SF3B4 expression and immune cell infiltration in HCC. Conclusions: SF3B4 may be associated with tumor immune infiltration in HCC, and EV-SF3B4 shows potential as a novel non-invasive diagnostic biomarker of HCC. [ABSTRACT FROM AUTHOR]

Cancer-associated fibroblasts (CAFs) contribute to tumor progression, and microRNAs (miRs) play an important role in regulating the tumor-promoting properties of CAFs. The objectives of this study were to clarify the specific miR expression profile in CAFs of hepatocellular carcinoma (HCC) and identify its target gene signatures. Small-RNA-sequencing data were generated from nine pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively. Bioinformatic analyses were performed to identify the HCC-CAF-specific miR expression profile and the target gene signatures of the deregulated miRs in CAFs. Clinical and immunological implications of the target gene signatures were evaluated in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA_LIHC) database using Cox regression and TIMER analysis. The expressions of hsa-miR-101-3p and hsa-miR-490-3p were significantly downregulated in HCC-CAFs. Their expression in HCC tissue gradually decreased as HCC stage progressed in the clinical staging analysis. Bioinformatic network analysis using miRWalks, miRDB, and miRTarBase databases pointed to TGFBR1 as a common target gene of hsa-miR-101-3p and hsa-miR-490-3p. TGFBR1 expression was negatively correlated with miR-101-3p and miR-490-3p expression in HCC tissues and was also decreased by ectopic miR-101-3p and miR-490-3p expression. HCC patients with TGFBR1 overexpression and downregulated hsa-miR-101-3p and hsa-miR-490-3p demonstrated a significantly poorer prognosis in TCGA_LIHC. TGFBR1 expression was positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and M2 macrophages in a TIMER analysis. In conclusion, hsa-miR-101-3p and hsa-miR-490-3p were substantially downregulated miRs in CAFs of HCC, and their common target gene was TGFBR1. The downregulation of hsa-miR-101-3p and hsa-miR-490-3p, as well as high TGFBR1 expression, was associated with poor clinical outcome in HCC patients. In addition, TGFBR1 expression was correlated with the infiltration of immunosuppressive immune cells. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. Wiskott–Aldrich syndrome protein family member 2 (WASF2) is an integral member of the actin cytoskeleton pathway, which plays a crucial role in cell motility. In this study, we aimed to explore the role of WASF2 in HCC carcinogenesis and its regulatory mechanism. Methods: WASF2 expression in HCC was analyzed using six public RNA-seq datasets and 66 paired tissues from patients with HCC. The role of WASF2 in normal hepatocyte cell phenotypes was evaluated using a WASF2 overexpression vector in vitro; it was evaluated in HCC cell phenotypes using small interfering RNA (siRNA) in vitro and in vivo. Epigenetic regulatory mechanism of WASF2 was assessed in the Cancer Genome Atlas liver hepatocellular carcinoma project (TCGA_LIHC) dataset and also validated in 38 paired HCC tissues. Site mutagenesis, bisulfite sequencing polymerase chain reaction (BSP), methylation-specific polymerase chain reaction (MSP), and quantitative MSP (qMSP) were used for evaluating WASF2 methylation status. Results: WASF2 is overexpressed in HCC and is clinically correlated with its prognosis. WASF2 overexpression promoted normal hepatocyte proliferation. WASF2 inactivation decreased the viability, growth, proliferation, migration, and invasion of Huh-7 and SNU475 HCC cells by inducing G2/M phase arrest. This induced cell death and inhibited epithelial–mesenchymal transition, hindering actin polymerization. In addition, WASF2 knockdown using siWASF2 in a xenograft mouse model and a lung metastasis model exerted tumor suppressive effect. There was a negative correlation between WASF2 methylation status and mRNA expression. The methylation pattern of CpG site 2 (− 726 bp), located in the WASF2 promoter, plays an important role in the regulation of WASF2 expression. Furthermore, the cg242579 CpG island in the WASF2 5′ promoter region was hypomethylated in HCC compared to that in the matched non-tumor samples. Patients with high WASF2 methylation and low WASF2 expression displayed the highest overall survival. Conclusions: WASF2 is overexpressed and hypomethylated in HCC and correlates with patient prognosis. WASF2 inactivation exerts anti-tumorigenic effects on HCC cells in vitro and in vivo, suggesting that WASF2 could be a potential therapeutic target for HCC. [ABSTRACT FROM AUTHOR]

Liquid biopsy can provide a strong basis for precision medicine. We aimed to identify novel single-nucleotide variants (SNVs) in circulating tumor DNA (ctDNA) in patients with hepatocellular carcinoma (HCC). Deep sequencing of plasma-derived ctDNA from 59 patients with HCC was performed using a panel of 2924 SNVs in 69 genes. In 55.9% of the patients, at least one somatic mutation was detected. Among 25 SNVs in 12 genes, four frequently observed SNVs, MLH1 (13%), STK11 (13%), PTEN (9%), and CTNNB1 (4%), were validated using droplet digital polymerase chain reaction with ctDNA from 62 patients with HCC. Three candidate SNVs were detected in 35.5% of the patients, with a frequency of 19% for MLH1 chr3:37025749T>A, 11% for STK11 chr19:1223126C>G, and 8% for PTEN chr10:87864461C>G. The MLH1 and STK11 SNVs were also confirmed in HCC tissues. The presence of the MLH1 SNV, in combination with an increased ctDNA level, predicted poor overall survival among 107 patients. MLH1 chr3:37025749T>A SNV detection in ctDNA is feasible, and thus, ctDNA can be used to detect somatic mutations in HCC. Furthermore, the presence or absence of the MLH1 SNV in ctDNA, combined with the ctDNA level, can predict the prognosis of patients with HCC. [ABSTRACT FROM AUTHOR]

The aim of this study was to explore novel genomic biomarkers predicting hepatocellular carcinoma (HCC) prognosis by integrative analysis of DNA copy number aberrations (CNAs) and gene expression profiles. Array comparative genomic hybridization and expression array were performed on 45 and 31 HCC samples, respectively. To identify functionally important genes, concordant results of DNA copy number and gene expression were retrieved by integrative analysis. Cox regression analysis indicated that the CNAs in 192 genomic regions were significantly associated with overall survival (OS; p < 0.05). Integrative analysis capturing concordant results demonstrated that the low expression of TLE4 ( p = 0.041) and XPA ( p = 0.006) was associated with poor OS. In the analysis of tumor recurrence, 514 genomic regions with CNAs were associated with recurrence. Integrative analysis revealed that the overexpression of 16 genes, including FGR ( p = 0.003), RELA ( p = 0.049), LTBP3 ( p = 0.050), and RIN1 ( p = 0.023), was significantly associated with shorter time to tumor recurrence. On multivariate analysis, FGR and XPA were independent risk factors of early recurrence and poor OS, respectively. Integrated analysis of CNAs and gene expression profiles correlated with long-term follow-up data successfully identified potential prognostic markers predicting survival and tumor recurrence in patients with HCC who underwent surgical resection. [ABSTRACT FROM AUTHOR]

Simple Summary: A high percentage of patients who undergo surgical resection for hepatocellular carcinoma (HCC) experience recurrence. Therefore, identification of accurate molecular markers for predicting recurrence of HCC is important. We analyzed recurrence and non-recurrence HCC tissues using two public omics datasets comprising microarray and RNA-sequencing and found novel gene signatures associated with recurrent HCC. These molecules might be used to not only predict for recurrence of HCC but also act as potential prognostic indicators for patients with HCC. Hepatocellular carcinoma (HCC) has a high rate of cancer recurrence (up to 70%) in patients who undergo surgical resection. We investigated prognostic gene signatures for predicting HCC recurrence using in silico gene expression analysis. Recurrence-associated gene candidates were chosen by a comparative analysis of gene expression profiles from two independent whole-transcriptome datasets in patients with HCC who underwent surgical resection. Five promising candidate genes, CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB were identified, and the expression of these genes was evaluated using quantitative reverse transcription PCR in the validation set (n = 57). The genes CETN2, HMGA1, RACGAP1, and SNRPB, but not MPZL1, were upregulated in patients with recurrent HCC. In addition, the combination of HMGA1 and MPZL1 demonstrated the best area under the curve (0.807, 95% confidence interval [CI] = 0.681–0.899) for predicting HCC recurrence. In terms of clinicopathological correlation, CETN2, MPZL1, RACGAP1, and SNRPB were upregulated in patients with microvascular invasion, and the expression of MPZL1 and SNRPB was increased in proportion to the Edmonson tumor differentiation grade. Additionally, overexpression of CETN2, HMGA1, and RACGAP1 correlated with poor overall survival (OS) and disease-free survival (DFS) in the validation set. Finally, Cox regression analysis showed that the expression of serum alpha-fetoprotein and RACGAP1 significantly affected OS, whereas platelet count, microvascular invasion, and HMGA1 expression significantly affected DFS. In conclusion, HMGA1 and RACGAP1 may be potential prognostic biomarkers for predicting the recurrence of HCC after surgical resection. [ABSTRACT FROM AUTHOR]

Reverse transcription-quantitative (RT-q) PCR is the most feasible and useful technique for identifying and evaluating cancer biomarkers; however, the method requires suitable reference genes for gene expression analysis. The aim of the present study was to identify the most suitable reference gene for the normalization of relative gene expression in human hepatocellular carcinoma (HCC) tissue and blood samples. First, 14 candidate reference genes were selected through a systematic literature search. The expression levels of these genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPLP0, RPL13A, SDHA, TBP, TFRC and YWHAZ) were evaluated using human multistage HCC transcriptome data (dataset GSE114564), which included normal liver (n=15), chronic hepatitis (n=20), liver cirrhosis (n=10), and early (n=18) and advanced HCC (n=45). From the 14 selected genes, five genes, whose expression levels were stable in all liver disease statuses (ACTB, GAPDH, HMBS, PPIA and RPLP0), were further assessed using RT-qPCR in 40 tissues (20 paired healthy tissues and 20 tissues from patients with HCC) and 40 blood samples (20 healthy controls and 20 samples from patients with HCC). BestKeeper statistical algorithms were used to identify the most stable reference genes, of which HMBS was found to be the most stable in both HCC tissues and blood samples. Therefore, the results of the present study suggest HMBS as a promising reference gene for the normalization of relative RT-qPCR techniques in HCC-related studies. [ABSTRACT FROM AUTHOR]

Objectives Aim of this study was to investigate the prognostic potential of plasma microRNA-122 levels in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma after hepatic resection or radiofrequency ablation (RFA). Design and methods A total of 120 patients with HBV-related hepatocellular carcinoma who underwent hepatic resection (n = 63) or RFA (n = 57) were included. The pretreatment plasma microRNA-122 level was assessed using quantitative real time polymerase chain reaction, and the correlation between microRNA-122 expression and various clinical parameters was investigated. Results Multivariate Cox regression analysis demonstrated that, in all patients, a low platelet count (< 100 × 10 9 /L), low albumin level (≤ 3.5 g/dL.), and advanced tumor stage (modified Union for International Cancer Control stage III/IV) were independent prognostic factors for disease-free survival, while a low albumin level and advanced tumor stage were independent prognostic factors for overall survival (OS). In a subgroup analysis of patients who underwent RFA, the patients with high miR-122 expression (> 100) had significantly lower OS on Kaplan–Meier analysis ( P = 0.042). Furthermore, high microRNA-122 expression (hazard ratio [HR] = 2.67; 95% confidence interval [CI] = 1.12–6.35; P = 0.026) and advanced tumor stage (HR = 2.27; 95% CI = 1.23–4.18; P = 0.009) were independent risk factors for poor OS in patients treated with RFA. The combination of microRNA-122 and tumor stage resulted in an area under the curve of 0.818 for predicting 1-year OS in patients who underwent RFA. Conclusions High plasma microRNA-122 expression was associated with poor OS in patients with HBV-related hepatocellular carcinoma who underwent RFA. [ABSTRACT FROM AUTHOR]