Circulating small extracellular vesicle-derived splicing factor 3b subunit 4 as a non-invasive diagnostic biomarker of early hepatocellular carcinoma.

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.

Tumor Endothelial Cells-Associated Integrin Alpha-6 as a Promising Biomarker for Early Detection and Prognosis of Hepatocellular Carcinoma.

HCC remains a lethal cancer type, with early detection being critical for improved patient outcomes. This study introduces a comprehensive methodological approach to identify the ITGA6 gene as a potential blood marker for early HCC (eHCC) detection. We initially analyzed the GSE114564 dataset encompassing various stages of liver disease, identifying 972 differentially expressed genes in HCC. A refined analysis yielded 59 genes specifically differentially expressed in early HCC, including ITGA6. Subsequent validation in multiple datasets confirmed the consistent upregulation of ITGA6 in HCC. In addition, when analyzing progression-free survival (PFS) within the entire patient cohort and overall survival (OS) specifically among patients classified as tumor grade G1, the group of patients characterized by high expression levels of ITGA6 displayed an elevated risk ratio in relation to prognosis. Further analyses demonstrated the predominant expression of ITGA6 in TECs and its enrichment in angiogenesis-related pathways. Additionally, positive correlations were found between ITGA6 expression and pro-tumorigenic immune cells, but not with anti-tumorigenic immune cells. Our study elucidates the potential of ITGA6 as a blood-based marker for HCC early detection and diagnosis and its complex interplay with the tumor microenvironment. Further research may lead to novel strategies for HCC management and patient care.

Aberrantly Expressed MicroRNAs in Cancer-Associated Fibroblasts and Their Target Oncogenic Signatures in Hepatocellular Carcinoma.

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.

Cancer-associated fibroblast-derived secreted phosphoprotein 1 contributes to resistance of hepatocellular carcinoma to sorafenib and lenvatinib.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the induction of chemo-resistance. This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors (TKIs), sorafenib and lenvatinib, and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma (HCC). METHODS: We performed a systematic integrative analysis of publicly available gene expression datasets and whole-transcriptome sequencing data from 9 pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively, to identify key molecules that might induce resistance to TKIs. We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms. The associations of plasma secreted phosphoprotein 1 (SPP1) expression levels before sorafenib/lenvatinib treatment with progression-free survival (PFS) and overall survival (OS) of 54 patients with advanced HCC were evaluated using Kaplan-Meier and Cox regression analysis. RESULTS: Bioinformatic analysis identified CAF-derived SPP1 as a candidate molecule driving TKI resistance. SPP1 inhibitors reversed CAF-induced TKI resistance in vitro and in vivo. CAF-derived SPP1 activated rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) through the integrin-protein kinase C-alpha (PKCα) signaling pathway and promoted epithelial-to-mesenchymal transition (EMT). A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS (P = 0.026) and OS (P = 0.047) in patients with advanced HCC after TKI treatment. CONCLUSIONS: CAF-derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion. Its inhibition represents a promising therapeutic strategy against TKI resistance in HCC. Moreover, plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.

Hypomethylation-mediated upregulation of the WASF2 promoter region correlates with poor clinical outcomes in hepatocellular carcinoma.

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.

HMBS is the most suitable reference gene for RT-qPCR in human HCC tissues and blood samples.

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.

Overexpressed Proteins in HCC Cell-Derived Exosomes, CCT8, and Cofilin-1 Are Potential Biomarkers for Patients with HCC.

Protein markers of hepatocellular carcinoma (HCC)-derived exosomes (HEX) have not yet been fully evaluated. Here, we identified novel protein contents of HEX and their clinical significance as biomarkers. Exosomes were isolated from human HCC cell lines and an immortalized normal hepatocyte cell line. Proteomic analyses revealed 15 markedly overexpressed proteins in HEX. The clinical relevance of the 15 proteins was analyzed in public RNA-sequencing datasets, and 6 proteins were selected as candidate of potential biomarkers. Serum CCT8 and CFL1 were markedly overexpressed in test cohort (n = 8). In the validation cohort (n = 224), the area under the curve (AUC) of serum CCT8 and CFL1 for HCC diagnosis was calculated as 0.698 and 0.677, respectively, whereas that of serum alpha-fetoprotein (AFP) was 0.628. The combination of three serum markers (CCT8, CFL1, and AFP) demonstrated the highest AUC for HCC diagnosis. (AUC = 0.838, 95% confidence interval = 0.773-0.876) Furthermore, higher serum CCT8 and CFL1 concentrations were significantly associated with the presence of vascular invasion, advanced tumor stage, poor disease-free survival, and poor overall survival. Cofilin-1 and CCT8, enriched proteins in HEX, were identified as potential diagnostic and prognostic serum biomarkers for HCC patients.

Circulating Exosomal MicroRNA-1307-5p as a Predictor for Metastasis in Patients with Hepatocellular Carcinoma.

Exosomal microRNAs (exo-miRs) contribute to cancer metastasis. To identify pro-metastatic circulating exo-miRs in hepatocellular carcinoma (HCC), next-generation sequencing-based plasma exo-miR profiles of 14 patients with HCC (eight non-metastatic and six with metastasis within 1 year of follow-up) were analyzed. Sixty-one miRs were significantly overexpressed among patients with metastatic HCC. Candidate miRs were selected through integrative analyses of two different public expression datasets, GSE67140 and The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA_LIHC). Integrative analyses revealed 3 of 61 miRs (miR-106b-5p, miR-1307-5p, and miR-340-5p) commonly overexpressed both in metastasis and vascular invasion groups, with prognostic implications. Validation was performed using stored blood samples of 150 patients with HCC. Validation analysis showed that circulating exo-miR-1307-5p was significantly overexpressed in the metastasis group (p = 0.04), as well as in the vascular invasion and tumor recurrence groups. Circulating exo-miR-1307-5p expression was significantly correlated with tumor stage progression (p < 0.0001). Downstream signaling pathways of miR-1307 were predicted using TargetScan and Ingenuity Pathway Analysis. On comprehensive bioinformatics analysis, the downstream pathway of miR-1307-5p, promoting epithelial-mesenchymal transition (EMT), showed SEC14L2 and ENG downregulation. Our results show that circulating exo-miR-1307-5p promotes metastasis and helps predict metastasis in HCC, and SEC14L2 and ENG are target tumor suppressor genes of miR-1307 that promote EMT.

Detection of Allergen Specific Antibodies From Nasal Secretion of Allergic Rhinitis Patients.

PURPOSE: Allergic rhinitis (AR) is a common and increasing disease in which Dermatophagoides (D.) farinae is one of the most common causative allergens. The aims of this study were to confirm the presence of locally produced antibodies to D. farinae in nasal secretions between nasal provocation test (NPT)-positive and -negative groups of AR patients, to evaluate their relationships with the levels of inflammatory mediators, and to determine adaptive and innate immune responses in nasal mucosa. METHODS: Sixty AR patients sensitive to house dust mites confirmed by skin prick test or serum specific IgE to D. farinae underwent NPT for D. farinae. Nasal packs were placed in both nasal cavities of the patients for 5 minutes to obtain nasal secretions after NPT. The levels of total IgE, specific IgE to D. farinae, eosinophil cationic protein (ECP), and tryptase in nasal secretions were detected by using ImmunoCAP. The levels of specific IgE, IgA, and secretory IgA antibodies to D. farinae in nasal secretions were measured by using ELISA. The levels of IL-8, VEGF, IL-25, and IL-33 were also measured by using ELISA. RESULTS: High levels of total IgE, specific IgE, specific IgA, and secretory IgA to D. farinae, as well as inflammatory mediators, such as ECP, IL-8, VEGF and tryptase, were detected in nasal secretions, although the differences were not statistically significant between the NPT-positive and NPT-negative groups. Levels of all immunoglobulins measured in this study significantly correlated with ECP, IL-8, and VEGF (P<0.05), but not with tryptase (P>0.05). IL-33 and IL-25 were also detected, and IL-25 level significantly correlated with IL-8 (r=0.625, P<0.001). CONCLUSIONS: These findings confirmed the presence of locally produced specific antibodies, including D. farinae-specific IgE and IgA, in nasal secretions collected from D. farinae-sensitive AR patients in both the NPT-positive and NPT-negative groups, and close correlations were noted between antibodies and nasal inflammatory mediators, including such as ECP, IL-8 and VEGF, indicating that locally produced antibodies may be involved in the nasal inflammation of AR.