HBV-related Hepatocellular Carcinoma Tissues Research Articles

Overexpression of tousled-like kinase 2 predicts poor prognosis in HBV-related hepatocellular carcinoma patients after radical resection.

Background: Tousled-like kinase 2 (TLK2) is integral to DNA repair, replication, and cell cycle regulation, crucial for maintaining genome stability and integrity. However, the expression and prognostic value of TLK2 in hepatitis B viral (HBV) -related hepatocellular carcinoma (HCC) remains unclear. Methods: We examined TLK2 expression and prognostic implications in pan-cancer by using diverse databases. Subsequently, TLK2 expression in HBV-related HCC tissues and adjacent tissues was assessed using quantitative real-time PCR and immunohistochemistry. The prognostic value of TLK2 was assessed through ROC curves, time-dependent ROC curves, Cox regression, Kaplan-Meier curve, and decision curve analysis. Additionally, analyses of immune infiltration, protein-protein interactions, key molecules of tumor-related signaling pathways, molecular subtypes, and TLK2-associated differentially expressed genes (DEGs) were conducted, along with GO/KEGG and GSEA enrichment analyses. Results: TLK2 expression was significantly higher in HCC tissues compared to adjacent tissues and correlated with gender, AFP levels, albumin-bilirubin (ALBI) grade, microvascular invasion (MVI), maximum tumor diameter, tumor number, and TNM stage. TLK2 overexpression emerged as an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) in HBV-related HCC patients. An integrated OS nomogram model, incorporating TLK2, age, ALBI grade, MVI, and tumor number, displayed enhanced prognostic capability (C-index: 0.765, 95% CI: 0.732-0.798) in predicting OS and has a higher net benefit than the TNM stage. Moreover, TLK2 expression correlated closely with immune cell infiltration and key molecules of signaling pathways. Functional enrichment analyses highlighted significant associations with DNA duplex unwinding, double-strand break repair, DNA replication, cell cycle, E2F targets, G2M checkpoint, and MYC targets V1. Conclusion: TLK2 is notably overexpressed in HBV-related HCC and emerges as a promising prognostic biomarker, necessitating further validation.

Hepatitis B Virus-Encoded MicroRNA (HBV-miR-3) Inhibits FIH-1 Expression to Promote Tumor Angiogenesis in HBV-Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a solid tumor with a rich blood supply, and anti-angiogenesis has important clinical significance. Hepatitis B Virus-Encoded MicroRNA 3 (HBV-miR-3) has recently been reported to be involved in HCC development. In this study, we aim to elucidate the role of HBV-miR-3 in promoting HBV-related HCC angiogenesis through Factor Inhibiting Hypoxia-inducible factor 1 (FIH-1). By analyzing HBV-related HCC tissue samples, we found that high expression of HBV-miR-3 was associated with poor overall survival and HBV-miR-3 expression was significantly correlated with VEGFR2 and FIH-1 expressions. In vitro, HBV-miR-3 agomir repressed FIH-1 expression and promoted HIF-1α/VEGFA signaling activation in HepG2 cells, resulting in increased HUVEC lumen formation in HepG2-HUVEC co-culture model. Conversely, HBV-miR-3 antagomir induced FIH-1 expression and inhibited HIF-1α/VEGFA signaling activation in HepG2.2.15 cells, resulting in decreased HUVEC lumen formation in HepG2.2.15-HUVEC co-culture model. The effect of HBV-miR-3 to HCC angiogenesis was also confirmed by a mouse tumor bearing model. We also confirmed that HBV-miR-3 repressed FIH-1 expression via targeting the 3'-UTR of FIH-1 mRNA by luciferase activity assay. HBV-miR-3 was related to HCC patients' overall survival and it promoted angiogenesis by repressing FIH-1 expression. HBV-miR-3 may be a new marker for predicting prognosis and a novel target for anti-angiogenic treatment of HBV-related HCC.

COL15A1 interacts with P4HB to regulate the growth and malignancy of HepG2.2.15 cells

Liver cancer is the fourth most common cause of cancer related deaths, ranking sixth in terms of incidence rate, and hepatocellular carcinoma (HCC) is the main type of liver cancer. Hepatitis B virus (HBV) infection is the main cause of HCC, and currently, HBV related HCC has become an important public health issue. COL15A1 encodes the alpha chain of collagen XV, a member of the FACIT collagen family, which has anti-angiogenic and anti-tumoral properties and play a vital role in tissue homeostasis in the liver, and its specific function in HBV-related HCC still needs further exploration. This study aimed to determine the regulatory role of COL15A1 in HBV-related HCC and explored the underlying mechanisms at the cellular level. Firstly, the biochip analysis results showed that the expression of COL15A1 was increased in human HBV-related HCC tissues. Furthermore, HBV induction also could significantly increase the expression of COL15A1 in hepatoma cell lines. Functionally, it found that COL15A1 silencing could significantly inhibit apoptosis and promote proliferation, migration, invasion and growth of HepG2.2.15. Mechanically, it found that COL15A1 could interact with P4HB，and its silencing could significantly increase the expression level of P4HB, thereby inhibiting the GRP76 expression and promoting growth and malignancy of HepG2.2.15 cells, revealing COL15A1 might play an anticancer role in HBV-related HCC.

Intratumoral microbial heterogeneity affected tumor immune microenvironment and determined clinical outcome of HBV-related HCC.

The intratumoral microbiome has been reported to regulate the development and progression of cancers. We aimed to characterize intratumoral microbial heterogeneity (IMH) and establish microbiome-based molecular subtyping of HBV-related HCC to elucidate the correlation between IMH and HCC tumorigenesis. A case-control study was designed to investigate microbial landscape and characteristic microbial signatures of HBV-related HCC tissues adopting metagenomics next-generation sequencing. Microbiome-based molecular subtyping of HCC tissues was established by nonmetric multidimensional scaling. The tumor immune microenvironment of 2 molecular subtypes was characterized by EPIC and CIBERSORT based on RNA-seq and verified by immunohistochemistry. The gene set variation analysis was adopted to explore the crosstalk between the immune and metabolism microenvironment. A prognosis-related gene risk signature between 2 subtypes was constructed by the weighted gene coexpression network analysis and the Cox regression analysis and then verified by the Kaplan-Meier survival curve.IMH demonstrated in HBV-related HCC tissues was comparably lower than that in chronic hepatitis tissues. Two microbiome-based HCC molecular subtypes, defined as bacteria- and virus-dominant subtypes, were established and significantly correlated with discrepant clinical-pathologic features. Higher infiltration of M2 macrophage was detected in the bacteria-dominant subtype with to the virus-dominant subtype, accompanied by multiple upregulated metabolism pathways. Furthermore, a 3-gene risk signature containing CSAG4 , PIP4P2 , and TOMM5 was filtered out, which could predict the clinical prognosis of HCC patients accurately using the Cancer Genome Atlas data. Microbiome-based molecular subtyping demonstrated IMH of HBV-related HCC was correlated with a disparity in clinical-pathologic features and tumor microenvironment (TME), which might be proposed as a biomarker for prognosis prediction of HCC.

Cell cycle kinases (AUKA, CDK1, PLK1) are prognostic biomarkers and correlated with tumor-infiltrating leukocytes in HBV related HCC

Hepatocellular carcinoma (HCC) is one of the high incidence cancers and third leading cause of cancer-related mortality. HBV is the top most risk factor accounting for 50–80% of the HCC cases. Kinases: Aurora kinase A (AURKA), cyclin-dependent kinase (CDK1) and Polo-like kinase 1 (PLK1), the key regulators of cell mitosis are overexpressed in varieties of cancers including HCC. However, the exact role of these genes in prognosis of HCC is not fully unveiled. In addition, there is no such an accurate prognostic biomarker for HBV-related HCC. To address this issue, we performed a multidimensional analysis of AURKA, CDK1 and PLK1 with a series of publicly available databases in multiple cancers and with experimental validation in HBV-related HCC tissues. Overexpression of AURKA, CDK1 and PLK1 was found in multiple cancers including HCC. Elevated expression of these genes could result from lowered DNA methylation and genomic alterations. Transcriptional overexpression was significantly correlated with poor prognosis of HCC patients. The expression levels were also significantly positively associated with tumor grades and stages. Furthermore, the expression levels of these genes had a strong correlation with infiltration of immune cells. Our analysis shows that AURKA, CDK1 and PLK1 are correlated with immune infiltration and are the prognostic biomarkers for HBV-induced HCC. Communicated by Ramaswamy H. Sarma

Hepatitis B virus promotes hepatocellular carcinoma development by activating GP73 to repress the innate immune response

BackgroundHepatitis B virus (HBV) causes acute and chronic infection in the clinic. Hepatocellular carcinoma (HCC) is closely linked to HBV infection. Serum Golgi protein 73 (GP73) increases during HBV infection. However, the role of GP73 during HBV infection and the occurrence of HBV-related HCC is still poorly understood.MethodsThe underlying role of HBV-induced GP73 in regulating HCC development was investigated in this study. GP73 expression in HBV-related clinical HCC tissues and in HBV-infected hepatoma cells and primary human hepatocytes was evaluated by immunohistochemistry, ELISAs, Western blotting and quantitative real-time PCR (qRT-PCR) analysis. Tumorigenicity of GP73 overexpressed cells was detected by flow cytometry, qRT-PCR, xenograft nude mouse analyses and sphere formation assays. The effects of GP73 and HBV infection on host innate immune responses in hepatocytes were further investigated by Western blotting and qRT-PCR analysis.ResultsInitially, we confirmed that HBV-positive HCC tissues had significantly higher expression of GP73. Ectopic expression of the HBV gene could induce GP73 expression in primary human hepatocytes and hepatoma cells in vitro. In addition, we discovered that GP73 promotes HCC in both normal liver cells and hepatoma cells. We also found that ectopic expression of HBV genes increases GP73 expression, suppressing the host's innate immune responses in hepatocytes.ConclusionsOur results demonstrate that HBV facilitates HCC development by activating GP73 to repress the host's innate immune response. This study adds to our understanding of the pathogenesis of HBV infection-induced HCC. The findings also provide preclinical support for GP73 as a potential HCC prevention or treatment target.

Network pharmacology and in vitro experiments-based strategy to investigate the mechanisms of KangXianYiAi formula for hepatitis B virus-related hepatocellular carcinoma.

The Chinese traditional medicine KangXianYiAi formula (KXYA) is used to treat hepatic disease in the clinic. Here we aim to confirm the therapeutic effects and explore the pharmacological mechanisms of KXYA on hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We first collected and analyzed clinical data of 40 chronic hepatitis B (CHB) patients with precancerous liver lesions under KXYA treatment. Then, the cell viability, migration, cell cycle, and apoptosis of HepAD38 cells with KXYA treatment were examined. Next, we performed network pharmacological analysis based on database mining to obtain the key target pathways and genes of KXYA treatment on HBV-related HCC. We finally analyzed the expression of the key genes between normal and HBV-related HCC tissues in databases and measured the mRNA expression of the key genes in HepAD38 cells after KXYA treatment. The KXYA treatment could reduce the liver nodule size of CHB patients, suppress the proliferation and migration capabilities, and promote apoptosis of HepAD38 cells. The key pathways of KXYA on HBV-related HCC were Cancer, Hepatitis B, Viral carcinogenesis, Focal adhesion, and PI3K-Akt signaling, and KXYA treatment could regulate the expression of the key genes including HNF4A, MAPK8, NR3C1, PTEN, EGFR, and HDAC1. The KXYA exhibited a curative effect via inhibiting proliferation, migration, and promoting apoptosis of HBV-related HCC and the pharmacological mechanism was related to the regulation of the expression of HNF4A, MAPK8, NR3C1, PTEN, EGFR, and HDAC1.

Acyl-CoA Binding Domain Containing 4 Polymorphism rs4986172 and Expression Can Serve as Overall Survival Biomarkers for Hepatitis B Virus-Related Hepatocellular Carcinoma Patients After Hepatectomy.

BackgroundThe aim of our study was to evaluate the potential of expression and single nucleotide polymorphism of Acyl-CoA binding domain containing 4 (ACBD4) gene as prognosis biomarkers in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after hepatectomy.MethodsHBV-related HCC patients from the First Affiliated Hospital of Guangxi Medical University and GSE14520 were included in the current study, as well as The Cancer Genome Atlas (TCGA) HCC verification cohort. Prognostic analysis and multiple functional enrichment analysis methods were used to evaluate the prognostic value and potential biological functions of the ACBD4 gene in HBV-related HCC.ResultsWe found that ACBD4 gene is highly expressed in normal liver tissues and markedly down-regulated in HBV-related HCC tissues. ACBD4 gene was significantly related to overall survival (OS) of HCC in TCGA and GSE14520 cohorts, and patients with low ACBD4 expression were markedly related to poor OS. Rs4986172 was observed as an OS biomarker after hepatectomy in the Guangxi HBV-related HCC cohort. The OS of rs4986172 GG genotype was worse than that of HCC patients with A allele (AA and AG genotypes). Multifunctional enrichment analysis suggested that ACBD4 gene is closely related to the metabolic, peroxisome proliferator-activated receptor and cytochrome P450 pathway. Through connectivity map, we also identified eight compounds that may be used as targeted therapeutic agents for ACBD4 gene in HBV-related HCC; these compounds were scopoletin, alfaxalone, bephenium hydroxynaphthoate, apramycin, 4,5-dianilinophthalimide, DL-thiorphan, aminohippuric acid and quinidine. Immune microenvironment analysis revealed that there were significant differences in immune scores of HBV-related HCC tumor tissues with different ACBD4 expression levels.ConclusionOur study reveals that ACBD4 expression and rs4986172 can be serve as biomarkers of OS in HBV-related HCC patients after hepatectomy.

Long noncoding RNA RP11-241J12.3 targeting pyruvate carboxylase promotes hepatocellular carcinoma aggressiveness by disrupting pyruvate metabolism and the DNA mismatch repair system

Accumulating evidence indicates that hepatitis B virus X protein (HBx) plays a key role in HBV-related hepatocellular carcinoma (HCC) aggressiveness; however, the underlying mechanisms are not entirely clear. Long non-coding RNAs (lncRNAs), which participate in the regulation of diverse biological processes, may be critical for the function of HBx. Our research indicated that HBx induced changes in the expression of numerous lncRNAs and implicated the novel lncRNA RP11-241J12.3 in HBx-mediated HCC aggressiveness. Although RP11-241J12.3 expression was downregulated in transient HBx-expressing HCC cells (similar to the early stage of HBV infection), its oncogenic properties remained. The results showed that RP11-241J12.3 not only accelerated DNA synthesis and upregulated the expression of pyruvate carboxylase (PC) and MSH3, which is a key protein in pyruvate metabolism and DNA mismatch repair (MMR), but also promoted tumor growth in vitro and in vivo, thus promoting HCC aggressiveness. More importantly, we revealed that RP11-241J12.3 may interact with PC and identified its location in the cytoplasm close to the nucleus using fluorescence in situ hybridization (FISH). We also observed RP11-241J12.3 expression was upregulated in HCC tissues compared with the paracarcinomatous tissues. Furthermore, RP11-241J12.3 expression levels showed a close relationship with clinical stage and tumor size and that low RP11-241J12.3 expression was significantly correlated with longer HCC patient survival. These results further our understanding of the lncRNAs regulated by HBx in HCC, and provide evidence that dysregulation of RP11-241J12.3 contributes to HCC aggressiveness.

LncRNA LINC00924 upregulates NDRG2 to inhibit epithelial-mesenchymal transition via sponging miR-6755-5p in hepatitis B virus-related hepatocellular carcinoma.

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is a life-threatening cancer. Long noncoding RNAs participate in HBV-related HCC progression. Based on the bioinformatics analysis, LINC00924 downregulation is positively related to unfavorable outcomes in patients with HBV-related HCC. Herein, we detected the biological functions and regulatory system of LINC00924 in HCC. The LINC00924 downregulation in HBV-related HCC tissues and cells was revealed by reverse transcription-quantitative polymerase chain reaction. Functionally, as Transwell assays and western blotting indicated, LINC00924 elevation inhibited HCC cell invasion and epithelial-mesenchymal transition (EMT). The binding site between LINC00924 and miR-6755-5p was determined by luciferase reporter assays. miR-6755-5p was confirmed to target NDRG2. miR-6755-5p upregulation decreased NDRG2 messenger RNA (mRNA) and protein levels. The mRNA and protein levels of NDRG2 were downregulated in tissues and cells. NDRG2 knockdown attenuated the inhibition induced by LINC00924 overexpression on invasion and EMT of HCC cells. In summary, LINC00924 increases NDRG2 expression to inhibit EMT by targeting miR-6755-5p in HBV-related HCC.

CDK1, CCNB1 and NDC80 are associated with prognosis and progression of hepatitis B virus-associated hepatocellular carcinoma: a bioinformatic analysis

To identify the key genes involved in the transformation of hepatitis B virus (HBV) into hepatocellular carcinoma (HCC) and explore the underlying molecular mechanisms. We analyzed the mRNA microarray data of 119 HBV-related HCC tissues and 252 HBV-related non-tumor tissues in GSE55092, GSE84044 and GSE121248 from the GEO database, and the "sva" R package was used to remove the batch effects. Integration analysis was performed to identify the differentially expressed genes (DEGs) in HBV-related liver cancer and liver tissues with HBV infection. The significant DEGs were functionally annotated using GO and KEGG analyses, and the most important modules and hub genes were explored with STRING analysis. Kaplan-Meier and Oncomine databases were used to verify the HCC gene expression data in the TCGA database to explore the correlations of the hub genes with the occurrence, progression and prognosis of HCC. We also examined the expressions of the hub genes in 17 pairs of surgical specimens of HCC and adjacent tissues using RT-qPCR. We identified a total of 121 DEGs and 3 genetic markers in HCC (P < 0.01). These DEGs included cyclin1 (CDK1), cyclin B1 (CCNB1), and nuclear division cycle 80 (NDC80), which participated in cell cycle, pyrimidine metabolism and DNA replication and were highly correlated (P < 0.05). Analysis of the UALCAN database confirmed high expressions of these 3 genes in HCC tissues, which were correlated with a low survival rate of the patients, as shown by Kaplan-Meier analysis of the prognostic data from the UALCAN database. CDK1, CCNB1 and NDC80 were all correlated with the clinical grading of HCC (P < 0.05). The results of RT-qPCR on the surgical specimens verified significantly higher expressions of CDK1, CCNB1 and NDC80 mRNA in HCC tissues than in the adjacent tissues. CDK1, CCNB1 and NDC80 genes can be used as prognostic markers of HBV-related HCC and may serve as potential targets in preclinical studies and clinical treatment of HCC.

Hepatocellular carcinoma progression mediated by hepatitis B virus-encoded circRNA HBV_circ_1 through interaction with CDK1

Hepatitis B virus (HBV) produces circular RNA (circRNA), whose functions have not yet been clearly elucidated. In this study, a novel circRNA HBV_circ_1 produced by HBV was identified in HBV-positive HepG2.2.15 cells and HBV-related hepatocellular carcinoma (HCC) tissue (HCCT). Microarray analysis of 68 HCCT samples showed that HBV_circ_1 abundance was significantly higher than that in paracancerous tissues. In addition, survival rate of HBV_circ_1-positive patients was significantly lower compared with HBV_circ_1-negative patients. Transient expression indicated that HBV_circ_1 enhanced cell proliferation, migration, and invasion and inhibited apoptosis in vitro. Furthermore, ectopical HBV_circ_1 expression increased tumor size in vivo. HBV_circ_1 was confirmed to interact with cyclin-dependent kinase 1 (CDK1) to regulate cell proliferation. These results suggest that HCC progression may be promoted by interaction of HBV_circ_1 with CDK1. Our data not only showed a novel clue to understand carcinogenesis and progress of HBV-related HCC but also provided a new target for the development of therapeutic drugs.

Circ_0027089 regulates NACC1 by targeting miR-136-5p to aggravate the development of hepatitis B virus-related hepatocellular carcinoma.

Hepatitis B virus (HBV) infection is the main trigger of hepatocellular carcinoma (HCC). Circular RNA plays an indispensable role in cancer development, and this study aimed to disclose the function and mechanism of circ_0027089 in HBV-related HCC. The expression levels of circ_0027089, miR-136-5p and nucleus accumbens associated protein 1 (NACC1) mRNA were measured by quantitative real-time PCR, and the protein level of NACC1 was detected by western blot. For functional analyses, cell proliferation was assessed by cell counting kit-8 assay and colony formation assay. Cell apoptosis and cell cycle were detected by flow cytometry assay, and cell apoptosis was also assessed by caspase 3/7 activity. The capacities of migration and invasion were evaluated by wound healing assay and transwell assay, respectively. The predicted relationship between miR-136-5p and circ_0027089 or NACC1 was validated by dual-luciferase reporter assay and RNA binding protein immunoprecipitation assay. Animal experiments were performed in nude mice to explore the role of circ_0027089 in vivo. Circ_0027089 expression and NACC1 expression were elevated, while miR-136-5p expression was decreased in HBV-related HCC tissues and cells. In function, circ_0027089 knockdown inhibited HepG2.2.15 and HepAD38 (tet-off) cell proliferation, migration and invasion but induced cell cycle arrest and apoptosis, while circ_0027089 overexpression played the reversed effects. For mechanism exploration, miR-136-5p was a target of circ_0027089, and miR-136-5p deficiency could reverse the role of circ_0027089 knockdown. Circ_0027089 functioned as an oncogene to promote the development of HBV-related HCC by regulating NACC1 via competitively targeting miR-136-5p.

Urine miR-93-5p is a promising biomarker for early detection of HBV-related hepatocellular carcinoma

IntroductionThe mortality rate of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC)continues to increase because sensitive, early and readily available diagnostic tools are lacking. To address this problem, we aimed to identify diagnosticbio markers to be used for early detection of HCC. Materials and methodsmiR-93–5p was selected as a candidate biomarker based on the analyses of relevant Gene Expression Omnibus (GEO) datasets; it was validated using qPCR to quantify its expression levels in tissue, plasma and saliva sample sets. ResultsmiR-93–5p was significantly upregulated in HBV-related HCC tissue. Notably, miR-93–5p in plasma and urine was also significantly increased in patients with early HBV-related HCC. The expression of miR-93–5p was significantly and positively correlated in pairwise comparisons of samples (tissue vs. plasma, tissue vs. urine, plasma vs. urine). Moreover, after curative hepatectomy,miR-93–5p in plasma and urine decreased significantly over one month after the curative hepatectomy and returned to normal levels. Furthermore, receiver operating characteristic (ROC) analysis indicated that both plasma and urine miR-39–5p could detect be used to early, advanced and overall HBV-related HCC cases with more than 85% sensitivities and 93% of specificities. Finally, urine miR-93–5p could be used to predict progress-free survival for early HCC patients who received curative hepatectomy and overall survival for advanced HCC patients without curative treatments. ConclusionsPlasma and urine miR-93–5p show great promise as potential novel biomarkers for early detection of HBV-related HCC. Moreover, urine miR-93–5p could be used to predict the prognosis of patients with HBV-related HCC.

A positive-feedback loop between HBx and ALKBH5 promotes hepatocellular carcinogenesis

BackgroundHepatitis B Virus (HBV) contributes to liver carcinogenesis via various epigenetic mechanisms. The newly defined epigenetics, epitranscriptomics regulation, has been reported to involve in multiple cancers including Hepatocellular Carcinoma (HCC). Our previous study found that HBx, HBV encodes X protein, mediated H3K4me3 modification in WDR5-dependent manner to involve in HBV infection and contribute to oncogene expression. AlkB Homolog 5 (ALKBH5), one of epitranscriptomics enzymes, has been identified to be associated with various cancers. However, whether and how ALKBH5 is dysregulated in HBV-related HCC remains unclear yet. This study aims to investigate ALKBH5 function, clinical significance and mechanism in HBV related HCC (HBV-HCC) patients derived from Chinese people.MethodsThe expression pattern of ALKBH5 was evaluated by RT-qPCR, Western blot, data mining and immunohistochemistry in total of 373 HBV-HCC tissues and four HCC cell lines. Cell Counting Kit 8 (CCK8) assay, Transwell and nude mouse model were performed to assess ALKBH5 function by both small interference RNAs and lentiviral particles. The regulation mechanism of ALKBH5 was determined in HBx and WDR5 knockdown cells by CHIP-qPCR. The role of ALKBH5 in HBx mRNA N6-methyladenosine (m6A) modification was further evaluated by MeRIP-qPCR and Actinomycin D inhibitor experiment in HBV-driven cells and HBx overexpression cells.ResultALKBH5 increased in tumor tissues and predicts a poor prognosis of HBV-HCC. Mechanically, the highly expressed ALKBH5 is induced by HBx-mediated H3K4me3 modification of ALKBH5 gene promoter in a WDR5-dependent manner after HBV infection. The increased ALKBH5 protein catalyzes the m6A demethylation of HBx mRNA, thus stabilizing and favoring a higher HBx expression level. Furthermore, there are positive correlations between HBx and ALKBH5 in HBV-HCC tissues, and depletion of ALKBH5 significantly inhibits HBV-driven tumor cells’ growth and migration in vitro and in vivo.ConclusionsHBx-ALKBH5 may form a positive-feedback loop to involve in the HBV-induced liver carcinogenesis, and targeting the loop at ALKBH5 may provide a potential way for HBV-HCC treatment.

LncRNA XIST upregulates TRIM25 via negatively regulating miR-192 in hepatitis B virus-related hepatocellular carcinoma

BackgroundLong non-coding RNA (lncRNA) XIST has been implicated in the progression of a variety of tumor diseases. The purpose of this study was to explore the molecular role of lncRNA XIST in human hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).MethodsThe expression levels of lncRNA XIST, miR-192 and TRIM25 in HBV-related HCC tissues and HepG2.2.15 cells were detected by qRT-PCR. Biological information and luciferin gene reporter assay were performed to detect the interaction among lncRNA XIST, miR-192 and TRIM25. CCk-8 assay, wound healing assay and colony formation assay were conducted to detect the proliferation and migration ability of HepG2.2.15 cells.ResultsqRT-PCR results showed that the expression levels of lncRNA XIST were remarkably increased in HBV-related HCC tissues and HepG2.2.15 cells. In addition, miR-192 was a direct target gene of lncRNA XIST, and the expression of miR-192 and lncRNA XIST were negatively correlated. Moreover, overexpression of miR-192 observably inhibited the proliferation and migration of HCC cells, while overexpression of lncRNA XIST showed an opposite effect. Furthermore, TRIM25 was a direct target of miR-192, and lncRNA XIST could up-regulate the expression of TRIM25 by targeting miR-192.ConclusionLncRNA XIST could up-regulate the expression of TRIM25 by targeting and binding to miR-192, thus accelerating the occurrence and development of HCC.

Hepatitis B virus X protein promotes vimentin expression via LIM and SH3 domain protein 1 to facilitate epithelial-mesenchymal transition and hepatocarcinogenesis

BackgroundHepatitis B virus (HBV) X protein (HBX) has been reported to be responsible for the epithelial-mesenchymal transition (EMT) in HBV-related hepatocellular carcinoma (HCC). Vimentin is an EMT-related molecular marker. However, the importance of vimentin in the pathogenesis of HCC mediated by HBX has not been well determined.MethodsThe expression of vimentin induced by HBX, and the role of LIM and SH3 domain protein 1 (LASP1) in HBX-induced vimentin expression in hepatoma cells were examined by western blot and immunohistochemistry analysis. Both the signal pathways involved in the expression of vimentin, the interaction of HBX with vimentin and LASP1, and the stability of vimentin mediated by LASP1 in HBX-positive cells were assessed by western blot, Co-immunoprecipitation, and GST-pull down assay. The role of vimentin in EMT, proliferation, and migration of HCC cells mediated by HBX and LASP1 were explored with western blot, CCK-8 assay, plate clone formation assay, transwell assay, and wound healing assay.ResultsVimentin expression was increased in both HBX-positive hepatoma cells and HBV-related HCC tissues, and the expression of vimentin was correlated with HBX in HBV-related HCC tissues. Functionally, vimentin was contributed to the EMT, proliferation, and migration of hepatoma cells mediated by HBX. The mechanistic analysis suggested that HBX was able to enhance the expression of vimentin through LASP1. On the one hand, PI3-K, ERK, and STAT3 signal pathways were involved in the upregulation of vimentin mediated by LASP1 in HBX-positive hepatoma cells. On the other hand, HBX could directly interact with vimentin and LASP1, and dependent on LASP1, HBX was capable of promoting the stability of vimentin via protecting it from ubiquitination mediated protein degradation. Besides these, vimentin was involved in the growth and migration of hepatoma cells mediated by LASP1 in HBX-positive hepatoma cells.ConclusionTaken together, these findings demonstrate that, dependent on LASP1, vimentin is crucial for HBX-mediated EMT and hepatocarcinogenesis, and may serve as a potential target for HBV-related HCC treatment.34DcRyoit96E3KKn7g7GiGVideo abstract

Identification and validation of three core genes in p53 signaling pathway in hepatitis B virus-related hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a common cancer and the leading cause is persistent hepatitis B virus (HBV) infection. We aimed to identify some core genes and pathways for HBV-related HCC.MethodsGene expression profiles of GSE62232, GSE121248, and GSE94660 were available from Gene Expression Omnibus (GEO). The GSE62232 and GSE121248 profiles were the analysis datasets and GSE94660 was the validation dataset. The GEO2R online tool and Venn diagram software were applied to analyze commonly differentially expressed genes between HBV-related HCC tissues and normal tissues. Then, functional enrichment analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Gene and Genome (KEGG) as well as the protein-protein interaction (PPI) network was conducted. The overall survival rates and the expression levels were detected by Kaplan-Meier plotter and Gene Expression Profiling Interactive Analysis (GEPIA). Next, gene set enrichment analysis (GSEA) was performed to verify the KEGG pathway analysis. Furthermore, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to validate the levels of these three core genes in tumor tissues and adjacent non-tumor liver tissues from 12 HBV related HCC patients, HBV-associated liver cancer cell lines and normal liver cell lines, and HepG2 with p53 knockdown or deletion, respectively.ResultsFifteen highly expressed genes associated with significantly worse prognoses were selected and CCNB1, CDK1, and RRM2 in the p53 signaling pathway were identified as core genes. GSEA results showed that samples highly expressing three core genes were all enriched in the p53 signaling pathway in a validation dataset (P < 0.0001). The expression of these three core genes in tumor tissue samples was higher than that in relevant adjacent non-tumor liver tissues (P < 0.0001). Furthermore, we also found that the above genes were highly expressed in liver cancer cell lines compared with normal liver cells. In addition, we found that the expression of these three core genes in p53 knockdown or knockout HCC cell lines was lower than that in negative control HCC cell lines (P < 0.05).ConclusionsCCNB1, CDK1, and RRM2 were enriched in the p53 signaling pathway and could be potential biomarkers and therapeutic targets for HBV-related HCC.

Hepatitis B virus core protein promotes the expression of neuraminidase 1 to facilitate hepatocarcinogenesis

AbstractNeuraminidase 1 (NEU1) has been reported to be associated with hepatocellular carcinoma (HCC). However, the function and associated molecular mechanisms of NEU1 in hepatitis B virus (HBV)-related HCC have not been well investigated. In the present study, the expression of NEU1 mediated by HBV and HBV core protein (HBc) was measured in hepatoma cells. The expression of NEU1 protein was detected via immunohistochemical analysis in HBV-associated HCC tissues. The role of NEU1 in the activation of signaling pathways and epithelial-mesenchymal transition (EMT) and the proliferation and migration of hepatoma cells mediated by HBc was assessed. We found that NEU1 was upregulated in HBV-positive hepatoma cells and HBV-related HCC tissues. HBV promoted NEU1 expression at the mRNA and protein level via HBc in hepatoma cells. Mechanistically, HBc was able to enhance the activity of the NEU1 promoter through NF-κB binding sites. In addition, through the increase in NEU1 expression, HBc contributed to activation of downstream signaling pathways and EMT in hepatoma cells. Moreover, NEU1 facilitated the proliferation and migration of hepatoma cells mediated by HBc. Taken together, our findings provide novel insight into the molecular mechanism underlying the oncogenesis mediated by HBc and demonstrate that NEU1 plays a vital role in HBc-mediated functional abnormality in HCC. Thus, NEU1 may serve as a potential therapeutic target in HBV-associated HCC.

Small hepatitis B virus surface antigen promotes malignant progression of hepatocellular carcinoma via endoplasmic reticulum stress-induced FGF19/JAK2/STAT3 signaling.

Chronic hepatitis B virus (HBV) infection is one of the major global health problems. Although the small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV viral protein, its pathogenic role and molecular mechanism in malignant progression of HBV-related hepatocellular carcinoma (HCC) remain largely unknown. Here we reported that SHBs expression induced epithelial-mesenchymal transition (EMT) process in HCC cells and significantly increased their migratory and invasive ability as well as metastatic potential. Mechanistically, SHBs expression in HCC cells induced endoplasmic reticulum (ER) stress that activated the activating transcription factor 4 (ATF4) to increase the expression and secretion of fibroblast growth factor 19 (FGF19). The autocrine released FGF19 in turn activated JAK2/STAT3 signaling for induction of EMT process in HCC. Notably, SHBs was positively correlated with the expression of mesenchymal markers, the phosphorylation status of JAK2 and STAT3 as well as FGF19 levels in human HCC samples. HCC patients with SHBs positive had a more advanced clinical stage and worse prognosis. These results suggest an important role of SHBs in the metastasis and progression of HCC and may highlight a potential target for preventive and therapeutic intervention of HBV-related HCC and its malignant progression.