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Motivation: Hepatocellular carcinoma (HCC) is a primary malignancy with a poor prognosis. Recently, multi-omics molecular-level measurement enables HCC diagnosis and prognosis prediction, which is crucial for early intervention of personalized therapy to diminish mortality. Here, we introduce a novel strategy utilizing DNA methylation and RNA expression data to achieve a multi-omics gene pair signature (GPS) for HCC discrimination., Results: The immune genes with negative correlations between expression and promoter methylation are enriched in the highly connected cancer-related pathway network, which are considered as the candidates for HCC detection. After that, we separately construct a methylation GPS (mGPS) and an expression GPS (eGPS), and then assemble them as a meGPS with five gene pairs, in which the significant methylation and expression changes occur between HCC tumor and non-tumor groups. Reliable performance has been validated by independent tissue (age, gender and etiology) and blood datasets. This study proposes a procedure for multi-omics GPS identification and develops a novel HCC signature using both methylome and transcriptome data, suggesting potential molecular targets for the detection and therapy of HCC., Availability and Implementation: Models are available at https://github.com/bioinformaticStudy/meGPS.git., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Mutational signature analyses are an effective tool in identifying cancer etiology. Humans are frequently exposed to pyrrolizidine alkaloids (PAs), the most common carcinogenic phytotoxins widely distributed in herbal remedies and foods. However, due to the lack of human epidemiological data, PAs are classified as group II hepatocarcinogens by the World Health Organization. This study identified a PA mutational signature as the biomarker to investigate the association of PA exposure with human liver cancer., Approach and Results: Pyrrole-protein adducts (PPAs), the PA exposure biomarker, were measured and found in 32% of surgically resected specimens from 34 patients with liver cancer in Hong Kong. Next, we delineated the mode of mutagenic and tumorigenic actions of retrorsine, a representative PA, in mice and human hepatocytes (HepaRG). Retrorsine induced DNA adduction, DNA damage, and activation of tumorigenic hepatic progenitor cells, which initiated hepatocarcinogenesis. PA mutational signature, as the unique molecular fingerprint of PA-induced mutation, was derived from exome mutations in retrorsine-exposed mice and HepaRG cells. Notably, PA mutational signature was validated in genomes of patients with PPA-positive liver cancer but not patients with PPA-negative liver cancer, confirming the specificity of this biomarker in revealing PA-associated liver cancers. Furthermore, we examined the established PA mutational signature in 1,513 liver cancer genomes and found that PA-associated liver cancers were potentially prevalent in Asia (Mainland China [48%], Hong Kong [44%], Japan [22%], South Korea [6%], Southeast Asia [25%]) but minor in Western countries (North America [3%] and Europe [5%])., Conclusions: This study provides a clinical indication of PA-associated liver cancer. We discovered an unexpectedly extensive implication of PA exposure in patients with liver cancer, laying the scientific basis for precautionary approaches and prevention of PA-associated human liver cancers., (© 2021 by the American Association for the Study of Liver Diseases.)

Hepatocellular carcinoma (HCC) is heterogeneous, rendering its current curative treatments ineffective. The emergence of single-cell genomics represents a powerful strategy in delineating the complex molecular landscapes of cancers. In this study, we demonstrated the feasibility and merit of using single-cell RNA sequencing to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare cell subpopulations of significance. Exploration of the inter-relationship among liver cancer stem cell markers showed two distinct major cell populations according to EPCAM expression, and the EPCAM + cells had upregulated expression of multiple oncogenes. We also identified a CD24 + /CD44 + -enriched cell subpopulation within the EPCAM + cells which had specific signature genes and might indicate a novel stemness-related cell subclone in HCC. Notably, knockdown of signature gene CTSE for CD24 + /CD44 + cells significantly reduced self-renewal ability on HCC cells in vitro and the stemness-related role of CTSE was further confirmed by in vivo tumorigenicity assays in nude mice. In summary, single-cell genomics is a useful tool to delineate HCC intratumoral heterogeneity at better resolution. It can identify rare but important cell subpopulations, and may guide better precision medicine in the long run., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Long noncoding RNA (lncRNA) is a kind of RNAi molecule composed of hundreds to thousands of nucleotides. There are several major types of functional lncRNAs which participate in some important cellular pathways. LncRNA-RNA interaction controls mRNA translation and degradation or serves as a microRNA (miRNA) sponge for silencing. LncRNA-protein interaction regulates protein activity in transcriptional activation and silencing. LncRNA guide, decoy, and scaffold regulate transcription regulators of enhancer or repressor region of the coding genes for alteration of expression. LncRNA plays a role in cellular responses including the following activities: regulation of chromatin structural modification and gene expression for epigenetic and cell function control, promotion of hematopoiesis and maturation of immunity, cell programming in stem cell and somatic cell development, modulation of pathogen infection, switching glycolysis and lipid metabolism, and initiation of autoimmune diseases. LncRNA, together with miRNA, are considered the critical elements in cancer development. It has been demonstrated that tumorigenesis could be driven by homeostatic imbalance of lncRNA/miRNA/cancer regulatory factors resulting in biochemical and physiological alterations inside the cells. Cancer-driven lncRNAs with other cellular RNAs, epigenetic modulators, or protein effectors may change gene expression level and affect the viability, immortality, and motility of the cells that facilitate cancer cell cycle rearrangement, angiogenesis, proliferation, and metastasis. Molecular medicine will be the future trend for development. LncRNA/miRNA could be one of the potential candidates in this category. Continuous studies in lncRNA functional discrepancy between cancer cells and normal cells and regional and rational genetic differences of lncRNA profiles are critical for clinical research which is beneficial for clinical practice.

Background: Circular RNAs are a class of regulatory RNA transcripts, which are ubiquitously expressed in eukaryotes. In the current study, we evaluate the function of a novel circRNA derived from the β-catenin gene locus, circβ-catenin., Results: Circβ-catenin is predominantly localized in the cytoplasm and displays resistance to RNase-R treatment. We find that circβ-catenin is highly expressed in liver cancer tissues when compared to adjacent normal tissues. Silencing of circβ-catenin significantly suppresses malignant phenotypes in vitro and in vivo, and knockdown of this circRNA reduces the protein level of β-catenin without affecting its mRNA level. We show that circβ-catenin affects a wide spectrum of Wnt pathway-related genes, and furthermore, circβ-catenin produces a novel 370-amino acid β-catenin isoform that uses the start codon as the linear β-catenin mRNA transcript and translation is terminated at a new stop codon created by circularization. We find that this novel isoform can stabilize full-length β-catenin by antagonizing GSK3β-induced β-catenin phosphorylation and degradation, leading to activation of the Wnt pathway., Conclusions: Our findings illustrate a non-canonical function of circRNA in modulating liver cancer cell growth through the Wnt pathway, which can provide novel mechanistic insights into the underlying mechanisms of hepatocellular carcinoma.

Background and Aim: Hepatocellular carcinoma (HCC) is a complicated disease with low survival rate partially due to frequent recurrence and no efficient therapy. Promoter hypermethylation of tumor suppressor genes has been demonstrated as one of the molecular mechanisms contributing to tumorigenesis and progression in HCC. This study aims to investigate regulation of NKAPL expression by promoter methylation and its clinical relevance as a biomarker for HCC., Methods: We measured mRNA expression of NKAPL in 5 HCC cell lines and a cohort of 62 pairs of primary HCC tumor and their adjacent non-cancer liver tissues. NKAPL protein expression on HCC cell lines and clinical samples was assessed by Western blot and immunohistochemistry, respectively. Association analyses between NKAPL expression and clinicopathologic characteristics in the cohort were conducted. Methylation statuses of NKAPL promoter in 18 pairs of tumor and adjacent non-tumor HCC samples were studied using methylation-specific PCR. Biological functions of NKAPL in HCC were investigated by ectopic expression of NKAPL in HCC cells, and cell viability and cell cycle analyses were performed., Results: Our present study showed suppressed expression and promoter hypermethylation are common events in HCC. Demethylation experiment in HCC cells demonstrated that the NKAPL expression was regulated by promoter methylation. In addition, high methylation level of NKAPL and its low expression predict poor outcome. Furthermore, ectopic expression of NKAPL in the HCC cells inhibited cell growth., Conclusions: Our findings suggest that methylation of NKAPL is a frequent event and is a potential prognosis biomarker in HCC.

The Krüppel-like factor 4 (KLF4) gene is related to various biological processes including stem cell reprogramming and tumorigenesis. In this study, we identified and characterized a non-coding transcript of KLF4, which was designated KLF4‑003, in human liver tissue samples. KLF4‑003 was identified in a number of cell lines by reverse transcription PCR and DNA sequencing. Its expression levels were determined in 54 pairs of human hepatocellular carcinoma (HCC) tissues and a number of HCC cell lines by real-time PCR (RT-PCR). Methylation status of KLF4‑003 CpG islands was determined by bisulfite sequencing. The regulatory effect of KLF4‑003 CpG islands hypermethylation in Hep3B cells was then validated by the 5-aza-dC demethylation treatment, followed by RT-PCR analysis. Receiver operating characteristic (ROC) curve was created to evaluate the diagnostic value for differentiating between HCC cancer and benign diseases. The association study between KLF4‑003 expression level and clinical traits of HCC patients was performed with SPSS. We found that KLF4‑003 was downregulated in 46 out of 54 HCC samples compared with their adjunct normal tissues. The reduced KLF4‑003 expression was significantly associated with HCC recurrence (P=0.045) in the follow-up of 31 HCC patients. Significant differences were detected between the methylation status of HCC specimens and their adjacent normal controls. Demethylation treatment significantly rescued the expression of KLF4‑003 in Hep3B cells. Such observation indicated that the CpG island hypermethylation was at least partially responsible for the downregulation of KLF4‑003 in HCC. The area under ROC curve for the prediction of HCC reached 0.803 (95% CI=0.719-0.886, P<0.001). Our results suggested that the expression of KLF4‑003 was epigenetically regulated by methylation status of a KLF4‑003 CpG island in HCC. The differential expression of KLF4‑003 might play an important role in HCC development and might serve as a potential biomarker for the diagnosis of HCC.

Promyelocytic leukemia zinc finger (PLZF) acts as a tumor-suppressor gene in a series of cancers including prostate, melanoma, colon cancer and leukemia. However, its role in hepatocellular carcinoma (HCC) has not yet been illustrated. The present study aimed to investigate the expression and epigenetic regulation of PLZF as well as its clinical significance in HCC. We found that the expression of PLZF was significantly downregulated in HCC samples at both the RNA level (P<0.001) and protein level compared with these levels in adjacent normal tissues. The relative expression level of PLZF was also positively correlated with the ALP level (P=0.026) noted in the HCC patients. However, hypermethylation was only detected in one out of 5 paired HCC samples, indicating that methylation of the selected promoter region (from -1702 to -1388) may not be the major regulatory mechanism for the downregulation of PLZF in HCC. A receiver operating characteristic (ROC) curve was created to evaluate the diagnostic value for differentiating between HCC and benign diseases. The area under the ROC curve (AUC) for indicating the value of PLZF as an HCC biomarker was 0.794 (95% CI, 0.697-0.892; P<0.001). Taken together, our results suggest that PLZF may play an important role in HCC development and may be a potential biomarker for the diagnosis of HCC.

The putative tumor suppressor Mst1, when cleaved to its 36kDa cleaved form, amplifies apoptotic signals. We found that Mst1 was predominantly expressed in its full-length form in 76% (17/25 cases) of hepatocellular carcinoma (HCC) tumors. Mst1 cleavage was basically absent in HCC cells. Ectopic full-length Mst1 expression increased the growth of HCC cells by 55-80% within 3days after transfection. Expression of exogenous NORE1B, a tumor suppressor commonly lost in HCC tumors (~56% of our cohort), was sufficient to suppress the growth promotion of full-length Mst1. Hence, Mst1 exhibits a growth promoting activity in HCC cells upon NORE1B downregulation., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The four-and-a-half LIM protein 2 (FHL2) was originally identified to be expressed abundantly in the heart, as well as in a wide range of tissues demonstrated in various studies. The human FHL2 gene expresses different transcripts which are known to differ only in the 5'UTR region. However, little is known about the functional role of the different variants and the mechanism of gene regulation. In the present study, we characterized the different alternative spliced transcripts of FHL2 by in silico analysis and RT-PCR analysis. A novel transcript variant was identified. The FHL2 gene produces transcripts by different 5' exons, which may be responsible for tissue-specific regulation. To study the mechanism of FHL2 gene regulation, the potential promoter region was investigated. We have identified a functional promoter region upstream of the transcription start site. Deletion mutation analysis of 5' flanking region showed that the fragment from -138 to +292 bp have positive regulatory effect. We identified the binding sites of Pax-5/ZF5 in this region and found that Pax-5 and ZF5 expression in HCC samples had a significant positive correlation with FHL2 expression, suggesting a possible role for these transcription factors in the regulation of FHL2 expression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

FHL2 displays tumor promoting or tumor suppressing activities depending on the types of tumor cells. In this study, we demonstrated that FHL2 overexpression inhibits the proliferation of human HCC cells Hep3B through cell cycle regulation by decreasing cyclin D1 expression while increasing the expressions of p21 and p27. FHL2 overexpression also inhibits migration and invasion of Hep3B cells through the regulation of epithelial-mesenchymal transition. Surprisingly, we also demonstrated an antiapoptotic function for FHL2 overexpression with increased resistance to doxorubicin-induced apoptosis, which indicates the separation of anti-proliferative and anti-apoptotic role of FHL2. Taken together, our results indicate FHL2 could exert anti-apoptotic effect independent of tumor growth suppression., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Multidrug resistance (MDR) is frequently observed after prolonged treatment in human hepatoma with conventional anti-tumor drugs, and photodynamic therapy (PDT) is a recently suggested alternative to overcome MDR. The therapeutic potential of PDT was evaluated in a multidrug resistance (MDR) human hepatoma cell line R-HepG2 with photosensitizer pheophorbide a (Pa)., Results: Our results demonstrated that intracellular accumulation of Pa was not reduced by the overexpression of P-glycoprotein. Pa-based PDT (Pa-PDT) significantly inhibited the growth of R-HepG2 cells with an IC50 value of 0.6 microM. Mechanistic study demonstrated that genomic DNA fragmentation and phosphatidylserine externalization occurred where increase of intracellular singlet oxygen level triggers the phosphorylation of c-Jun N-terminal Kinase (JNK) and leads to activation of intrinsic apoptotic caspases cascade during the Pa-PDT treatment. The cytotoxicity of Pa-PDT, accumulation of sub-G1 population, and depolarization of mitochondrial membrane could be inhibited by JNK inhibitor in the Pa-PDT treated cells. Interestingly, the Pa-PDT induced JNK activation showed inhibitory effect on MDR by the down-regulation of P-glycoprotein in R-HepG2 cells in a dose-dependent manner. In addition, significant reduction of tumor size was obtained in Pa-PDT treated R-HepG2-bearing nude mice with no significant damages in liver and heart., Conclusion: In summary, our findings provided the first evidence that PDT could inhibit the MDR activity by down-regulating the expression of P-glycoprotein via JNK activation using pheophorbide a as the photosensitizer, and our work proved that Pa-PDT inhibited the growth of MDR hepatoma cells by mitochondrial-mediated apoptosis induction.

Scutellaria barbata, a Traditional Chinese Medicine native in southern China, has been widely used for treating liver diseases. In this study, the anti-proliferative effect of Pheophorbide a (Pa), an active component from S. barbata, was examined on a multi-drug resistant (MDR) human hepatoma cell line R-HepG2. Our study showed that Pa could significantly inhibit the growth of R-HepG2 cells with an IC50 value at 25.0 microM after 48 hours treatment. When compared with the parental HepG2 cells, Pa showed weak resistance to R-HepG2. Efflux of Pa out of R-HepG2 cells was not observed as its cellular uptake level showed no significant difference comparing with HepG2 cells. Interestingly, significant reduction of P-glycoprotein expression on Pa-treated R-HepG2 cells was found at both transcriptional and translational levels, leading to reduction of P-glycoprotein activity. In addition, mechanistic study elucidated that Pa induced cell cycle arrest at G2/M phase and inhibited the expressions of G2/M phase cell cycle regulatory proteins, cyclin-A1 and cdc2 in a dose-dependent manner.

Photodynamic therapy (PDT) is an effective treatment for cancer by inducing apoptosis or necrosis in the target cells. Pheophorbide a (Pa), a chlorophyll derivative, is a photosensitzier which can induce significant anti-proliferative effects in a number of human cancer cell lines. This study investigated the action mechanism of Pa-mediated photodynamic therapy (Pa-PDT) on the human hepatocellular carcinoma, Hep3B cells. Pa-PDT significantly inhibited the growth of Hep3B cells with an IC50 value of 1.5 microM. Intracellular ROS level was increased in Pa-PDT treated cells and the cytotoxic effect could be reversed when ascorbic acid was applied. Pa was found to be localized in the mitochondria and then induced the target cells to undergo apoptosis, which was confirmed by propidium iodide staining and DNA fragmentation assay. Pa-PDT treatment also led to the depolarization of mitochondrial membrane potential (Deltapim) and a release of cytochrome c from mitochondria to the cytosol. The caspase cascade was activated as shown by a significant decrease of procaspase-3 and -9 in Pa-PDT treated cells in a dose-dependent manner. Furthermore, in nude mice model, Pa-PDT treatment could reduce the tumor size by 57% after 14 days treatment.

Hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC) worldwide. The pathogenesis of HBV-associated HCC has been studied extensively, and molecular changes during malignant transformation have been identified. It has been proposed that the insertion of HBV DNA into the human genome results in chromosomal instability and inactivation of tumor suppressor genes. Transactivation of oncogenes, inactivation of tumor suppressor genes, and alteration of the cell cycle by HBV proteins are also involved in the progression of hepatocellular carcinogenesis. Traditional clinical examinations of HCC, such as biopsy, computer tomography, ultrasonic imaging, and detection of such biomarkers as a-fetoprotein, are currently the "gold standard" in diagnosis. These tests diagnose HCC only in the late stages of disease. This limitation has greatly reduced the chance of survival of HCC patients. To resolve this problem, new biomarkers that can diagnose HCC in earlier stages are necessary. Based on recent molecular studies of the effects of HBV on cellular transformation, differentially expressed biomarkers of HBV infection have been elucidated. With the analyses of the HBV replication profile, the viral load (HBV DNA levels) of patients, and the viral protein expression, the severity of hepatitis in the preneoplastic stages can be assessed. In the future, with the molecular profiles identified by genomic and proteomic approaches, stage-specific biomarkers should be identified to monitor the progression and prognosis of HCC.

Chronic hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma. HBV encodes an oncogenic hepatitis B virus X protein (HBx), which can transactivate host cell transcriptional machinery and mediate cellular transformation. To disclose the early genetic response in HBx-mediated transformation process, we constructed a conditional HBx-expressing hepatocyte cell line, which allows us to compare the gene expression profiles under controllable HBx induction. A cDNA microarray containing more than 8700 mouse genes and ESTs was utilized to examine the gene expression profiles. We identified 260 candidate genes and 259 ESTs which have shown aberrant expression under HBx induction. Most of them are involved in signal transduction pathway, cell cycle control, metastasis, transcriptional regulation, immune response, and metabolism. These results provide additional insight into early cellular targets of HBx, which could give us a better understanding of the function of HBx and their progressive changes during HBx-mediated hepatocarcinogenesis.