ENPP1 inhibits the transcription activity of the hepatitis B virus pregenomic promoter by upregulating the acetylation of LMNB1.

Current therapies for hepatitis B virus (HBV) infection can slow disease progression but cannot cure the infection, as it is difficult to eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The interaction between host factors and cccDNA is essential for their formation, stability, and transcriptional activity. Here, we focused on the regulatory role of the host factor ENPP1 and its interacting transcription factor LMNB1 in HBV replication and transcription to better understand the network of host factors that regulate HBV, which may facilitate the development of new antiviral drugs. Overexpression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in Huh7 cells decreased HBV pregenomic RNA (pgRNA) and hepatitis B core antigen (HBcAg) expression levels, whereas knockdown of ENPP1 increased them. A series of HBV promoter and mutant plasmids were constructed, and a luciferase reporter assay showed that overexpression of ENPP1 caused inhibition of the HBV promoter and its mutants. A DNA pull-down assay showed that lamin B1 (LMNB1), but not ENPP1, interacts directly with the HBV enhancer II/ basic core promoter (EnhII/BCP). ZDOCK and PyMOL software were used to predict the interaction of ENPP1 with LMNB1. Overexpression of LMNB1 inhibited the activity of the HBV promoter and its mutant. The acetylation levels at the amino acids 111K, 261K, and 483K of LMNB1 were reduced compared to the control, and an LMNB1 acetylation mutant containing 111R, 261Q, 261R, 483Q, and 483R showed increased promoter activity. In summary, ENPP1 together with LMNB1 increased the acetylation level at 111K and 261K, and LMNB1 inhibited the activity of HBV promoter and downregulated the expression of pregenomic RNA and HBcAg. Our follow-up studies will investigate the expression, clinical significance, and relevance of ENPP1 and LMNB1 in HBV patient tissues, explore the effect of LMNB1 on post-transcriptional progression, and examine whether ENPP1 can reduce cccDNA levels in the nucleus.

HBx Regulation on HBV Pregenome Promoter in the Episomal Form Versus the Integrated Form.

BACKGROUND: Hepatitis B virus (HBV) genome structure is an incomplete closed double stranded circular DNA and it uses covalently closed circular DNA (cccDNA) as template for replication. To study the antiviral effect on different HBV replication forms, a stable cell line expressing HBV using Huh7 cells with shuttle plasmid to imitate the real HBV replication form was stablished. Unlike the HepG2.2.15 cells, the replication of HBV-expressing Huh7 cells present significant decrease after 9 days of interferon-α (IFN-α) treatment. This study aimed to verify whether hepatitis B virus X (HBx) epigenetic regulation by HBV promoter is affected by the DNA form and discuss the differences between the episomal form and the integrated form. MATERIAL AND METHODS: Huh7 cells were used with two different plasmids containing HBV genome to imitate HBV-expressing cells with the episomal form and the integrated form. Luciferase reporting system was used to determine the activation of the promoter after treatment with IFN-α with different concentrations and promoter regulation factor HBx. HBx-expressing plasmid was transfected to evaluate its effect on HBV replication in the episomal form. HBV DNA and pregenomic RNA (pgRNA) in HBx knockdown cell line was determined and HBx-expressing plasmid was transfected to evaluate its effect on HBx in the episomal form. RESULTS: The two cell lines were established successfully and used for further experiments after selection. IFN-α showed significant inhibition effect on HBV pregenome promoter in the episomal form DNA while was not observed in the integrated form. After HBx-expressing plasmid was transfected, HBV pregenome promoter activity was higher in the episomal form rather than the integrated form. HBx showed a concentration-dependant activation on HBV replication in the episomal form. HBx knockdown reduced HBV production and HBV concentration significantly increased after transfection by HBx-expressing plasmid. CONCLUSIONS: HBx regulation effect on HBV pregenome promoter is influenced by the HBV genome form. The epigenetic regulation effect on HBV pregenome promoter is more active in the episomal form rather than the integrated form.

Protein Acetylation Increased Risk of Fibrosis-Related Liver Cancer.

Objective: The occurrence of liver fibrosis and fibrosis-related liver cancer is the reason for the increase in morbidity and mortality worldwide. Transforming growth factor-ß2 (TGF-ß2) is an important mediator of chronic liver fibrosis. This study aims to find the molecular mechanism that mediates HBV infection and induces TGF-ß2 and verifies that CREB binding protein acetylation mediates HBV infection and induces TGF-ß2 expression. Methods: The acetylated proteins were extracted from HepG2-NTCP cells and HBV-infectedHepG2-NTCP cells. The acetylated proteins were screened by modification enrichment technology and database search. Protein annotation, motif analysis of modification sites, and protein function enrichment analysis of these proteins were performed to roughly clarify the location and function of these acetylated modification proteins in cells. Acylated proteins enriched in the TGF-ß pathway were obtained by KEGG pathway enrichment analysis. The effect of the selected acetylated modification protein on the TGF-ß pathway was verified by experiments, that is, the target protein gene was knocked out by siRNA, and the expression level of the TGF-ß2 was detected by qRT-PCR. Results: Proteins were extracted from HepG2-NTCP cells and HepG2-NTCP cells infected with HBV, and differential acetylation modification proteins were screened. The target protein CREB binding protein was screened by modification enrichment technology and database search. The aggregation analysis of TGF-ß pathway showed that CREB binding protein was acetylated at amino acid positions 434 and 439, and enriched in the TGF-ß signaling pathway. siRNA targeting CREB binding protein was transfected, and the expression of TGF-ß2 in cells was detected by qRT-PCR and western blot, respectively. It was verified that HBV infection-inducedCREB-binding protein acetylation regulated the high expression of TGF-ß2. Conclusion: After HBV infection, CREBBP acetylation was up-regulated, which promoted the high expression of TGF-ß2.

Impaired Fanconi anemia pathway causes DNA hypomethylation in human angiosarcomas.

Angiosarcomas (AS) is a rare soft tissue sarcomas with poor treatment options and a dismal prognosis. The abnormal DNA methylation pattern has been determined as the certain clinical relevance with different angiosarcoma subtypes. However, the profound mechanism is not clear. In present study, we studied thirty-six AS with or without chronic lymphedema, and reported that DNA damage was an important factor causing DNA methylation abnormality. Furthermore, we determined that the impaired Fanconi anemia (FA) pathway contributed to severe DNA damage in AS with chronic lymphedema. We also observed that the activated FANCD2 could facilitate DNMT1 recruitment on genomic DNA. Our study uncovers a novel regulatory mechanism of FA pathway on DNA methylation, and is a benefit to advanced understanding the pathogenesis of AS, as well as providing the potential therapeutic targets for AS treatment.

Hydroxyacid Oxidase 2 (HAO2) Inhibits the Tumorigenicity of Hepatocellular Carcinoma and Is Negatively Regulated by miR-615-5p.

Background: Hepatocellular carcinoma (HCC) is the sixth most common kind of cancer worldwide and the third leading cause of cancer mortality. Although a few studies have shown that hydroxyacid oxidase 2 (HAO2) may prevent HCC development, the molecular mechanism is unclear. Methods: We examined the levels of HAO2 expression in 23 pairs of HCC/paracancerous tissues by quantitative real-time polymerase chain reaction (qRT-PCR) and evaluated HAO2's expression in The Cancer Genome Atlas (TCGA) database. Furthermore, we examined the biological activity of HAO2 utilizing cell-based functional assays. Additionally, we evaluated the relationship between miR-615-5p and HAO2 in Hep3B cells using a dual-luciferase reporter system and assessed the downstream regulatory mechanisms of miR-615-5p on HAO2. Finally, the nude mice tumor formation experiment was used to determine the impact of HAO2 on the tumorigenicity of HCC cells. Results: HAO2 expression was considerably underexpression in HCC tissues and cells, and patients with low HAO2 expression had poorer disease-free survival. Inhibition of cell proliferation, migration, and invasion was observed when HAO2 was overexpressed. miR-615-5p had a negative relation with HAO2, and miR-615-5p restored HAO2's biological activity in HCC cells. Additionally, the tumor volume and weight were considerably reduced in the OV-HAO2 group compared to the OV-NC group. Conclusion: HAO2 was found to be underexpressed in HCC tissues and cells, and HAO2 overexpression inhibited HCC cell motility, which was negatively regulated by miR-615-5p. Exogenous expression of HAO2 reduced the tumorigenicity of HCC cells in vivo in nude mice.

Novel protein kinase C phosphorylated kinase inhibitor-matrine suppresses replication of hepatitis B virus via modulating the mitogen-activated protein kinase signal.

HBV (hepatitis B virus) infection still threatens human health. Therefore, it is essential to find new effective anti-HBV compounds. Here, we identified matrine as a novel inhibitor of PKC (protein kinase C) phosphorylated kinase by screening a natural compound library. After HepG2.215 cells were treated with matrine, we carried out a phosphorylated proteomics sequence study and analyzed the prediction of related kinase expression level. In the case of HBV infection, it was found that PKC kinase mediates the activation of mitogen-activated protein kinase (MAPK) signaling pathway known as son of sevenless (SOS) activation. It was also found that PKC kinase inhibits the expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) by inhibiting the activity of activating transcription factor 2/ cAMP response element binding protein (ATF2/CREB), and this effect is independent of its activated MAPK signaling pathway. Finally, Western blot was used to detect the expression of MAPK, ATF2, CREB3 phosphorylation and nonphosphorylation in matrine-treated cells and PKC-treated cells. PKC phosphorylated kinase inhibitor-matrine suppresses the replication of HBV via modulating the MAPK/ATF2 signal. Matrine is a good clinical drug to enhance the autoimmunity in the adjuvant treatment of chronic HBV infection.

ENO3 promoted the progression of NASH by negatively regulating ferroptosis via elevation of GPX4 expression and lipid accumulation.

BACKGROUND: ENO3 expression is upregulated in Non-alcoholic fatty liver disease (NAFLD) patient tissues, demonstrated that ENO3 might play crucial roles in NAFLD. However, the mechanism of ENO3 in NAFLD remains unclear. Therefore, this study aimed to investigate the regulatory mechanism of ENO3 in the progression of non-alcoholic steatohepatitis (NASH) in vivo and vitro NASH model. METHODS: In vivo and vitro NASH model were established by methionine-choline deficient (MCD)-diet feeding and high free fatty acid (HFFA) induction in L02 cells. Loss and gain function of ENO3 and GPX4 was performed to study the mechanism in NASH. Western blot was used to detect the expression of ENO3 and GPX4. Hematoxylin and eosin (H&E), picrosirius Red and Oil Red O staining was used to evaluate histopathology of liver in NASH model. Ferroptosis indicators were measured by assay kits according to the manufacturer's instructions. RESULTS: NASH mouse model was successfully established induced by MCD diet with steatosis, inflammatory infiltration, ballooning and fibrosis observed in the liver tissue. The expression of ENO3 and GPX4 was significantly elevated while ferroptosis was inhibited in NASH mice and cell model. Upregulation of both ENO3 and GPX4 could promote the lipid accumulation in L02 cells. In addition, overexpressed ENO3 attenuated the status of ferroptosis. CONCLUSIONS: In the present study, we demonstrate that ENO3 promoted the progression of NASH by negatively regulating ferroptosis via elevating GPX4 expression and lipid accumulation. These findings provided solid foundation for the mechanism of ferroptosis on the progression of NASH regulated by ENO3, suggesting that ENO3 may be a potential therapeutic target for NASH.

Sirtuin 1 ameliorates defenestration in hepatic sinusoidal endothelial cells during liver fibrosis via inhibiting stress-induced premature senescence.

OBJECTIVE: Premature senescence is related to progerin and involves in endothelial dysfunction and liver diseases. Activating sirtuin 1 (SIRT1) ameliorates liver fibrosis. However, the mechanisms of premature senescence in defenestration of hepatic sinusoidal endothelial cells (HSECs) and how SIRT1 affects HSECs fenestrae remain elusive. METHODS: We employed the CCl4 -induced liver fibrogenesis rat models and cultured primary HSECs in vitro, administered with the SIRT1-adenovirus vector, the activator of SIRT1 and knockdown NOX2. We measured the activity of senescence-associated ß-galactosidase (SA-ß-gal) in HSECs. Meanwhile, the protein expression of SIRT1, NOX2, progerin, Lamin A/C, Ac p53 K381 and total p53 was detected by Western blot, co-immunoprecipitation and immunofluorescence. RESULTS: In vivo, premature senescence was triggered by oxidative stress during CCl4 -induced HSECs defenestration and liver fibrogenesis, whereas overexpressing SIRT1 with adenovirus vector lessened premature senescence to relieve CCl4 -induced HSECs defenestration and liver fibrosis. In vitro, HSECs fenestrae disappeared, with emerging progerin-associated premature senescence; these effects were aggravated by H2 O2 . Nevertheless, knockdown of NOX2, activation of SIRT1 with resveratrol and SIRT1-adenovirus vector inhibited progerin-associated premature senescence to maintain fenestrae through deacetylating p53. Furthermore, more Ac p53 K381 and progerin co-localized with the abnormal accumulation of actin filament (F-actin) in the nuclear envelope of H2 O2 -treated HSECs; in contrast, these effects were rescued by overexpressing SIRT1. CONCLUSION: SIRT1-mediated deacetylation maintains HSECs fenestrae and attenuates liver fibrogenesis through inhibiting oxidative stress-induced premature senescence.

Knockdown of lncRNA ABHD11-AS1 Suppresses the Tumorigenesis of Pancreatic Cancer via Sponging miR-1231.

BACKGROUND: Pancreatic cancer ranks first among the most aggressive malignancies. Long non-coding RNA (LncRNA) ABHD11-AS1 is known to be upregulated in pancreatic cancer. However, the mechanism by which ABHD11-AS1 mediates the tumorigenesis of pancreatic cancer remains unclear. METHODS: Gene and protein expressions in pancreatic cancer cells were detected by qRT-PCR and Western blot, respectively. Cell viability was measured by CCK-8 assay. Cell apoptosis and cycle were tested by flow cytometry. In addition, cell migration and invasion were tested by wound healing and transwell assay, respectively. The correlation between ABHD11-AS1, miR-1231 and cyclin E1 was confirmed by dual-luciferase report and RNA pull-down. Finally, xenograft mice model was established to investigate the role of ABDH-AS1 in pancreatic cancer in vivo. RESULTS: ABHD11-AS1 was found to be negatively correlated with the survival rate of patients with pancreatic cancer. ABHD11-AS1 silencing significantly inhibited the proliferation and induced the apoptosis of pancreatic cancer cells. Additionally, knockdown of ABHD11-AS1 greatly inhibited the migration and invasion of pancreatic cancer cells. Meanwhile, ABHD11-AS1 bound to miR-1231 and cyclin E1 was found to be the target of miR-1231. Moreover, ABHD11-AS1 knockdown-induced G1 arrest in pancreatic cancer cells was reversed by miR-1231 antagomir. Finally, knockdown of ABHD11-AS1 obviously inhibited the tumor growth of pancreatic cancer in vivo. CONCLUSION: ABHD11-AS1 silencing significantly inhibited the tumorigenesis of pancreatic cancer in vitro and in vivo. Thus, ABHD11-AS1 may serve as a potential target for the treatment of pancreatic cancer.

A novel circular RNA circ-LRIG3 facilitates the malignant progression of hepatocellular carcinoma by modulating the EZH2/STAT3 signaling.

BACKGROUND: Circular RNA (circRNA) is emerging as an important player in human diseases, especially cancer. In our previous study, we identified a series of deregulated circRNAs in hepatocellular carcinoma (HCC) by performing circRNA microarray expression profile. Here, we aimed to explore the role of circ-LRIG3 (hsa_circ_0027345) in HCC. METHODS: qRT-PCR and western blot were used to asses gene and protein expression, respectively. CCK-8, EdU and Transwell assays were used to detect cell proliferation, migration and invasion. GSEA software was applied to analyze the pathway related to circ-LRIG3. Co-IP, RIP and ChIP assays were used to identify the positive feedback axis of circ-LRIG3/EZH2/STAT3. Animal study was carried to test the role of circ-LRIG3 in vivo. RESULTS: Circ-LRIG3 was notably upregulated in HCC and promoted HCC cell proliferation, migration, invasion and reduced apoptosis. Circ-LRIG3 formed a ternary complex with EZH2 and STAT3, facilitating EZH2-induced STAT3 methylation and subsequent phosphorylation, resulting in the activation of STAT3 signaling. In turn, activated STAT3 could directly bind to circ-LRIG3 promoter to increase circ-LRIG3 transcription activity, thus forming a positive feedback loop. The animal models showed that exogenous expression of circ-LRIG3 enhanced tumorigenicity and metastasis in vivo, whereas these effects were blocked after treatment with C188-9, a specific STAT3 small-molecule inhibitor. Clinically, high circ-LRIG3 was closely linked with aggressive clinicopathological features and was identified as an independent risk prognostic factor of overall survival. Importantly, plasma circ-LRIG3 was found to be a highly sensitive and specific non-invasive diagnostic indicator for HCC. CONCLUSIONS: Our study reveals the carcinogenic role of circ-LRIG3 in HCC, which may provide a new therapeutic target for HCC patients.

ACADSB regulates ferroptosis and affects the migration, invasion, and proliferation of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most pressing health issues in today's society. As such, it is imperative that the scientific community devise effective methods to inhibit the proliferation and metastasis of CRC cells. Ferroptosis is a recently discovered regulatory cell death mode mainly manifested by dysregulation of cellular iron metabolism and mitochondrial lipid peroxidation. ACADSB is a member of the acyl-CoA dehydrogenase. This study finds that ACADSB is lowly expressed in CRC tissues. Its expression is negatively correlated with N- and M-stage CRC but positively correlated with the overall survival rate of CRC patients. In addition, it finds that ACADSB is found in the mitochondria of cells. Overexpression of ACADSB inhibits CRC cell migration, invasion, and proliferation, while ACADSB knockdown has the opposite effect. More importantly, the study finds that ACADSB negatively regulates expression of glutathione reductase and glutathione peroxidase 4, the two main enzymes responsible for clearing glutathione (GSH) in CRC cells. ACADSB overexpression enhances the concentration of malondialdehyde, Fe+ , superoxide dismutase, and lipid peroxidation in CRC cells, but reduces the concentration of GSH. This is significant, as all of these are important indicators of ferroptosis. Evaluating the data as a whole, this paper speculates that ACADSB affects CRC cell migration, invasion, and proliferation by regulating CRC cell ferroptosis.

Proteomic Analysis of Human Esophageal Cancer Using Tandem Mass Tag Quantifications.

Esophageal cancer (EC) is a type of extremely aggressive gastrointestinal cancer with high incidences in China and other Asian countries. EC does not have specific symptoms and is relatively easy to metastasize, which makes it difficult in early diagnosis. Thus, novel noninvasive diagnostic method is urgently needed in clinical practice. In this study, mass spectrometry with tandem mass tags and differential protein analysis were applied for identifying esophageal cancer-related proteins. The identified proteins were annotated based on their enrichment in Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, hierarchical clustering was applied based on differentially expressed proteins. As a result, a total of 5131 quantifiable proteins were identified from our liquid chromatography-tandem mass spectrometry with tandem mass tags (LC-MS/MS-TMT) method with 63 upregulated and 97 downregulated differential proteins between esophageal cancer and controlled normal samples. The differentially expressed proteins were highly enriched in GO terms associated with mitochondrial dissemble and apoptosis, and blood vessel regulation, and the upregulated differentially expressed proteins in EC samples were significantly enriched in major histocompatibility complex MHC-class I/II pathway of immune system. The functional clustering analysis revealed potential protein-protein interactions among tetraspanin, myosin, and S-100. In summary, our study provided a practical technological procedure of proteomic analysis for discovering novel biomarkers of a specific cancer type.

Downregulation of miR-486-5p Enhances the Anti-Tumor Effect of 5-Fluorouracil on Pancreatic Cancer Cells.

BACKGROUND: 5-Fluorouracil (5-Fu) has been applied to treat pancreatic cancer, which is one of the most common types of digestive system tumors. Evidence has shown that miR-486-5p could promote the proliferation of pancreatic cancer cells. Therefore, this study aimed to investigate whether downregulation of miR-486-5p could enhance the anti-tumor effect of 5-Fu on pancreatic cancer cells. METHODS: Cell Counting Kit 8 assay, flow cytometry and wound healing assays were used to detect proliferation, apoptosis and migration in PANC-1 cells. The expressions of Bcl-2, Bax, cleaved caspase 3, PTEN, p-Akt and p-ERK in PANC-1 cells were detected with Western blot assay. RESULTS: In this study, the inhibitory effects of 5-Fu on the proliferation, migration and invasion of PANC-1 cells were significantly enhanced following transfection with miR-486-5p antagonist. In addition, downregulation of miR-486-5p markedly enhanced the pro-apoptosis effect of 5-Fu on PANC-1 cells. Moreover, bioinformatics analysis and luciferase reporter assay identified that PTEN was the directly binding target of miR-486-5p. Meanwhile, downregulation of miR-486-5p markedly enhanced the anti-tumor effect of 5-Fu in PANC-1 cells via upregulation of the level of PTEN, and downregulation of the expressions of p-ERK and p-Akt. In vivo experiments confirmed that knockdown of miR-486-5p could enhance the anti-tumor effect of 5-Fu in PANC-1 xenograft model. CONCLUSION: We found that the downregulation of miR-486-5p could enhance the anti-tumor effect of 5-Fu on pancreatic cancer cells. Therefore, miR-486-5p antagonist plus 5-Fu might be considered as a potential therapeutic strategy for the treatment of pancreatic cancer.

Establishment of stable cell lines in which the HBV genome replicates episomally for evaluation of antivirals.

INTRODUCTION: Due to the increasing resistance to nucleot(s)ide analogs in patients with chronic hepatitis B, development of new antiviral drugs to eradicate hepatitis B virus is still urgently needed. MATERIAL AND METHODS: To date, most studies on evaluating anti-HBV drugs have been performed using cell lines where the HBV genomic DNA is chromosomally integrated, e.g. Hep2.2.15 in HBV-infected livers of the viral episomal genome replicates in the nucleus and covalently closed circular DNA (cccDNA) serves as a transcriptional template. Another option involves the use of HBV-infected cells of HepaRG or NTCP-overexpressing cells. However, the development of the infection system is expensive and laborious, and its HBV expression level remained low. RESULTS: Compared to HuH7 cells, the established stable cell lines based on episomal-type pEB-Multi vectors can been expressed HBV wild-type by qRT-PCR and immunoblotting (p < 0.05). These two vectors are also sensitive to Entecavir and against nucleoside analog Lamivudine in mutants cellines. CONCLUSIONS: It is worth demonstrating how useful the established cell system is for evaluating antiviral agents and their mechanisms of action.

Circular RNA circ-FOXP1 induced by SOX9 promotes hepatocellular carcinoma progression via sponging miR-875-3p and miR-421.

Circular RNA (circRNA) is a special type of endogenous non-coding RNA that plays an important role in carcinogenesis. However, its biological relevance in hepatocellular carcinoma (HCC) is still largely uncharacterized. Here, we aimed to explore the function and clinical implication of circ-FOXP1 in HCC. We found that circ-FOXP1 was significantly upregulated in HCC tissues, serum and cell lines., which was attributed to the upregulation of oncogenic transcription factor SOX9. Depletion of circ-FOXP1 significantly inhibited HCC cell proliferation, invasion and induced apoptosis. Nevertheless, overexpression of circ-FOXP1 displayed the opposite trend. Further mechanismic study revealed that circ-FOXP1 was preferentially located in the cytoplasm and could concurrently sponge miR-875-3p and miR-421, resulting in increasing levels of a cohort of their target oncogenes, including SOX9. Moreover, knockdown of circ-FOXP1 evidently retarded tumor growth in vivo, but this effect was significantly abolished after silencing of miR-875-3p or miR-421. Clinically, high circ-FOXP1 was closely correlated with larger tumor size, microvascular invasion, advanced TNM stage, and predicted poor prognosis. In addition, serum circ-FOXP1 level could effectively discriminate HCC patients from healthy controls. Collectively, our data clearly suggest that circ-FOXP1 is a novel driver for the tumorigenesis and aggressive progression of HCC, which provides a potential therapeutic target for patients with HCC.

Anti-TNF-α Monoclonal Antibody Therapy Improves Anemia through Downregulating Hepatocyte Hepcidin Expression in Inflammatory Bowel Disease.

Anemia is one of the most common complications in patients with inflammatory bowel disease (IBD). Hepcidin as a key regulator of iron metabolism is pivotal in mediating the occurrence of anemia of chronic disease. Herein, we analyzed the levels of hepcidin in sera from IBD patients by enzyme-linked immunosorbent assay and investigated its potential role in regulating the anemia in IBD. We observed that the levels of serum hepcidin were increased in active IBD patients compared with those in remitted IBD patients and healthy controls and that serum hepcidin was associated with disease activity, CRP, and ESR, respectively. Importantly, we found that the increased levels of serum hepcidin were positively correlated with the severity of anemia and the imbalance of iron metabolism in anemic UC and CD patients. Proinflammatory factors (e.g., IL-6, IL-17, and TNF-α) were positively correlated with the concentrations of serum hepcidin in IBD patients. Interestingly, hepcidin was found to be decreased in patients with Crohn's disease after successful therapy with anti-TNF-α mAb (i.e., infliximab), indicating the underlying association between TNF-α and hepcidin expression. To investigate the specific mechanisms involved, we cultured LO2 and HepG2 cell lines in vitro under stimulation with TNF-α and observed that the levels of hepcidin mRNA were markedly upregulated in caspase-3/8- and NF-κB-dependent manners. Therefore, our data suggest that TNF-α stimulates the expression of hepcidin in IBD patients, resulting in aggravated anemia and that blockage of TNF-α or the caspase-3/8 and NF-κB pathways could downregulate hepcidin expression. This study provides inspiration for the therapy and management of anemia in IBD.

A comprehensive genome-wide profiling comparison between HBV and HCV infected hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, especially in East Asia. Even with the progress in therapy, 5-year survival rates remain unsatisfied. Chronic infection with the hepatitis B virus (HBV) or hepatitis C virus (HCV) has been epidemiologically associated with HCC and is the major etiology in the East Asian population. The detailed mechanism, especially the changes of DNA methylation and gene expression between the two types of virus-related HCC, and their contributions to the HCC development, metastasis, and recurrence remain largely unknown. METHODS: In this integrated analysis, we characterized genome-scale profiles of HBV and HCV infected HCC by comparing their gene expression pattern, methylation profiles, and copy number variations from the publicly accessible data of The Cancer Genome Atlas Program (TCGA). RESULTS: The HLA-A, STAT1, and OAS2 genes were highly enriched and up-regulated discovered in the HCV-infected HCC. Hypomethylation but not copy number variations might be the major factor for the up-regulation of these immune-related genes in HCV-infected HCC. CONCLUSIONS: The results indicated the different epigenetic changes of HBV/HCV related hepatocarcinogenesis. The top up-regulated genes in HCV group were significantly clustered in the immune-related and defense response pathways. These findings will help us to understand the pathogenesis of HBV/HCV associated hepatocellular carcinoma.

Circular RNA circ-ADD3 inhibits hepatocellular carcinoma metastasis through facilitating EZH2 degradation via CDK1-mediated ubiquitination.

Emerging evidence suggests that circular RNA (circRNA) plays a fundamental role in tumorigenesis. However, its contribution to hepatocellular carcinoma (HCC) malignancy remains largely unknown. Here, we performed circRNA microarray expression profile in four paired HCC and normal tissues, and found that circ-ADD3, a novel circRNA derived from linear ADD3 exon 4 to exon 12, was significantly downregulated in HCC, which was further validated in 112 matched HCC and paracancerous tissues. High circ-ADD3 expression was negatively correlated with vascular invasion, intrahepatic metastasis as well as distant metastasis. Moreover, it was identified as an effective biomarker for diagnosis and prognosis of HCC. Functionally, exogenous expression of circ-ADD3 dramatically weakened HCC cell invasion and metastasis both in vitro and in vivo. Mechanistically, circ-ADD3 was capable of reinforcing the interaction between CDK1 and EZH2, resulting in increased EZH2 ubiquitination and subsequent degradation via phosphorylation at Thr-345 and Thr-487 sites. The decreased EZH2 markedly increased the expression of a cohort of anti-metastatic genes, including circ-ADD3, by reducing H3K27me3 levels on their promoter regions, which formed a regulatory circuit, thereby dampening HCC metastasis. Taken together, our findings unveil the essential role of circ-ADD3 in inhibiting HCC metastasis through regulation of EZH2 stability.

Insulin-like growth factor-1 attenuates oxidative stress-induced hepatocyte premature senescence in liver fibrogenesis via regulating nuclear p53-progerin interaction.

Stress-induced premature senescence (SIPS), a state of cell growth arrest due to various stimuli, is implicated in the pathogeneses of hepatic fibrogenesis. Progerin, a permanently farnesylated mutant lamin A protein, likely leads to premature senescence to influent liver diseases. The previous reports showed that activation of insulin-like growth factor-1 (IGF-1) signaling could enhance cell longevity and attenuate liver fibrosis. However, the underlying mechanisms about hepatocyte premature senility in liver fibrosis, and how IGF-1 regulates cell premature aging and fibrogenesis, remain poorly understood. In the present study, we found the augment of hepatocyte oxidation and premature aging, along with the decrease of plasm IGF-1 level in patients with liver fibrosis and CCl4-induced liver injury rat models. Nevertheless, IGF-1 gene transfer to CCl4 rats to overexpress intrahepatic IGF-1 relieved hepatocyte oxidative stress and premature senescence, which was likely mediated by the p53/progerin pathway, to improve hepatic steatosis and fibrogenesis. In vitro, H2O2 caused abnormal accumulation of progerin in nuclear and activation of nuclear p53-progerin interaction to trigger primary rat hepatocyte premature senescence through the p21-independent pathway; while these effects were rescued by prolonged exogenous IGF-1 or the IGF-1 adenovirus vector. Furthermore, the IGF-1 adenovirus vector, transfected to H2O2-treated hepatocytes, reversed oxidative stress-induced premature senescence via enhancing cytoplasmic AKT1-p53 interaction and subsequently inhibiting nuclear p53-progerin interaction. Consequently, our data illuminate a novel role of IGF-1 in regulating stress-induced hepatocyte premature senescence in liver fibrosis: prolonged IGF-1 relieves oxidative stress-initiated hepatocyte premature senescence via inhibition of nuclear p53-progerin interaction to ameliorate hepatic steatosis and fibrogenesis.

Cell Type Diversity in Hepatitis B Virus RNA Splicing and Its Regulation.

Although RNA splicing of hepatitis B virus (HBV) is a commonly observed in livers of hepatitis B patients as well as in the cultured cells replicating the viral genome, its biological significance in the HBV life cycle and the detailed regulatory mechanisms are still largely unclear. In this study, we found cell-type dependency of HBV splicing of the 3.5 kb pregenomic RNA, which is efficiently spliced in human hepatoma cells but not in cells derived from human hepatic stellate, mouse hepatoma and human non-hepatic cells. It may be likely that RNA splicing is one of the determinants of host range restriction of HBV. Given the finding indicating the difference in cell-type dependency of the splicing efficiency between HBV and simian virus 40, we carried out intron-swapping experiments. The results suggest the presence of putative exonic splicing enhancer that possibly works in the cell-type dependent fashion. Together with further mutational analyses, a novel 50-nt intronic splicing silencer, whose secondary structure is well conserved among the HBV strains, was identified. It appears that this intronic silencer functions effectively independent of cell backgrounds.