Background: Hepatitis 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. Methods: The 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. Result: ALKBH5 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. Conclusions: HBx-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.

BACKGROUND:CD8+ cytotoxic T lymphocytes (CTLs) have been proved to exert crucial roles in immunological rejection. Exosomes (EXOs) secreted by CD4+CD25+ regulatory T (Treg) cells is believed to be deeply involved in immune regulation. Nevertheless, w...

Hepatic ischemia reperfusion (I/R) injury (HIRI) HIRI is a complex, multifactorial pathophysiological process and in liver surgery has been known to significantly affect disease prognosis, surgical success rates, and patient survival. Ginsenoside Rgl (Rgl) monomer is one of the main active ingredients of ginseng. Previous studies have demonstrated that Rgl exerts various pharmacological effects through several mechanisms including suppression of apoptosis-related proteins levels, downregulation of inflammatory mediators and as well as antioxidant, which effectively exerts an organ protective effect I/R-induced damage. However, the exact mechanisms of Rg1 on HIRI remain to be elucidated. In the present study, we investigated the protective effect of Rg1 on hepatic ischemia-reperfusion (I/R) injury (HIRI) and explored its underlying molecular mechanism. A rat warm I/R injury model in vivo and an oxygen-glucose deprivation/reperfusion (OGD/R)-treated BRL-3A cell model in vitro were established after pretreating with Rg1(20 mg/kg). The results showed that Rg1 reduced the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). TUNEL staining showed that pretreated with Rg1 inhibited the apoptosis rate compared with the I/R group. Moreover, pretreated with Rg1 significantly reduced the expression of Cyt-C, Caspase-9 and Caspase-3 to inhibit the cell apoptosis. Flow cytometry analysis showed the MMP in the I/R group was significantly increased, whereas pretreated with Rg1 effectively stabilized the MMP compared with the I/R group. in vitro, the proliferation of BRL-3A cells was significantly decreased by the OGD/R treatment, while Rg1 effectively reversed this phenomenon. In addition, western blotting showed that the increase of Cyt-C, Caspase-9 and Caspase-3 was inhibited by H2O2. These observations suggest that Rg1 exerts the protective effect by inhibiting the CypD protein-mediated mitochondrial apoptotic pathway.

AIM: To investigate the efficacy and molecular mechanisms of induced heme oxygenase (HO)-1 in protecting liver from warm ischemia/reperfusion (I/R) injury. METHODS: Partial warm ischemia was produced in the left and middle hepatic lobes of SD rats for 75 min, followed by 6 h of reperfusion. Rats were treated with saline, cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP) at 24 h prior to the ischemia insult. Blood and samples of ischemic lobes subjected to ischemia were collected at 6 h after reperfusion. Serum transaminases level, plasma lactate dehydrogenase and myeloperoxidase activity in liver were measured. Liver histological injury and inflammatory cell infiltration were evaluated by tissue section and liver immunohistochemical analysis. We used quantitative reverse transcription polymerase chain reaction to analyze liver expression of inflammatory cytokines and chemokines. The cell lysates were subjected to immunoprecipitation with anti-Toll-IL-1R-containing adaptor inducing interferon-β (TRIF) and anti-myeloid differentiation factor 88 (MyD88), and then the immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. RESULTS: HO-1 protected livers from I/R injury, as evidenced by diminished liver enzymes and well-preserved tissue architecture. In comparison with ZnPP livers 6 h after surgery, CoPP treatment livers showed a significant increase inflammatory cell infiltration of lymphocytes, plasma cells, neutrophils and macrophages. The Toll-like receptor (TLR)-4 and TANK binding kinase 1 protein levels of rats treated with CoPP significantly reduced in TRIF-immunoprecipitated complex, as compared with ZnPP treatment. In addition, pretreatment with CoPP reduced the expression levels of TLR2, TLR4, IL-1R-associated kinase (IRAK)-1 and tumor necrosis factor receptor-associated factor 6 in MyD88-immunoprecipitated complex. The inflammatory cytokines and chemokines mRNA expression rapidly decreased in CoPP-pretreated liver, compared with the ZnPP-treated group. However, the expression of negative regulators Toll-interacting protein, suppressor of cytokine signaling-1, IRAK-M and Src homology 2 domain-containing inositol-5-phosphatase-1 in CoPP treatment rats were markedly up-regulated as compared with ZnPP-treated rats. CONCLUSION: HO-1 protects liver against I/R injury by inhibiting TLR2/TLR4-triggered MyD88- and TRIF-dependent signaling pathways and increasing expression of negative regulators of TLR signaling in rats.

Background: Upregulation of Stathmin 1 (STMN1), a cytoplasmic phosphoprotein that controls the dynamics of cellular microtubules, is linked to malignant behavior and poor prognosis in a range of malignancies. However, little research has been done on STMN1’s potential role in HCC as a single factor in DNA methylation, m⁶A, or immunological modulation. Results: STMN1 is overexpressed in hepatocellular carcinoma, where it is related to clinicopathological parameters and affects the prognosis of HCC patients. STMN1 overexpression plays an important role in the diagnosis and prognosis of hepatocellular carcinoma. Meanwhile, methylation of 7 CpG sites of STMN1 in HCC was correlated with prognosis, and STMN1 expression was closely related to m⁶A modification. In addition, STMN1 expression is associated with immune cell infiltration, immune molecules, and immune checkpoints in HCC. Conclusion: STMN1 has a significant role in hepatocellular carcinoma diagnosis and prediction. STMN1 is implicated not just in the onset and course but also in the immunological modulation of the disease. DNA methylation and m⁶A are both linked to STMN1. Therefore, STMN1 could be used as a diagnostic and prognostic biomarker for HCC, as well as a target for immunotherapy.

To examine whether heme oxygenase (HO)-1 overexpression would exert direct or indirect effects on Kupffer cells activation, which lead to aggravation of reperfusion injury. Donors were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4 degrees C for 24 h before transplantation. Kupffer cells were isolated and cultured for 6 h after liver reperfusion. Postoperatively, serum transaminases were significantly lower and associated with less liver injury when donors were pretreated with CoPP, as compared with the ZnPP group. Production of the cytokines tumor necrosis factor-alpha and interleukin-6 generated by Kupffer cells decreased in the CoPP group. The CD14 expression levels (RT-PCR/Western blots) of Kupffer cells from CoPP-pretreated liver grafts reduced. The study suggests that the potential utility of HO-1 overexpression in preventing ischemia/reperfusion injury results from inhibition of Kupffer cells activation.