Editor’s Note: From November 10th to 14th, 2023, the American Association for the Study of Liver Diseases Annual Meeting (AASLD2023) was held in Boston, USA. Professor Hong Tang’s team from West China Hospital of Sichuan University presented multiple research studies in the fields of hepatitis B, hepatitis C, and non-alcoholic fatty liver disease (NAFLD) at the conference. The research was included in the conference proceedings and presented in the form of posters. Congratulations to the team! Hepatology Digest reports on these research findings, and the relevant content is summarized below.

Research One:

HBV-encoded microRNA Inhibits FIH-1 Expression, Promoting Tumor Angiogenesis in HBV-Related Hepatocellular Carcinoma

▲ AASLD2023 Poster Presentation (1503-C)

HBV-cccDNA can generate microRNAs (miRNAs) that, when binding to target mRNA, inhibit post-transcriptional gene expression. Recent reports suggest that in HBV-related hepatocellular carcinoma (HCC), HBV-encoded MicroRNA 3 (HBV-miR-3) can control HBV replication and regulate host gene expression. Hypoxia-inducible factor-1 (HIF-1) inhibitory factor (FIH-1) suppresses HIF-1α hydroxylation, inhibiting the transcription of vascular endothelial growth factor A (VEGFA). Interestingly, the 3′ untranslated region (3′-UTR) of FIH-1 contains the target sequence for HBV-miR-3. Professor Hong Tang’s research team conducted a series of studies exploring the role of HBV-miR-3 in promoting angiogenesis in HBV-related HCC.

Researchers used stem-loop quantitative PCR (stem-loop qPCR) to detect the expression of HBV-miR-3 in HBV-related HCC tissues and conducted survival analysis. They examined the expression of VEGFR2 in HCC tissues using qPCR and analyzed its correlation with HBV-miR-3. HepG2 cells were transfected with HBV-miR-3 agomir (agomir is a chemically modified miRNA agonist), and HepG2.2.15 cells were transfected with HBV-miR-3 antagomir (antagomir is a chemically modified miRNA antagonist) to measure the expression levels of FIH-1, HIF-1α, and VEGFA. A co-culture model of HepG2/HepG2.2.15 with human umbilical vein endothelial cells (HUVEC) was established to analyze the impact of transfecting HBV-miR-3 agomir into HepG2 cells or HBV-miR-3 antagomir into HepG2.2.15 cells on HUVEC tube formation. The binding site and nucleotide sequence of HBV-miR-3 targeting FIH-1 mRNA 3′-UTR were validated using a luciferase reporter gene assay. A mouse xenograft model was established with HepG2 and HepG2.2.15 cells, and HBV-miR-3 agomir and antagomir were injected into the tumors to analyze the expression of FIH-1, HIF-1α, VEGFA, and VEGFR2.

Results showed that the expression of HBV-miR-3 was significantly correlated with angiogenesis-related genes VEGFR2 and FIH-1, indicating that HBV-miR-3 may be involved in angiogenesis in HBV-related HCC. In vitro, HBV-miR-3 agomir inhibited FIH-1 expression in HCC cells, promoting HIF-1α and VEGFA expression, leading to increased HUVEC tube formation. HBV-miR-3 antagomir induced FIH-1 expression in HCC cells, inhibiting HIF-1α and VEGFA expression, resulting in decreased HUVEC tube formation. These results suggest that HBV-miR-3 affects angiogenesis in HBV-related HCC by upregulating HIF-1α/VEGFA expression. Luciferase activity assays also confirmed the binding site of HBV-miR-3 to FIH-1 mRNA 3′-UTR. The role of HBV-miR-3 in regulating tumor angiogenesis was further validated in a mouse xenograft model. Thus, HBV-miR-3 promotes angiogenesis in HBV-related HCC through targeting FIH-1 mRNA 3′-UTR, revealing a new mechanism by which HBV enhances angiogenesis in HBV-related HCC.

The above research indicates that HBV-miR-3, by inhibiting FIH-1 post-transcriptionally, promotes angiogenesis in HBV-related HCC, representing a novel mechanism by which HBV enhances angiogenesis in HBV-related HCC.

Research Two:

Sofosbuvir/Velpatasvir with or without Ribavirin is Safe and Effective in Treating Chinese HCV-Infected Patients

▲ AASLD2023 Poster Presentation (1868-A)

Sofosbuvir/Velpatasvir (SOF/VEL) is an effective pan-genotypic combination antiviral drug for treating chronic HCV-infected patients. However, real data on the effectiveness and safety of SOF/VEL in the treatment of chronic HCV-infected patients in the southwestern region of China, where HCV genotypes (GTs) are diverse, especially with a high prevalence of HCV genotype 3 (GT3), are limited. This prospective observational cohort study included adult patients with chronic HCV infection treated at West China Hospital of Sichuan University and Chengdu Public Health Clinical Medical Center. It included patients with any HCV genotype and those with cirrhosis, hepatocellular carcinoma (HCC), or co-infection with HIV/HBV. The SOF/VEL (400/100 mg) single-tablet regimen was administered once daily for 12 weeks, with or without ribavirin (RBV) depending on the patient’s GT3 infection, cirrhosis, and liver dysfunction. After completing the 12-week treatment, all patients were assessed for sustained virological response at 12 weeks (SVR12), defined as HCV RNA below 15 IU/mL after 12 weeks of treatment. Adverse events during treatment were also assessed.

Results showed that 452 patients were included in the study, with the distribution of HCV genotypes as follows: GT1, 40.3% (182/452); GT2, 7.3% (33/452); GT3, 37.4% (169/452); GT6, 11.3% (51/452); uncertain GT, 3.8% (17/452). RBV was used in 36.7% (166/452) of patients.

The overall SVR12 rate for all patients was 99.1% (448/452). The SVR12

rates for patients with HCV GT3b infection, cirrhosis or HCC, and those with HIV/HBV co-infection were 99.2% (130/131), 99.4% (162/163), and 97.6% (40/41), respectively. The SVR12 rate for patients receiving RBV combination therapy was 98.8% (164/166). Among patients receiving RBV combination therapy, 68.7% (114/166) had HCV GT3b infection, 40.4% (67/166) had cirrhosis, and 7.2% (12/166) had co-infection with HBV/HIV.

After treatment with SOF/VEL ± RBV, the serum albumin-bilirubin score (ALBI, based on ALBI score = 0.66 × lg total bilirubin [μmol/L] – 0.085 × albumin [g/L], categorizing liver function into three grades: ≤-2.60 as grade 1, -2.60 to -1.39 as grade 2, >-1.39 as grade 3) significantly decreased from baseline to SVR12 (-2.91 vs. -3.11, P < 0.001), indicating improved liver function. Additionally, the fibrosis evaluation indices, FIB4 index (based on aspartate aminotransferase, alanine aminotransferase, platelet count, and patient age) and aspartate aminotransferase/platelet ratio index (APRI), also significantly decreased [3.67 vs. 2.81 (P = 0.004) and 1.42 vs. 0.72 (P < 0.001), respectively]. No grade 3-5 adverse events were reported in the study population.

The above research suggests that despite a high proportion of HCV GT3b infection, cirrhosis, HCC, or co-infection with HIV/HBV, the effectiveness of SOF/VEL ± RBV for treating chronic HCV patients in southwestern China is highly significant, and the treatment is well-tolerated.

Research Three:

CircZBTB46 Alleviates NAFLD by Regulating the miR-326/FGF1/AMPK Signaling Axis

▲ AASLD2023 Poster Presentation (2286-C)

Non-alcoholic fatty liver disease (NAFLD) is currently the most common cause of chronic liver disease worldwide. Due to its complex pathogenesis and limited treatment options, further research is urgently needed. Circular RNA (circRNA) is increasingly found to play a role in NAFLD, but the underlying mechanisms remain unclear. Professor Hong Tang’s research team explored the role and molecular mechanism of circZBTB46 in NAFLD by regulating the miR-326/FGF1/AMPK signaling pathway.

Researchers performed RNA sequencing and bioinformatics analysis on liver tissues from three healthy controls, three NAFLD patients, and three NASH patients to identify differentially expressed circRNA. In vivo studies used mice fed with a Western diet as an NAFLD animal model, while in vitro studies used LO2 cells treated with free fatty acids as an NAFLD cell model. miRanda and TargetScan were used to predict circRNA-miRNA binding sites, and interactions were verified through dual luciferase reporter gene assays, RNA immunoprecipitation, and RNA pull-down experiments. qRT-PCR was used to detect mRNA expression levels, Western blot, immunohistochemistry, and immunofluorescence were used to detect protein expression levels, and ELISA was used to determine triglyceride and cholesterol levels. Oil Red O and BODIPY 493/503 staining were used to detect lipid deposition.

Results revealed the identification of a circular RNA named circZBTB46, derived from the ZBTB46 gene, which was downregulated in NAFLD patients as well as in in vitro and in vivo NAFLD models. Overexpression of circZBTB46 significantly reduced hepatic lipid deposition and triglyceride secretion. Mechanistically, circZBTB46 delayed NAFLD progression through the circZBTB46/miR-326/FGF1 pathway. miR-326 was upregulated in NAFLD, inhibiting FGF1 expression by binding to the 3′ untranslated region (3’UTR) of FGF1, leading to hepatic lipid deposition. CircZBTB46 acted as a sponge for miR-326 to counteract its inhibitory effect on FGF1/AMPK, promoting FGF1 expression and alleviating hepatic lipid deposition.

The above research indicates that the circZBTB46-miR326-FGF1/AMPK signaling axis plays a crucial role in NAFLD, and circZBTB46 can alleviate NAFLD by regulating the miR-326/FGF1/AMPK signaling axis. Exploring the expression of circZBTB46 in the blood of NAFLD patients may provide a new method for the non-invasive diagnosis of the disease.

Reference:

[1] Chen H, Cao D, Tang H. Hepatitis B Virus-Encoded MicroRNA (HBV-miR-3) Promotes Tumor Angiogenesis in HBV-related Hepatocellular Carcinoma via FIH-1. AASLD 2023. Poster 1503-C.

[2] Wang J, Du L, Zhang D, et al. Real-life study on the safety and efficacy of sofosbuvir (SOF)-based antiviral agents for chronic hepatitis C patients in China. AASLD 2023. Poster 1868-A.

[3] Zeng Q, Liu CH, Jiang W, et al. CircZBTB46 alleviates non-alcoholic fatty liver disease by targeting miR-326/FGF1/AMPK axis. AASLD 2023. Poster 2286-C.