Complete genome sequencing and clinical analysis of intrahepatic hepatitis B virus cccDNA from HCC.

BACKGROUND: More than half of hepatocellular carcinomas (HCCs) are etiologically attributed to hepatitis B virus (HBV) infection, but it remains unclear whether HBV mutations are virological factors that contribute to formation of HCC or instead reflect accumulation during the progression of HBV-related disease. METHODS: Rolling-cycle amplification and PCR sequencing were used to characterize covalently closed circular DNA (cccDNA) mutations in tumor tissues. Paired non-tumor tissues were used as controls. RESULTS: High frequencies of C1653T, T1753V, and A1762T/G1764A cccDNA mutations were observed in both tumor and non-tumor tissues. T1719G, C1329A, and T3098C mutations were related to the overall survival of HCC patients. Patients with G1719 tended to be in the high Barcelona Clinic Liver Cancer stage and had lower levels of total DNA and cccDNA per cell than patients with T1719. Additionally, in vitro analysis revealed that T1719G mutation reduced viral replication efficacy. Finally, significantly higher levels of preoperative alpha-fetoprotein were observed in patients harboring the G1078T, C1653T, G1727A, C1913A, T1978C, or C3116T mutations at the cccDNA level. CONCLUSIONS: We speculated that HBV cccDNA mutations accumulated over the course of HBV-related disease development, and that some key mutations had prognostic value for patients with HBV-related HCC.

One health systems strengthening in countries: Tripartite tools and approaches at the human-animal-environment interface.

Unexpected pathogen transmission between animals, humans and their shared environments can impact all aspects of society. The Tripartite organisations-the Food and Agriculture Organization of the United Nations (FAO), the World Health Organization (WHO), and the World Organisation for Animal Health (WOAH)-have been collaborating for over two decades. The inclusion of the United Nations Environment Program (UNEP) with the Tripartite, forming the 'Quadripartite' in 2021, creates a new and important avenue to engage environment sectors in the development of additional tools and resources for One Health coordination and improved health security globally. Beginning formally in 2010, the Tripartite set out strategic directions for the coordination of global activities to address health risks at the human-animal-environment interface. This paper highlights the historical background of this collaboration in the specific area of health security, using country examples to demonstrate lessons learnt and the evolution and pairing of Tripartite programmes and processes to jointly develop and deliver capacity strengthening tools to countries and strengthen performance for iterative evaluations. Evaluation frameworks, such as the International Health Regulations (IHR) Monitoring and Evaluation Framework, the WOAH Performance of Veterinary Services (PVS) Pathway and the FAO multisectoral evaluation tools for epidemiology and surveillance, support a shared global vision for health security, ultimately serving to inform decision making and provide a systematic approach for improved One Health capacity strengthening in countries. Supported by the IHR-PVS National Bridging Workshops and the development of the Tripartite Zoonoses Guide and related operational tools, the Tripartite and now Quadripartite, are working alongside countries to address critical gaps at the human-animal-environment interface.

Horizontal Integration and Financing Reform of Rural Primary Care in China: A Model for Low-Resource and Remote Settings.

Primary health care (PHC) systems are compromised by under-resourcing and inadequate governance, and fail to provide high-quality health care services in most low- and middle-income countries (LMICs). As a response to solve the problems of underfunding and understaffing, Pengshui County, an impoverished area in rural Chongqing, China, implemented a profound reform of its PHC delivery system in 2009, focusing on horizontal integration and financing mechanisms. This paper aims to present new evidence from the Pengshui model, and to assess the relevant changes over the past 10 years (2009-2018). An inductive approach was adopted, based on analysis of national and local policy documents and administrative data. From 2009 to 2018, the proportion of outpatients who sought first-contact care in rural community or township health centers increased from 29% (522,700 of 1,817,600) in 2009, to 40% (849,900 of 2,147,800) in 2018 (the national average in 2018 was 23%). Our findings suggest that many positive results have been achieved through the reform, and that innovations in financial governance and incentive mechanisms are the main driving forces behind the improvement. Pengshui County's experience has proven to be a successful experiment, particularly in rural and low-income areas.

RFX1 participates in doxorubicin-induced hepatitis B virus reactivation.

Cytotoxic chemotherapy drugs, including doxorubicin, can directly promote hepatitis B virus (HBV) replication, but the mechanism has not been fully clarified. This study investigated the potential mechanism underlying the cytotoxic chemotherapy-mediated direct promotion of HBV replication. We found that HBV replication and regulatory factor X box 1 gene (RFX1) expression were simultaneously promoted by doxorubicin treatment. The amount of RFX1 bound to the HBV enhancer I was significantly increased under doxorubicin treatment. Furthermore, the activity of doxorubicin in promoting HBV replication was significantly attenuated when the expression of endogenous RFX1 was knocked down, and the EP element of HBV enhancer I, an element that mediated the binding of RFX1 and HBV enhancer I, was mutated. In addition, two different sequences of the conserved EP element were found among HBV genotypes A-D, and doxorubicin could promote the replication of HBV harboring either of the conserved EP elements. Here, a novel pathway in which doxorubicin promoted HBV replication via RFX1 was identified, and it might participate in doxorubicin-induced HBV reactivation. These findings would be helpful in preventing HBV reactivation during anticancer chemotherapy.

The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication.

The CRISPR/Cas9 system is a novel genome editing technology which has been successfully used to inhibit HBV replication. Here, we described a novel gRNA-microRNA (miRNA)-gRNA ternary cassette driven by a single U6 promoter. With an anti-HBV pri-miR31 mimic integrated between two HBV-specific gRNAs, both gRNAs could be separated from the long transcript of gRNA-miR-HBV-gRNA ternary cassette through Drosha/DGCR8 processing. The results showed that the gRNA-miR-HBV-gRNA ternary cassette could efficiently express two gRNAs and miR-HBV. The optimal length of pri-miRNA flanking sequence in our ternary cassette was determined to be 38 base pairs (bp). Besides, HBV-specific gRNAs and miR-HBV in gRNA-miR-HBV-gRNA ternary cassette could exert a synergistic effect in inhibiting HBV replication and destroying HBV genome in vitro and in vivo. Most importantly, together with RNA interference (RNAi) approach, the HBV-specific gRNAs showed the potent activity on the destruction of HBV covalently closed circular DNA (cccDNA). Since HBV cccDNA is an obstacle for the elimination of chronic HBV infection, the gRNA-miR-HBV-gRNA ternary cassette may be a potential tool for the clearance of HBV cccDNA.

Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication.

AIM: To screen and investigate the effective gRNAs against hepatitis B virus (HBV) of genotypes A-D. METHODS: A total of 15 gRNAs against HBV of genotypes A-D were designed. Eleven combinations of two above gRNAs (dual-gRNAs) covering the regulatory region of HBV were chosen. The efficiency of each gRNA and 11 dual-gRNAs on the suppression of HBV (genotypes A-D) replication was examined by the measurement of HBV surface antigen (HBsAg) or e antigen (HBeAg) in the culture supernatant. The destruction of HBV-expressing vector was examined in HuH7 cells co-transfected with dual-gRNAs and HBV-expressing vector using polymerase chain reaction (PCR) and sequencing method, and the destruction of cccDNA was examined in HepAD38 cells using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these gRNAs was assessed by a mitochondrial tetrazolium assay. RESULTS: All of gRNAs could significantly reduce HBsAg or HBeAg production in the culture supernatant, which was dependent on the region in which gRNA against. All of dual gRNAs could efficiently suppress HBsAg and/or HBeAg production for HBV of genotypes A-D, and the efficacy of dual gRNAs in suppressing HBsAg and/or HBeAg production was significantly increased when compared to the single gRNA used alone. Furthermore, by PCR direct sequencing we confirmed that these dual gRNAs could specifically destroy HBV expressing template by removing the fragment between the cleavage sites of the two used gRNAs. Most importantly, gRNA-5 and gRNA-12 combination not only could efficiently suppressing HBsAg and/or HBeAg production, but also destroy the cccDNA reservoirs in HepAD38 cells. CONCLUSION: These results suggested that CRISPR/Cas9 system could efficiently destroy HBV expressing templates (genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV cccDNA in chronic HBV infection patients.

Integrative analysis of aberrant Wnt signaling in hepatitis B virus-related hepatocellular carcinoma.

AIM: To comprehensively understand the underlying molecular events accounting for aberrant Wnt signaling activation in hepatocellular carcinoma (HCC). METHODS: This study was retrospective. The HCC tissue specimens used in this research were obtained from patients who underwent liver surgery. The Catalogue of Somatic Mutations in Cancer (COSMIC) database was searched for the mutation statuses of CTNNB1, TP53, and protein degradation regulator genes of CTNNB1. Dual-luciferase reporter assay was performed with TOP/FOP reporters to detect whether TP53 gain-of-function (GOF) mutations could enhance the transcriptional activity of Wnt signaling. Methylation sensitive restriction enzyme-quantitative PCR was used to explore the methylation status of CpG islands located in the promoters of APC, SFRP1, and SFRP5 in HCCs with different risk factors. Finally, nested-reverse transcription PCR was performed to examine the integration of HBx in front of LINE1 element and the existence of HBx-LINE1 chimeric transcript in Hepatitis B virus-related HCC. All results in this article were analyzed with the software SPSS version 19.0 for Windows, and different groups were compared by χ(2) test as appropriate. RESULTS: Based on the data from COSMIC database, compared with other solid tumors, mutation frequency of CTNNB1 was significantly higher in HCC (P < 0.01). The rate of CTNNB1 mutation was significantly less frequent in Hepatitis B virus-related HCC than in other etiologies (P < 0.01). Dual-luciferase reporter system and TOP/FOP reporter assays confirmed that TP53 GOF mutants were able to enhance the transcriptional ability of Wnt signaling. An exclusive relationship between the status of TP53 and CTNNB1 mutations was observed. However, according to the COSMIC database, TP53 GOF mutation is rare in HCC, which indicates that TP53 GOF mutation is not a reason for the aberrant activation of Wnt signaling in HCC. APC and AXIN1 were mutated in HCC. By using methylation sensitive restriction enzyme-quantitative PCR, hypermethylation of APC was detected in HCC with different risk factors, whereas SFRP1 and SFRP5 were not hypermethylated in any of the HCC etiologies, which indicates that the mutation of APC and AXIN1, together with the methylation of APC could take part in the overactivation of Wnt signaling. Nested-reverse transcription PCR failed to detect the integration of HBx before the LINE1 element, or the existence of an HBx-LINE1 chimeric transcript, suggesting that integration could not play a role in the aberrant activation of Wnt signaling in HCC. CONCLUSION: In HCC, genetic/epigenetic aberration of CTNNB1 and its protein degradation regulators are the major cause of Wnt signaling overactivation.

Selection of reference genes for RT-qPCR analysis in tumor tissues from male hepatocellular carcinoma patients with hepatitis B infection and cirrhosis.

BACKGROUND: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has been widely used to quantify relative gene expression because of the high specificity, sensitivity and accuracy of this technique. However, its reliability is strongly depends on the expression stability of reference gene used for data normalization. Therefore, identification of reliable and condition specific reference genes is critical for the success of RT-qPCR. OBJECTIVE: Hepatitis B virus (HBV) infection, male gender and the presence of cirrhosis are widely recognized as the leading independent risk factors for the development of hepatocellular carcinoma (HCC). This study aimed to select reliable reference gene for RT-qPCR analysis in HCC patients with all of those risk factors. METHODS: Six candidate reference genes were analyzed in 33 paired tumor and non-tumor tissues from untreated HCC patients. The genes expression stabilities were assessed by geNorm and NormFinder. RESULTS: C-terminal binding protein 1(CTBP1) was the most stable gene among the 6 candidate genes evaluated by both geNorm and NormFinder. The expression stability values were 0.08 for CTBP1 and UBC, 0.09 for HPRT1, 0.12 for HMBS, 0.14 for GAPDH and 0.18 for 18S with geNorm analysis. The stability values suggested by NormFinder software were CTBP1: 0.044, UBC: 0.063, HMBS: 0.072, HPRT1: 0.072, GAPDH: 0.098 and 18S rRNA: 0.161. CONCLUSION: This is the first systematic analysis which suggested CTBP1 as the highest expression-stable gene in human male HBV infection related-HCC with cirrhosis. We recommend CTBP1 as the best candidate reference gene when RT-qPCR was used to determine gene(s) expression in HCC. This may facilitate the relevant HBV related HCC studies in the future.