| Background: Primary liver cancer(PLC)is the sixth most common cancer worldwide,the third leading cause of cancer mortality globally,and the second leading cause of cancer mortality in China.Hepatitis B virus(HBV)infection is responsible for 56% of PLC cases globally,while hepatitis C virus(HCV)accounts for 20% of cases.In East Asian countries,including China,chronic HBV infection is the primary cause of PLC,whereas HCV infection and nonalcoholic fatty liver disease are the primary causes in most European and American countries.Our previous large-scale epidemiological analysis revealed that HBVinduced integrated hepatocellular carcinoma(HCC)develops 10 years earlier,exhibits higher alpha-fetoprotein levels,and has a higher frequency of microvascular invasion compared to HCC caused by other etiologies.China,with a high prevalence of HBV infection,harbors more than half of the world’s HBV-infected patients,making the prevention and control of HBV-induced HCC a significant public health concern.A comprehensive understanding of the mechanisms underlying HBV-induced carcinogenesis is crucial for identifying effective biomarkers and therapeutic targets to address this public health issue.Previous studies,including our own,have identified key oncogenic mechanisms such as inflammatory imbalance and genetic repair-damage imbalance caused by chronic HBV infection,as well as critical HBV variants during HCC evolution.However,there are still significant gaps in our understanding of the mechanistic aspects of HBV-integrated carcinogenesis.Recent high-throughput sequencing data have shown that the telomerase reverse transcriptase(TERT)coding gene is the most common target of HBV integration,with HBV insertion in the TERT promoter occurring in approximately 26% of HBV-HCC cases.HBV integration in the TERT promoter region(HBV-TERT)has been associated with elevated TERT transcript levels and poor prognosis in HCC.However,the specific molecular mechanisms by which HBV-TERT promotes TERT transcription and HCC development remain unclear.Moreover,the current method for detecting HBV integration involves high-throughput sequencing of surgically resected tissues,lacking simplicity and minimally invasive characteristics.Therefore,finding a method to detect this marker in peripheral blood is crucial for HCC screening and prognosis prediction.Addressing the scientific challenges and practical application strategies mentioned above is of utmost urgency.Purpose:The purpose of this study is to analyze the death prevalence of primary liver cancer domestically and internationally,as well as the impact of HBV infection on the survival of HCC patients.We aim to explore the significant role of HBV infection in the development of hepatocellular carcinoma and investigate the specific mechanism of TERT transcription regulation by HBV-TERT integration in HBV infection,along with its impact on the progression of HCC.Additionally,we aim to identify HBV-TERT integration detection methods that can be used in clinical practice.Methods:1.Collect over 15 years of consecutive primary liver cancer death data from China and the United States.Calculate the age-standardized mortality rate(ASMR),annual percent change(APC),and average annual percent change(AAPC)indicators.Download the liver cancer deaths data for China and the United States in 2020 from the International Agency for Research on Cancer to compare the ASMRs for all age groups between the two countries.2.Obtain the HCC follow-up cohort data from The Cancer Genome Atlas(TCGA)and collect follow-up data of HCC patients at Navy Military Medical University Hospital.Use the life table method and Kaplan-Meier method to generate patient survival curves.Analyze the factors influencing the survival of HCC patients with HBV infection using the Cox proportional hazards regression model.3.Segment HBV viruses into functional fragments of varying sizes,namely HBV X(nt.1350-nt.2000),HBV pre S(nt.156-nt.835),HBV S(nt.1835-nt.2450),HBV P(nt.836-nt.1373),and HBV C/P2(nt.2450-nt.2847).Insert different HBV functional fragments into the TERT promoter region to construct dual fluorescein reporter plasmids.Screen HBV functional fragments that regulate TERT transcription through HBV-TERT integration in Huh7,Hep G2,and SK-Hep1 hepatoma cell lines.4.Segment the selected major functional HBV inserts into 19 bp sequences,synthesizing antigene duplex RNAs individually.Confirm the introduction of HBV functional regions into novel transcription factor binding sites using dual luciferase assays.5.Conduct reversion experiments on the Transcription Factor Prediction website to predict transcription factors potentially binding to HBV functional fragments introduced into new transcription factors.Subject the selected transcription factors to knockdown treatment and verify the key transcription factors bound by the HBV-TERT integration sequence using dual-luciferase assay.6.Investigate whether the HBV pre S-S functional protein,as well as the HBx protein,directly interact with the HBV-TERT integration sequence and affect TERT promoter activity.Verify the effect of hepatitis B virus protein on TERT expression through real-time PCR,Western blotting,and telomere length assay.7.Construct a cellular model of TERT insertion in the X region of HBV using CRISPR-Cas9 technology.Generate lentiviruses with single guide RNAs and Cas9 protein,as well as adenoviruses with HBV X fragments.The HBV-TERT integrated cell line was established through the co-transfection of the HBV X fragment into the TERT promoter-54 bp site.Monoclonal antibodies were employed to select integrating cell lines,which were then expanded.Cell migration and invasion were detected using the Transwell assay,while flow cytometry was utilized for cell apoptosis detection.The CCK8 assay was performed to evaluate the proliferation of cells.Telomere length and TERT expression were detected through q RT-PCR,and TERT protein expression was measured using Western blotting.8.TERT capture probes were specifically designed to detect sequences in the integrated part of TERT with HBV.DNA was extracted from clinical patient tissues and fresh blood tissues,which were then sequenced using a high-throughput sequencing platform to analyze and compare the consistency of TERT capture sequencing and HBV capture sequencing methods.Results:1.The ASMR of liver cancer in China generally showed a decreasing trend from 2004 to 2020.There was a significant difference in liver cancer ASMR between urban and rural areas,with higher rates observed in rural areas compared to cities.In the United States,the ASMR for liver cancer generally increased from 1990 to 2015 and then decreased after 2015.A comparison of liver cancer ASMRs between the two countries revealed that liver cancer ASMRs at all ages after the age of 20 and the overall ASMR of liver cancer in China were higher than those in the United States in 2020.2.The median survival of patients with hepatocellular carcinoma at the Naval Medical University Affiliated Hospital is 35.3 months.The risk of death for patients with HBV DNA viral load greater than 106 copies/ml is 1.503 times higher than that of patients without HBV infection(95% CI: 1.221,1.851).The risk of death for patients with tumor TNM stage IV is 4.422 times higher than that of patients with stage I(95% CI: 2.717,7.198).The median survival of the TCGA LIHC follow-up cohort is 52.2 months.The risk of death for HBV-infected patients is 1.474 times higher than that of patients without HBV infection(95% CI: 1.079,2.015).In tumor tissue,there is no statistically significant difference in TERT expression between patients with and without HBV infection.The expression of TERT in adjacent non-cancerous tissue is lower in patients with HBV infection compared to those without HBV infection(P=0.042).The expression of TERT is lower in patients with HBV infection compared to those without HBV infection in all tissues(P=0.023).3.The insertion of the HBV P fragment into TERT up-regulates TERT transcriptional activity in different hepatoma cell lines.Compared to the wild-type TERT,the transcriptional activity of TERT increased up to 800-fold after the insertion of the HBV P fragment,resulting in significantly higher functional activity than the control group(in Huh7,Hep G2 and SK-Hep1 cell lines P<0.05).Similarly,the HBV X fragment could upregulate TERT transcriptional activity in Huh7 and Hep G2 cell lines(P<0.001,P=0.0027),causing an increase in TERT transcriptional activity by more than 10-fold.However,this functional activity was not observed in SK-Hep1.4.Critical HBV insertion sequences,which mainly introduce novel transcription factor binding sites,were identified as nt.620-nt.638(HBV P),nt.670-nt.688(HBV P),nt.1239-nt.1257(HBV X),nt.1222-nt.1240(HBV X),and nt.1260-nt.1278(HBV X).5.The key transcription factors involved in HBV-TERT integration are CEBPA and XBP-1.Compared to the wild-type TERT promoter activity,after knocking down the expression levels of CEBPA and XBP-1,the activity of the integrated TERT promoter in the X region and P region both decreased by 0.8-fold,respectively(P=0.0099,P<0.0001).6.Reporter gene plasmids containing the combined A1762T/G1764 T mutation and the hydroxy-terminal truncation HBV X fragment inserted into the TERT promoter region were constructed and named M1 and CT1,respectively.HCC cell lines were overexpressed with wild-type pre S protein and pre S2 deleted pre S protein,and the interaction of pre S protein with mutant HBV-TERT integration was verified using dual-luciferase assay.Wildtype HCC cells were used as a blank control.It was found that both wild-type and mutant pre S protein could interact with CT1 and decrease TERT transcriptional activity compared to the blank control group(P<0.0001,P<0.0001).The mutant pre S protein,when compared to the blank control group,could interact with M1 and reduce TERT transcriptional activity(P=0.0008).Additionally,compared with the blank control group,HBV X fragment reverse insertion of TERT and C/P2 fragment insertion of TERT can upregulate TERT promoter activity(P<0.0001,P<0.0001),but after interaction with wild-type and mutant pre S proteins,there is no significant difference between TERT promoter activity and the blank control group.7.A cell model of HBV-TERT integration was constructed by inserting the HBV X fragment into the TERT promoter-54 bp site using CRISPR-Cas9 technology.Monoclonal cells were selected to create cell models of HBV-TERT integration.Huh7 cells were used as the blank control,while only cells infected with lentivirus were used as negative controls,named Huh7-CRISPR.Two cell lines with partial HBV-TERT integration,named Huh7-X1 and Huh7-X59,were selected to explore the effect of HBV-TERT integration on the malignant phenotype of HCC cells.Compared to Huh7 and Huh7-CRISPR,Huh7-X59 exhibited a significant increase in invasion(P=0.0136,P=0.0271);apoptosis was decreased in Huh7-X59 cells compared with Huh7-CRISPR(P=0.0009)and increased in Huh7-CRISPR cells compared with Huh7 cells(P=0.0035);compared with wild-type Huh7 cells,Huh7-CRISPR,Huh7-X1,and Huh7-X59 had significantly lower proliferation ability(P=0.0003,P=0.0363,P<0.0001);compared with Huh7,Huh7-X1 and Huh7-X59 had decreased TERT m RNA expression(P=0.0002,P<0.0001),compared with Huh7-CRISPR,Huh7-X1 and Huh7-X59 had decreased TERT m RNA expression(P=0.0004,P<0.0001).8.Compared to HBV capture sequencing,TERT capture sequencing demonstrated comparable efficiency in detecting HBV-TERT integration.It is capable of detecting HBVTERT integration in the peripheral blood of patients,making it a valuable method for early clinical screening of HCC with HBV-TERT integration.Furthermore,TERT capture sequencing has the ability to identify HBV full-length integration by enhancing sequencing depth,which has been validated through Sanger sequencing.Conclusion:1.The overall ASMR and liver cancer ASMR in China are higher than those in the United States at all ages after the age of 20,with a greater difference observed in patients over 40 years old.In the HCC cohort in China,HBV replication activity indicated an increased risk of death in HCC patients,while the TCGA LIHC cohort showed a higher risk of death in HBV-infected patients.Prevention and treatment of HBV infection and understanding the HBV carcinogenic mechanisms are crucial for tertiary prevention of HCC.2.HBV-TERT integration promotes TERT transcription by introducing novel transcription factor binding sites.Full-length HBV sequence insertion in the TERT promoter region may facilitate the maintenance of HBV infection and replication.The TERT capture sequencing method showed comparable efficiency to HBV capture sequencing for HBV-TERT integration detection,and it can detect HBV-TERT integration from peripheral blood.This method can be used as a detection method for early clinical screening and prognosis prediction of HBV-TERT integration in HCC.However,its specific efficacy needs to be confirmed by subsequent large-scale epidemiological studies. |
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