Part 1.AHR Mediates The Aflatoxin B1 Toxicity Associated With Hepatocellular Carcinoma Part 2.Genome-wide Profiling Of Epstein-Barr Virus Integration By Targeted Sequencing In Epstein-Barr Virus Associated Malignancies

BACKGROUND:Hepatocellular carcinoma(HCC)is a malignant tumor mainly derived from liver epithelial cells,ranking fifth and second in the world in terms of tumor morbidity and mortality,and is the fourth most common malignant tumor in China.At present,Chronic infection with Hepatitis B virus(HBV)and Hepatitis C virus(HCV),as well as dietary Aflatoxin exposure,are the main causes of liver cancer.Aflatoxin-associated HCC(AF-HCC)is a significant type of liver cancer patients in China that is different from patients with foreign disease.After the aflatoxin is ingested by the individual,it enters the liver cells and causes the cells to undergo malignant transformation,which in turn induces liver cancer with different processes and outcomes.Therefore,it is important to explore the key molecules related to aflatoxin metabolism and toxicity in liver cells.It is urgent to elaborate the regulatory mechanism that affects aflatoxin metabolism and find potential screening targets for susceptibility to aflatoxin liver cancer.AIMS:(1)To identify the binding molecules of Aflatoxin B1(AFB1)in liver cells,and to reveal the key steps after AFB1 enters the cells.(2)To clarify the regulatory effect of AFB1 binding with AHR on downstream genes and related pathways,and further clarify the molecular mechanism of AFB1-induced liver cancer.(3)To predict the binding conformation of AHR and AFB1,and clarify the specific binding site between AHR and AFB1.(4)To clarify the feasibility of AHR as an immunotherapy target for AF-HCC.METHODS:This study is based on the whole-genome CRISPR-Cas9 high-throughput screening technology,and liver cancer cell lines were treated with AFB1,the key genes resistant to AFB1 continuous induction were identified by next generation sequencing.Non-targeted metabolomics was used to detect the changes in the abundance of metabolites in liver cancer cells with AFB 1 treatment in AHR-WT or AHR-KD cells.The AHR protein molecule was expressed by the E.coli system,a direct interaction between AFB1 and the N-terminus of AHR was confirmed with Saturation-Transfer Difference(STD)NMR analysis.Molecular docking was used to predict the binding site of AFB1 and AHR,and the binding power of the AHR mutant protein at the key binding site to AFB1 was detected.Specimens from patients with AF-HCC in Qidong area were collected to detect the relationship between AHR expression level and PD-L1 expression.In vivo experiments were performed to verify the therapeutic effect of PD-L1 inhibitors on liver cancer with high AHR expression.RESULTS:Genome-wide CRISPR-Cas9 high-throughput screening revealed that AHR deficiency enhanced cell viability under AFB1 treatment.AFB1 increased the accumulation of long chain fatty acids during cell death and was mediated by AHR activity.AFB1 can promote AHR expression and activated AHR nuclear translocation,and then formed a dimer with ARNT to regulate the expression of genes related to the downstream P450 metabolic pathway.The N-terminus of AHR protein can directly bind to AFB1,and amino acid ILE208 was the key binding site to regulate the combination.AHR deficiency can significantly reduce the formation of AFB1-adducts in liver cancer cells in vitro and the high expression of AHR can promote PD-L1 expression,which makes AF-HCC is more sensitive to immunotherapy.CONCLUSIONS:This project has demonstrated that AHR is a potential intracellular shuttle receptor for AFB1,which played an important role in AFB1 metabolism and toxin-induced liver cancer,and can act as the candidate therapeutic targets for AF-HCC through genome-wide CRISPR-Cas9 high-throughput screening.And the activation of AHR can induce the expression of PD-L1,which made AF-HCC was more sensitive to immunotherapy.BACKGROUND:Epstein-Barr virus(EBV)is associated with multiple malignancies,continuous infection of EBV can cause a variety of malignancies,including Burkitt lymphoma,Hodgkin lymphoma,nasopharynx Cancer,gastric cancer,etc.Viral oncogenic proteins and chronic inflammation as major mechanisms contributing to tumor development.A less well-studied mechanism is the integration of EBV into the human genome possibly at sites which may disrupt gene expression or genome stability.AIMS:Our work provides an unbiased large-scale genome-wide analysis of the EBV integration landscape in multiple malignancies.We compare the differences between EBV integration sites in different tumors,and explore the effect of EBV integration on gene expression,trying to clarify the association between EBV integration and tumorigenesis and development.METHODS:Frozen samples were collected for isolation of genomic DNAs from the following tissue types:NPC(n=177;the Sun Yat-sen University Cancer Center and the First Affiliated Hospital of Guangxi Medical University);gastric carcinoma(n=39;the Sun Yat-sen University Cancer Center and the Affiliated Hospital of Qingdao University);NK/T cell lymphoma(n=25;the Sun Yat-sen University Cancer Center and Rui Jin Hospital);Hodgkin lymphoma(n=11;the Sun Yat-sen University Cancer Center);and nasopharyngitis(n=1;the Sun Yat-sen University Cancer Center).Genomic DNA was subjected to hybrid capture using an EBV-targeting single-stranded DNA probe,bioinformatics methods were used to analyze the integration sites and the number of integration sites across the genome.The changes of gene expression near common integration sites were detected,and the relationship between hot-spot genes and EBV was verified by immunohistochemistry.RESULTS:A total of 197 EBV integration breakpoints were identified from 33 tumors and the C666-1 cell line.The integration rates were higher in the gastric carcinomas(25.6%)than in the NPC tumors(9.6%).We identified a number of integration sites close to tumor suppressor genes and inflammation-related genes that regulates TNF-?,NF-?B signaling pathway.In the EBV genome,the breakpoints were frequently at oriP or terminal repeats.These breakpoints were surrounded by microhomology sequences,consistent with a mechanism for integration involving viral genome replication and microhomology-mediated recombination.CONCLUSIONS:EBV integration occurs preferentially within unstable chromosomal regions of the host genome,surrounding oriP or terminal repeats of the EBV genome.Several integration sites were located in the proximity of tumor suppressor genes that are frequently disrupted during cancer progression.These findings suggest that EBV integration is involved in EBV replication and microhomology-mediated gene recombination.