| BACKGROUNDSHepatocellular carcinoma(HCC)with yearly increasing morbidity is the most common primary liver malignancy and one of the leading causes of cancer deaths around the world.Hepatitis B virus(HBV)infection is one of the key risk factors for HCC.HBV integration with the underlying carcinogenesis mechanism of expression of virus protein,cis-activation,producing novel fusion transcription and inducing chromosome instability,is a driven factor for HCC development.Compared with those HCC patients without HBV infection,the HBV-HCC patients are characterized of lower diagnosis age and lower incidence of cirrhosis.Meanwhile,the patients with early-onset HBV-HCC often have a large number of HBV integration events and poor prognosis.Therefore,HBV integrations in this population have direct clinical significances.In recent years,the development of the third-generation sequencing technology with unique advantages in the detection of repetitive sequences and structural variants has shed light on the full-spectrum understanding of human genetic variations and is revolutionizing the studies of genomics.The application of the third-generation sequencing technology to detect and analyze the HBV integration events in early-onset HBV-HCC will provide new perspectives to understanding the characteristics of HBV integration events and HCC carcinogenesis mechanisms.OBJECTIVESTo describe the characteristics of HBV integration in patients with early-onset HBV-HCC by using the third-generation sequencing platform;to explore the role and mechanism of HBV integration as a major driver for HBV-HCC development;and to provide clues for early diagnosis and treatment of HBV-HCC.METHODSPart I: Sixteen pairs of tumor and para-tumor liver tissues and pathological data from earlyonset HBV-HCC patients were collected,and six pairs of tissues were randomly selected for whole-genome sequencing on Nanopore platform.Bioinformatic analysis: NANOQC for quality control,Porechop for junctions’ acquisition and deletion,Minimap2 for data comparison,Nanofilt for data filtration,Nanostate for quality control and reads length counting.The filtered results were statistically analyzed and validated.PCR amplification and Sanger sequencing were used for validation.Part II: The full-length sequence of HBx-SINE was obtained by genome walking combined with Sanger sequencing.The sequences of HBx-SINE,PVT1 promoter and c-Myc promoter were synthesized and the dual luciferase reporter gene plasmids were constructed as follows: HBx-SINE-p GL3-Promoter,c-Myc-p GL3-Basic,HBx-SINE-p GL3-Basic,HBxSINE-c-Myc-p GL3-Basic,PVT1-p GL3-Basic,HBx-SINE-PVT1-p GL3-Basic.The cell lines used in this part were as follows: HEK293 T,HCT116,HT29,Hep G2,Hu H7,LM3 and MHCC-97 H.By using the dual luciferase reporter gene system,we analyzed the effects of HBx-SINE on the transcription regulation of SV40 virus promoter,c-Myc promoter and PVT1 promoter in different cell lines.HBx-SINE was truncated to construct HBx-SINE truncated mutation reporter gene plasmids: HBx-SINE(1-519 bp)-p GL3-Promoter,HBx-SINE(520-1,258 bp)-p GL3-Promoter,HBx-SINE(364-690 bp)-p GL3-Promoter,HBx-SINE(907-1,258 bp)-p GL3-Promoter,HBx-SINE(796-1,116 bp)-p GL3-Promoter;HBx-SINE(1-519)-c-Myc-p GL3-Basic,HBx-SINE(520-1,258)-c-Myc-p GL3-Basic,HBx-SINE(364-690)-c-Myc-p GL3-Basic,HBxSINE(907-1,258)-c-Myc-p GL3-Basic,HBx-SINE(796-1,116)-c-Myc-p GL3-Basic.The HCC cell lines were transfected with the above truncated mutation plasmids and then analyzed for the core region of HBx-SINE for the transcriptional regulation effects on the activities of SV40 promoter and c-Myc promoter.Part III: The core region of HBx-SINE was labeled with biotin,and HBx-SINE core region binding proteins were detected and analyzed using DNA-pull down combined with LC-MS/MS assay.The specific binding proteins were verified by gel migration assay and Ch IP-q PCR.Further,PARP1 knockout cell lines and knockout control cell lines were constructed using CRISPR-Cas9 system as follows: Hu H7-PARP1-KO,Hu H7-KO-Control,Hep G2-PARP1-KO,Hep G2-KO-Control,and the above cells were transfected with the constructed luciferase reporter gene plasmids to verify the binding protein’s effect on transcriptional regulation activity of SV40 virus promoter and c-Myc promoter.Part IV: RT-q PCR was used to detect the expression of PARP1 and c-Myc in HBV-HCC cell lines: SNU-387,SNU-182,Hep G2.2.15,PLC\PRF\5 and Hep3 B,and to analyze whether there was a co-expression relationship between them.PARP1 knockout cell lines and knockout control cell lines were constructed using the CRISPR-Cas9 system: PLC\PRF\5-PARP1-KO,PLC\PRF\5-KO-Control;Hep G2.2.15-PARP1-KO,Hep G2.2.15-KO-Control.The CRISPRd Cas9 system was used to construct PARP1-activated cell lines and control cell lines:PLC\PRF\5-PARP1-Activation,PLC\PRF\5-Activation-Control;Hep G2.2.15-PARP1-Activation,Hep G2.2.15-Activation-Control.Western blotting was done for the validation of the co-expression of PARP1 and c-Myc in HBV-HCC cell lines.Then the above constructed cell lines were used for PARP1 function detection,namely,CCK8 test,clone formation test and Ed U incorporation test.RESULTSPart I:(1)The length of HBV sequences integrated in tumor tissues of early-onset HBVHCC patients range from tens of bases to more than three thousand bases,with a high frequency of integration in the 1600-2000 bp region of the HBV genomic sequence.(2)HBV integrations in tumor tissues were clonal,while HBV integrations in para-tumor tissues were mostly sporadic.(3)HBV integrations in human chromosome 1 in tumor tissues were of the highest frequency in the early-onset HBV-HCC tumor tissues.(4)HBV integrations were associated with the human chromosome CNV events.(5)HBV integration plays a bridging role in the fusion between different human chromosomes.Part II:(1)The whole sequence of HBx-SINE was obtained.(2)HBx-SINE served enhancer effects on the transcription regulatory activity of SV40 virus promoter in HCC cell lines: Hep G2,Hu H7,LM3 and MHCC-97 H,but not in the cell lines: HEK293 T,HCT116 and HT29.(3)The HBx-SINE(518-906 bp)region was the core region of HBx-SINE.(4)HBxSINE enhanced transcriptional regulation function of c-Myc promoter in HCC cell lines,and the HBx-SINE(518-906 bp)region was also the core region of HBx-SINE serving as the enhancer for transcription regulatory activity of c-Myc promoter.(5)HBx-SINE had no significant effects on the transcriptional regulatory activity of the PVT1 promoter in HCC cell lines.Part III:(1)PARP1 was the main HBx-SINE binding protein.(2)PARP1 was specifically binding to the HBx-SINE core region.(3)PARP1 was a key molecule affecting the intracellular transcriptional regulation of HBx-SINE.Part IV:(1)The expression of PARP1 and c-Myc in HBV-HCC cell lines were positively correlated.(2)Cell lines: SNU-387,SNU-182,Hep G2.2.15 and Hep3 B cells all had chromosome 8 integrations and the integrations involved multiple genes on different chromosomes,while PLC\PRF\5 cell line had fewer HBV integration sites.(3)There was a coexpression relationship between PARP1 and c-Myc in HBV-HCC cell lines.(4)There were interaction effects between PARP1 and c-Myc.(5)PARP1 was a key molecule for the proliferation regulation in HBV-HCC cell lines.CONCLUSIONSIn early-onset HBV-HCC patients,HBV integration was clonal in tumor tissues suggesting that dominant clonal amplification of HBV integration was an early driver event of HBV-HCC.HBV-mediated fusion events between different chromosomes in tumor tissues of HBV-HCC patients were high-frequency events and might promote the early onset of HBV-HCC.HBV integration was associated with genomic copy number variation.In tumor tissues,the integrated HBV sequence HBx-SINE in 8q24 region enhanced the transcriptional regulatory activity of cMyc promoter in HCC cell lines by binding to PARP1 leading to the high expression level of cMyc,which was the underlying mechanism for HBV-HCC early onset. |
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