肝细胞癌乙型肝炎病毒整合的靶基因与断点特征分析

Analysis of target genes and breakpoint characteristics of HBV integration in hepatocellular carcinoma

  • 摘要: 目的:寻找乙型肝炎病毒(hepatitis B virus,HBV)相关肝细胞癌(hepatocellular carcinoma,HCC)患者肝组织中常见的HBV整合靶基因及整合片段,为优化肝癌的早期筛查策略提供理论依据。方法:使用HBV探针液相杂交捕获测序的方法,在26例HBV相关HCC患者的癌组织与癌旁组织中,检测HBV整合,探索HBV整合的规律及其与早期肝癌发病的关系。对部分癌旁组织样本中FN1基因的HBV整合进行靶向扩增和Sanger测序验证。结果:癌组织检出HBV整合的断点数少于癌旁组织。早期肝癌病例中HBV整合多见于癌旁组织(P<0.05)。在癌组织中最常见的HBV整合靶基因是TERT,在癌旁组织中,最常见的HBV整合靶基因是FN1。HBV整合片段主要集中在HBV基因组的300~500 bp和1 300~1 900 bp这一区域内,该区域包含整个HBX基因、Precore/core基因以及部分S基因。最后,经PCR和Sanger测序验证了HBV在FN1基因上的精确整合位点。癌组织靶基因整合断点多位于外显子,癌旁组织多位于内含子。结论:HBV整合在早期肝癌的癌旁组织中显著富集,以FN1为最常见靶基因;由于整合的HBV片段可释放入血,本研究为后续探索基于血浆游离DNA(cell-free DNA,cfDNA)检测HBV整合的无创早筛技术提供了初步的理论依据。

     

    Abstract: Objective: To identify common hepatitis B virus(HBV) integration target genes and fragments in the liver tissues from patients with HBV-related hepatocellular carcinoma(HCC), thereby providing a theoretical basis for optimizing the early screening strategies of liver cancer. Methods: Liquid-phase hybridization capture sequencing with HBV probes was applied to detect HBV integration in the cancerous and adjacent noncancerous tissues from 26 patients with HBV-related HCC, and the patterns of HBV integration as well as their association with the pathogenesis of early-stage HCC were explored. Targeted amplification and Sanger sequencing were performed to validate HBV integration within the FN1 gene locus in a subset of samples from adjacent noncancerous tissues. Results: The number of HBV integration breakpoints detected in the cancerous tissues was significantly lower than that in the adjacent non-cancerous tissues. In early-stage HCC cases, HBV integration was predominantly observed in the adjacent non-cancerous tissues(P<0.05). TERT was the most common target gene of HBV integration in the cancerous tissues, whereas FN1 was the predominant target gene of HBV integration in the adjacent non-cancerous tissues. HBV integrated fragments were primarily clustered in the 300-500 bp and 1300-1900 bp regions of the HBV genome, and the regions encompassed the entire HBX gene, Precore/core gene, and partial S genes. Finally, the precise integration of HBV within the FN1 gene locus was validated using polymerase chain reaction(PCR) and Sanger sequencing. The integration breakpoints of target genes in the cancerous tissues were mostly located in exons, whereas those in the adjacent non-cancerous tissues were mostly located in introns. Conclusion: HBV integration is significantly enriched in the adjacent non-cancerous tissues of early-stage HCC, with FN1 being the most common target gene. Since the integrated HBV fragments can be released into the bloodstream, this study provides a preliminary theoretical basis for the subsequent development of non-invasive early screening techniques to detect HBV integration based on plasma cell-free DNA(cfDNA).

     

/

返回文章
返回