乙型肝炎病毒稳定转染后宿主细胞高级染色质结构改变分析

Structural changes of advanced chromatin in hepatitis B virus after stable transfection

目的研究乙型肝炎病毒(HBV)对肝细胞三维基因组学空间组织结构的影响。方法使用桥接高通量染色质构象捕获(BL‐Hi‐C)技术,获取HBV稳定转染细胞HepG2.CW与对照细胞HepG2的基因组三维空间互作信息,并联合转录组测序分析差异表达基因。通过CCK‐8实验及Kaplan‐Meier生存分析方法分别检测细胞增殖活力,分析差异表达基因功能。结果BL‐Hi‐C实验获取了2株细胞的基因组三维互作信息,在50 kb分辨率下,HBV稳定转染细胞HepG2.CW与HepG2相比,16号染色体与22号染色体发生了染色体易位以及易位点染色体拓扑相关结构域(TAD)的融合变异。结合转录组数据发现,染色体易位点及TAD变异关联区域的22号染色体开放阅读框34(C22ORF34)与肌醇加氧酶(MIOX)出现明显变化。细胞生长曲线表明,HepG2.CW具有更高的细胞增殖活力。生存分析发现C22ORF34及MIOX基因与患者的生存预期密切相关。结论HBV稳定转染可引起HepG2细胞发生染色体易位及三维基因组结构改变,并导致相关区域内C22ORF34和MIOX基因的表达改变。

Objective To investigate the three‐dimensional genomic spatial structural alterations in hepatocellular carcinoma cells which are stably transfected with hepatitis B virus(HBV)genome.Methods Bridge Linker Hi‐C(BL‐Hi‐C)was used to obtain information about genomic 3D spatial interactions in HepG2.CWcells stably transfected with HBV and in control cells HepG2.The differentially expressed genes affected by 3D structural alterations were analyzed via transcriptome sequencing.CCK‐8 assay and Kaplan‐Meier survival analysis were used to analyze the rate of cell proliferation and the function of genes affected by structural alterations of the 3D genome,respectively.Results Information about genomic3D interactions was obtained from BL‐Hi‐C assay of HepG2.CW and HepG2 cells.Translocations between chromosome 16and chromosome 22 and fusion variants of chromosomal topologically associated domains(TADs)were identified at 50kb resolution in HepG2.CW cells compared to HepG2 cells.Higher proliferation activity was detected in HepG2.CW cells as analyzed by the cell growth curve.The expression levels of chromosome 22 open reading frame 34(C22ORF34)and myo‐inositol oxygenase(MIOX)which were located in the chromosomal translocation and TAD‐associated regions were found to be significantly changed between HepG2.CW and HepG2 cells.Survival analysis suggested that C22ORF34 and MIOX were related to expected survival of patients.Conclusion Chromosome translocation and 3D genome structureare significantly changed in cells stably transfected with HBV,which may affect the expressions of C22ORF34 and MIOX.