肝癌作为中国及世界死亡率最高的癌症之一，在21世纪受到研究者们的广泛关注。肝细胞癌是最常见的原发性肝癌，其发生发展源于多种遗传因素和非遗传因素之间复杂的相互作用。这些因素会引起肝细胞内遗传和表观遗传致癌改变，诱发肝细胞各类生物学功能异常，进而导致患者体内的不良结节恶性转化，最终甚至使肿瘤在进化压力选择下进展到晚期，该过程中的病理进程极为复杂，因此距离人类克服肝癌的终点还需要很多努力。随着测序技术的发展，科学家们越来越关注基因组的变异与疾病之间的关系，拷贝数变异作为亚显微水平的基因组结构变异，与生物体的进化和许多复杂疾病的发生密切相关。在癌症中拷贝数变异与不同的分子、免疫学及临床特征相关，是肿瘤遗传变异的关键因素。通过查阅大量文献，本研究发现肝细胞癌的基因组变异可能影响肝细胞癌患者的化疗效果及预后，但肝细胞癌中拷贝数变异如何影响癌细胞基因表达、化疗药物应答及预后目前仍不明确。

在第3章本研究首先利用光学基因组图谱技术检测了人肝癌细胞系HepG2、Huh7的全基因组拷贝数变异，先分析了细胞拷贝数变异基因的功能，并根据富集通路对应的基因绘制蛋白质相互作用网络。同时选取两个细胞系核心网络中的关键基因，结合RNA-seq实验和GEPIA数据库分析肝细胞癌拷贝数变异与基因表达、肝细胞癌患者临床生存的关系。研究结果发现HepG2和Huh7基因组的拷贝数变异在变异数量和染色体分布情况差异很大，其中拷贝数变异基因如SRC、MAP3K7的拷贝数、表达水平和临床预后显示出细胞特异性，研究结果不仅支持了SRC、MAP3K7在肝细胞癌研究中的关键作用，也提示了基因组的拷贝数变异对肝细胞癌的高度异质性的作用，从而影响肿瘤细胞的发生发展、进化及患者临床治疗效果。

HepG2和Huh7基因组的变异可能影响着癌细胞对化疗药物的敏感性，第4章为进一步探究了肝细胞癌的异质性与化疗药物敏感性的关系。本研究用不同浓度的顺铂处理HepG2、Huh7细胞12h后，CCK8检测了顺铂对细胞的毒性，免疫荧光技术观测了细胞的损伤和衰老情况，RNA-seq检测了细胞的mRNA水平，并分析了差异表达基因在两细胞中的功能和浓度依赖的趋势差异。结果显示它们的细胞活力、细胞衰老损伤、基因表达趋势、及应答反应既有一致性又存在差异性。两种细胞随着顺铂浓度升高，均出现细胞活力降低，同时细胞的损伤和衰老增加。转录组水平上，随着顺铂浓度升高，两种细胞的DNA损伤修复增加，基因的表达呈现出动态的变化，基因表达的趋势与细胞拷贝数的变异也相关。基因功能分析显示，细胞的表观遗传修饰受到抑制，同时能量代谢和物质代谢显著升高，体现了肝癌细胞对顺铂应答的一致性反应。Huh7在顺铂浓度最高时，细胞的代谢、能量及物质供应等通路对应的基因表达显著降低，因此Huh7可能对顺铂更敏感。研究结果体现出了肝癌细胞系的特异性，这也与临床上肝细胞癌的高度异质一致，这种异质性可能与临床肝细胞癌患者顺铂耐药、预后不良相关。

综上所述，本研究通过肝细胞癌拷贝数变异和顺铂应答的基因表达谱分析，发现拷贝数变异与基因表达、临床预后显著相关，也揭示了拷贝数变异对于肝癌的发展和异质性的重要影响；两种细胞对化疗药物顺铂的基因表达应答存在差异，使我们也在分子水平上认识了肝细胞癌对顺铂应答的生物过程。但是，HepG2和Huh7两种细胞应答的差异是否与基因组拷贝数变异相关，拷贝数变异是否能影响肝细胞癌患者的顺铂耐药，还有待深入的研究。

As one of the cancers with the highest mortality rate in China and the world, liver cancer has attracted extensive attention from researchers in the 21st century. Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Its occurrence and development result from the complex interaction between many genetic and non-genetic factors. These factors will cause genetic and epigenetic carcinogenic changes in liver cells, induce various biological abnormalities of liver cells, and then lead to malignant transformation of adverse nodules in patients, and eventually even make tumors progress to advanced stages under evolutionary pressure selection. The pathological process in this process is extremely complex, so it still needs a lot of efforts to overcome the end point of liver cancer. With the development of sequencing technology, scientists are paying more and more attention to the relationship between genome variation and disease. As a submicroscopic genomic structural variation, copy number variation is closely related to the evolution of organisms and the occurrence of many complex diseases. In cancer, copy number variation is associated with different molecular, immunological and clinical characteristics, and is a key factor in the genetic variation of tumors. Through reviewing a large number of literatures, this study found that genomic variation in hepatocellular carcinoma may affect the chemotherapy effect and prognosis of patients with hepatocellular carcinoma. However, how copy number variation in hepatocellular carcinoma affects gene expression of cancer cells, response to chemotherapy drugs and prognosis remains unclear.

In Chapter 3, this study first detected the copy number variation of human hepatoma cell lines HepG2 and Huh7 using optical genome mapping technology, first analyzed the function of the gene with copy number variation, and mapped the protein interaction network according to the gene corresponding to the enrichment pathway. At the same time, key genes in the core network of two cell lines were selected to analyze the relationship between copy number variation, gene expression and clinical survival of patients with hepatocellular carcinoma combined with RNA-seq assay and GEPIA database. The results showed that the copy number variation of HepG2 and Huh7 genomes differed greatly in the number of variants and chromosome distribution, among which the copy number, expression level and clinical prognosis of the copy number variant genes such as SRC and MAP3K7 showed cell specificity. The results not only supported the key role of SRC and MAP3K7 in hepatocellular carcinoma research. It also suggests that hepatocellular carcinoma is highly heterogeneous, and the heterogeneity of hepatocellular carcinoma may affect the occurrence, development, evolution of tumor cells and the clinical therapeutic effect of patients.

The variation of HepG2 and Huh7 genomes may affect the sensitivity of cancer cells to chemotherapy drugs. Chapter 4 further explores the relationship between the heterogeneity of hepatocellular carcinoma and sensitivity to chemotherapy drugs. In this study, HepG2 and Huh7 cells were treated with different concentrations of cisplatin for 12h. The toxicity of cisplatin was detected by CCK8, the damage and senescence of cells were observed by immunofluorescence, mRNA levels were detected by RNA-seq, and the functional and concentration dependent trends of differentially expressed genes in the two cells were analyzed. The results showed that their cell vitality, cell aging damage, gene expression trend, and response were both consistent and different. With the increase of cisplatin concentration, the cell vitality of both kinds of cells decreased, while the cell damage and senescence increased. At the transcriptome level, with the increase of cisplatin concentration, DNA damage repair of the two types of cells increased, and gene expression showed dynamic changes, and the trend of gene expression was also related to the variation of cell copy number. Gene function analysis showed that epigenetic modification was inhibited, while energy and substance metabolism were significantly increased, reflecting the consistent response of HCC cells to cisplatin. Huh7 may be more sensitive to cisplatin when the concentration of cisplatin is the highest, and the expression of genes corresponding to metabolic, energy and material supply pathways of cells is significantly decreased. The results of this study reflect the specificity of hepatocellular carcinoma cell lines, which is also consistent with the high heterogeneity of clinical hepatocellular carcinoma. Such heterogeneity may be related to cisplatin resistance and poor prognosis of clinical hepatocellular carcinoma patients.

In summary, through the gene expression profile analysis of copy number variation and cisplatin response in hepatocellular carcinoma, this study found that copy number variation was significantly correlated with gene expression and clinical prognosis, and also revealed the important influence of copy number variation on the development and heterogeneity of hepatocellular carcinoma. The difference in the gene expression response of the two cells to the chemotherapy drug cisplatin also enables us to understand the biological process of hepatocellular carcinoma response to cisplatin at the molecular level. However, whether the differences in cellular responses of HepG2 and Huh7 are related to genomic copy number variation, and whether copy number variation can affect cisplatin resistance in hepatocellular carcinoma patients, remains to be further studied.