第一部分

目的：黑色素瘤是一种恶性程度很高的肿瘤。在黑色素瘤的不同亚型中，研究最为广泛的是欧洲人中高发的皮肤黑色素瘤（Cutaneous melanoma, CM），而黏膜黑色素瘤多发于亚洲人群且大多出现在身体不受阳光照射的区域。但迄今为止只有少数亚型黏膜黑色素瘤的分子特征被揭示。因此，我们利用外显子测序来探究妇科黏膜黑色素瘤（Gynecological mucosal melanoma, GM）的基因突变谱进而阐述其疾病发展的可能机制。

方法：1.对肿瘤组织进行二代测序（Next generation sequencing, NGS）以探究外显子区域突变；2.通过荧光原位杂交（Fluorescence in situ hybridization, FISH）和 C-circle实验验证端粒替代延长机制（Alternative lengthening of telomeres, ALT）的存在；3.免疫组化（Immunohistochemistry， IHC）研究 ATRX 蛋白的表达情况；4.液滴数字PCR（Droplet digital PCR, ddPCR）检测 TERT 拷贝数变异。

结果：通过对 32 例妇科黏膜黑色素瘤样本进行外显子测序，发现了与 ALT 相关的ATRX 的频繁突变。在所有 58 份样本中，ALT 阳性 33 例（56.9%），其中 23 例出现 ATRX 蛋白缺失。此外，GM 样本中均未检测到 TERT 启动子突变（n=40）；但在 20%（7/35）的标本中存在 TERT 区拷贝数增加，且 TERT 扩增与 ALT 阳性互斥（P<0.05）。Kaplan-Meier 分析进一步显示，相对于 TERT 扩增来讲，ALT 阳性与无转移 GM 患者的生存期延长有关。

结论：端粒维持机制（Telomere maintenance mechanisms, TMMs）在 GM 的发生发展中起重要作用，这可能有助于预测 GM 患者的临床预后和靶向治疗效果。

第二部分

目的：构建 PBRM1 敲除的肝癌细胞系并探究 PBRM1 在肝癌发生发展中的功能。

方法：应用 CRISPR/Cas9 系统在 Huh7 细胞中靶向敲除 PBRM1 基因。采用 Sanger测序检测目的区域的敲除情况，Western blot 检测蛋白的表达。通过转录本测序（RNA-seq）比较 Huh7 敲除(Knock-out, KO)细胞系与 Huh7 野生(Wild-type, WT)细胞系基因表达谱，富集差异表达基因。使用流式细胞术分析细胞周期的改变。CCK-8实验和克隆形成实验探究细胞增殖情况。此外对 Huh7-KO 细胞系和 Huh7-WT 细胞系进行不同浓度的γ-干扰素（Interferon-γ，IFN-γ）培养，验证 GO 富集中 IFN-γ应答通路的改变。

结果：测序结果显示 PBRM1 被成功敲除，且在 Huh7-KO 细胞系中未检测到 PBRM1蛋白表达。Huh7-KO 细胞的生长速度和克隆形成率均显著慢于 Huh7-WT 细胞（P<0.0001）。GO 和 KEGG 功能富集分析发现下调基因主要与细胞周期相关，包括 DNA 复制、染色体分离、核分裂等；上调基因主要与免疫应答相关，包括适应性免疫应答，对 IFN-γ应答等。Huh7-KO 细胞系种处于 G1期细胞的比例显著高于Huh7-WT（P=0.0166），而前者处于 S 期的细胞比例明显低于后者（P=0.0002）。另外，PBRM1 缺失使细胞对高浓度 IFN-γ的杀伤作用更敏感（P=0.0091），验证了 GO 富集中 Huh7-KO 细胞系对 IFN-γ应答的改变。

结论：肝癌细胞系 Huh7 中敲除 PBRM1 可以引起 G1/S 期阻滞从而抑制细胞增殖。同时，PBRM1 缺失影响肝癌细胞对 IFN-γ刺激的响应。这些发现可为 PBRM1 缺失型肝癌患者的免疫治疗提供新思路。

Purpose：Melanoma, a highly malignant tumor, has many subtypes, among which, themost widely studied one is cutaneous melanomas (CMs) with the high incidence in Europeans. Unlike CMs, mucosal melanoma is more common in Asians and mostly occurs in areas where the body is not exposed to sunlight. But only a few subtypes of mucosal melanomas have so far been molecularly characterized. Therefore, we performed exome sequencing and explored mutations in genetic landscape of gynecological mucosal melanoma (GM) to further elaborate possible mechanisms driving the development of the disease.

Methods：We performed next generation sequencing (NGS) on GMs to detect mutations in exome regions. Alternative lengthening of telomeres (ALT) pathway was further validated by Fluorescence in situ hybridization (FISH) and C-circle assay. Immunohistochemistry (IHC) was carried out to assess ATRX protein levels. We also used droplet digital PCR (ddPCR) to confirm copy number variations of TERT.

Results ： We performed exome sequencing on 32 GM samples and found frequent mutations in ATRX, which were associated with the ALT. In 58 samples, we identified 33(56.9%) ALT-positive cases and 23 of them showed loss of ATRX protein. In addition,TERT promoter mutation was not detected in GMs (n = 40); However, copy number variations in the TERT region were observed in 20% (7/35) of the samples, and the TERT amplification was mutually exclusive with ALT (P < 0.05). Kaplan-Meier analysis further showed that ALT relative to TERT amplification was associated with longer survival inGM patients without metastasis.

Conclusion： These findings indicate that telomere maintenance mechanisms (TMMs)play a critical role in the tumorigenesis of GMs and may aid in prediction of clinical prognosis and the development of targeted therapy for the treatment of GM.

Objective：PBRM1 knock-out hepatocellular carcinoma was constructed to explore the function of PBRM1 in hepatocellular carcinoma.

Method: We used CRISPR/cas9 technique to knock out PBRM1 gene in Huh7 cell line.Sanger sequencing was performed to verify the knockout of the target region. Western blot was used to detect the expression of PBRM1 protein. RNA-seq was performed to enrich the function of the differentially expressed gene between Huh7 knock-out (KO)cell line and Huh7 wild-type (WT) cell line. We also detected the change of cell cycle by flow cytometry. With CCK-8 assay and clone forming test, we explored the cell proliferation. Finally, we cultured Huh7-KO cell line and Huh7-WT cell line with Interferon-γ (IFN-γ ) respectively to verify the change of IFN-γ response pathway.

Result: Sequencing result showed that PBRM1 was successfully knocked out, and loss of PBRM1 expression was validated in Huh7-KO cell line. The growth rate and colony forming efficiency of Huh7-KO cells were significantly slower than Huh7-WT cells (P <0.0001). GO and KEGG functional enrichment analysis showed that down-regulated genes were mainly related to cell cycle, including DNA replication, chromosome separation, nuclear division, etc. While the up-regulated genes were involved in immune response, including adaptive immune response, cellular response to IFN-γ, etc. The proportion of Huh-KO cells in G1 phase was higher than that in Huh7-WT cells (P =0.0166), while the proportion of Huh7-KO cells in S phase was significantly less (P =0.0002). In addition, PBRM1 deficiency made Huh7 more sensitive to high concentration of IFN-γ (P = 0.0091), which also proved the change of cellular response to IFN-γ in Huh7-KO cell line by GO analysis.

Conclusion: PBRM1 knockout in Huh7 hepatocarcinoma cell line could inhibit cell proliferation through G1/S arrest. PBRM1 deficiency affects the response of hepatocellular carcinoma to IFN-γ stimulation. These findings may provide new ideas for immunotherapy of PBRM1 mutant liver cancer.