第一部分CEP170在肝癌中的表达及对肝癌细胞增殖的影响

肝癌具有高复发、易转移和预后差等显著特点，在我国每年因患肝癌而死亡的人数约占世界肝癌死亡总人数的50%，严重危害我国人民生命健康。尽管肝癌的研究在多个方面取得了一定的进展，但预后仍不能令人满意，寻找更有效的治疗靶点显得极其重要。近些年来，随着基于质谱的蛋白质组学技术迅速发展，促使人们对蛋白质组复杂性和动态性有了更加直观的理解，同时利用蛋白质组学数据能够更加精准的回答生物学问题。本研究首先从蛋白质组数据库CPTAC下载肝癌蛋白质组学数据及相关临床信息，利用R包limma分析差异蛋白表达与临床预后关系。通过UALCAN及cBioPortal在线分析TCGA数据库中CEP170转录水平及基因拷贝数变异情况。利用Kaplan-Meier法进行生存分析，再通过ROC曲线检验预测效应。接下来通过组织及细胞实验对数据分析结果进行验证。免疫组化实验验证癌组织与癌旁组织中CEP170的蛋白表达差异。用特异性siRNA敲降肝癌细胞株Huh-7的CEP170表达，利用CCK-8法检测细胞增殖能力，再利用平板克隆实验观察转染后细胞克隆形成能力差异，最后通过流式细胞仪检测瞬时敲降CEP170蛋白表达后肝癌细胞周期的改变。

通过CPTAC蛋白表达数据库分析发现，在肝癌的癌组织及其配对的癌旁组织中，人中心体蛋白CEP170表达差异显著（P＜0.001），高表达与预后不良正相关（P＜0.01），且CEP170高表达是患者预后不良的独立风险因素。TCGA数据库分析CEP170转录水平差异，结果显示癌组织中CEP170的mRNA水平显著高于癌旁组织（P<0.001），且与预后不良正相关（P<0.001）。cBioPortal数据库分析基因组DNA拷贝数的变化差异，发现在6%的癌组织中CEP170存在扩增。Human Protein Atlas数据库中的IHC结果及组织切片IHC验证均可见CEP170在癌组织中表达升高。通过特异性siRNA敲降肝癌细胞Huh-7的CEP170表达后，细胞增殖及克隆形成能力均显著降低（P<0.001），细胞周期发生G0/G1期阻滞。

综上所述，本研究首次揭示了CEP170对肝癌细胞增殖的影响，CEP170在肝癌组织中异常高表达且与预后不良正相关，特异性敲降CEP170表达能够显著抑制人肝癌细胞Huh-7的增殖和克隆形成能力且细胞周期发生G0/G1期阻滞。CEP170是肝癌治疗中潜在的治疗靶点。

第二部分天冬酰胺合成酶在子宫内膜癌中的表达及功能研究

子宫内膜癌是原发于子宫内膜上皮的恶性肿瘤，是女性生殖系统三大恶性肿瘤之一。近年来，子宫内膜癌的发病率逐渐升高并日趋年轻化，发病率持续增加，给我国社会和人民生活带来了沉重的负担。

蛋白质组学数据库整合了肿瘤蛋白质组和患者的临床信息，使得研究者通过高通量测序的方法探索与肿瘤治疗相关的分子靶点及可用于患者预后预测的生物标志物。本研究内容首先对CPTAC，TCPA及TCGA数据库中子宫内膜癌及癌旁进行差异基因和预后分析，筛选鉴定出了与子宫内膜癌预后不良正相关的分子天冬酰胺合成酶ASNS。选择ASNS高表达的子宫内膜癌细胞株AN3-CA和RL95-2，利用特异性siRNA敲降ASNS表达后，通过CCK-8法检测细胞增殖能力，平板克隆实验观察转染后细胞克隆形成能力差异。随后通过慢病毒干扰RNA质粒和慢病毒基因过表达质粒，分别构建了ASNS稳定敲除和过表达的稳转细胞株，开展裸鼠移植瘤实验观察ASNS表达改变后移植瘤形成能力的变化。

研究结果显示，ASNS在EC组织中蛋白表达及mRNA水平均显著升高(P<0.001)，且与患者预后不良正相关(P<0.01)。在蛋白表达及mRNA水平，ASNS与增殖相关分子Ki67及PCNA的表达均正相关。对配对的癌和癌旁组织进行qPCR及Western Blot检测，结果显示ASNS的转录及蛋白水平在EC组织中均显著升高(P<0.05)。敲降AN3-CA和RL95-2细胞ASNS表达后，细胞增殖及克隆形成能力显著降低（P<0.001）。裸鼠移植瘤实验结果显示，稳定敲降ASNS表达后裸鼠移植瘤形成能力显著降低（P<0.001），过表达则增强成瘤能力（P<0.05）。

综上所述，本研究首次探讨了ASNS在子宫内膜癌中的表达改变和患者预后的相关性，进一步通过体内外实验证实了ASNS过表达可促进子宫内膜癌细胞的恶性增殖，ASNS在子宫内膜中是可用于患者疾病风险预测和候选的药物治疗靶点。

Part ⅠAnalysis of CEP170 expression in liver cancer and its role in proliferation of liver cancer cells

Liver cancer has the remarkable characteristics of high recurrence, easy metastasis and poor prognosis. The number of deaths from liver cancer in our country accounts for about 50% of the total number of liver cancer deaths in the world, which seriously endangers the life and health of the Chinese people. Although the research of liver cancer has made some progress in many aspects, the prognosis is still unsatisfactory, and it is extremely important to find more effective therapeutic targets. In recent years, with the rapid development of mass spectrometry-based proteomics technology, people have a more intuitive understanding of the complexity and dynamics of the proteome, and the use of proteomic data can more accurately answer biological questions. In this study, the proteomic data and related clinical information of liver cancer were downloaded from the proteome database CPTAC, and the relationship between differential protein expression and clinical prognosis was analyzed using the R package limma. The transcript level and gene copy number variation of CEP170 in the TCGA database were analyzed online by UALCAN and cBioPortal. Survival analysis was performed using the Kaplan-Meier method, and the predictive effect was tested by ROC curve. Next, the data analysis results were verified by tissue and cell experiments. Immunohistochemical experiments confirmed the difference in protein expression of CEP170 in cancer tissues and adjacent tissues. The expression of CEP170 in liver cancer cell line Huh-7 was knocked down with specific siRNA, the cell proliferation ability was detected by CCK-8 method, and the difference in the ability of cell clone formation after transfection was observed by plate cloning experiment. Finally, the transient knockdown was detected by flow cytometry. Changes in the cell cycle of hepatocellular carcinoma after decreasing CEP170 protein expression.

Through the analysis of CPTAC protein expression database, it was found that the expression of human centrosome protein CEP170 was significantly different in the cancer tissue of liver cancer and its paired adjacent tissue (P<0.001), and high expression was associated with poor prognosis（P＜0.01）, and high expression of CEP170 was associated with the prognosis of patients Adverse independent risk factors. The TCGA database analyzed the differences in the transcription level of CEP170, and the results showed that the mRNA level of CEP170 in cancer tissues was significantly higher than that in adjacent tissues (P<0.001)，and high expression was associated with poor prognosis（P＜0.001）. The cBioPortal database analyzed the variation of genomic DNA copy number and found that CEP170 was amplified in 6% of cancer tissues. IHC results in the Human Protein Atlas database and IHC verification of tissue sections showed that CEP170 expression was elevated in cancer tissues. After knocking down the expression of CEP170 in Huh-7 liver cancer cells by specific siRNA, the cell proliferation and clone formation ability were significantly decreased (P<0.001), and the cell cycle was arrested in G0/G1 phase.

In conclusion, this study revealed for the first time the effect of CEP170 on the proliferation of hepatoma cells. CEP170 is abnormally highly expressed in hepatocellular carcinoma tissues and is positively correlated with poor prognosis. Specific knockdown of CEP170 expression can significantly inhibit the proliferation of human hepatoma cells Huh-7. and clonogenicity and cell cycle arrest in G0/G1 phase. CEP170 is a potential therapeutic target in the treatment of liver cancer.

Part ⅡExpression and function of asparagine synthase in endometrial carcinoma

Endometrial carcinoma is a malignant tumor originating from the endometrial epithelium and is one of the three major malignant tumors of the female reproductive system. In recent years, the incidence of endometrial carcinoma has gradually increased and become younger, and the incidence has continued to increase, which has brought a heavy burden to our society and people's lives.

The proteomic database integrates tumor proteome and clinical information of patients, allowing researchers to explore molecular targets related to tumor therapy and biomarkers that can be used to predict patient prognosis through high-throughput sequencing methods. In this study, the differential genes and prognosis of endometrial carcinoma and adjacent cancer in the CPTAC, TCPA and TCGA databases were firstly analyzed, and the molecular asparagine synthase ASNS, which was positively correlated with poor prognosis of endometrial carcinoma, was screened and identified. The endometrial carcinoma cell lines AN3-CA and RL95-2 with high ASNS expression were selected. After the ASNS expression was knocked down by specific siRNA, the cell proliferation ability was detected by CCK-8 method, and the cell clone formation ability after transfection was observed by plate cloning experiment. difference. Then, using lentiviral interfering RNA plasmids and lentiviral gene overexpression plasmids to construct stable ASNS knockout and overexpression stable cell lines, respectively, and carry out nude mice xenograft experiments to observe the changes in the ability of xenografts to form tumors after ASNS expression changes.

The results showed that the protein expression and mRNA level of ASNS in EC tissues were significantly increased (P<0.001), and it was positively correlated with the poor prognosis of patients(P<0.01). At protein expression and mRNA level, ASNS was positively correlated with the expression of proliferation-related molecules Ki67 and PCNA. The paired cancer and adjacent tissues were detected by qPCR and Western Blot, and the results showed that the transcription and protein levels of ASNS were significantly increased in EC tissues (P<0.05). Knockdown of ASNS expression in AN3-CA and RL95-2 cells significantly decreased cell proliferation and colony formation (P<0.001). The results of the nude mouse xenograft experiments showed that the ability of nude mice to form xenografts was significantly reduced after stably knocking down the expression of ASNS（P<0.001）, while overexpression enhanced the tumorigenic ability（P<0.05）.

In conclusion, this study is the first to explore the correlation between ASNS expression changes in endometrial carcinoma and patient prognosis. Further in vitro and in vivo experiments confirmed that ASNS overexpression can promote the malignant proliferation of endometrial carcinoma cells. ASNS in the endometrium is a potential target for patient disease risk prediction and drug therapy.