IQGAP3促进结直肠癌细胞增殖和迁移的作用及分子机制

发布时间：2018-03-02 10:53

本文关键词： 结直肠癌 IQGAP3 增殖迁移 KRAS　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的:结直肠癌(Colorectal Cancer,CRC)是世界范围内最常见的恶性肿瘤之一,其发病率位居所有恶性肿瘤第三位,死亡率位居第四位,仅次于肺癌、肝癌和胃癌。在中国,CRC的发病率和死亡率自2010年以来一直在持续增长,成为癌症患者死亡的主要原因,并呈现年轻化趋势。CRC不仅降低了患者的生活质量,也给社会带来了沉重的经济负担;然而,CRC发生和发展的分子机制目前尚不完全清楚。CRC的发生是一个多阶段、多步骤的复杂过程,遵循着“正常-增生-腺瘤-癌”等发展变化过程;主要机制有包括染色体不稳定(CNI)、微卫星不稳定(MSI)、CpG岛甲基化表型(CIMP)、锯齿状等发生途径,还有一部分CRC的发生发展涉及两条及以上方式。肿瘤转移是临床上导致患者死亡的主要原因,其过程主要包括局部浸润、渗入血管、随血循环流动、穿出血管,最终在远处器官定植并形成新的转移灶等过程,在这一系列过程中,涉及了许多复杂的因素。所以,深入研究CRC发生、发展和侵袭转移的分子机制,寻找有价值的分子标志物,对CRC的早期诊断、预后评估以及靶向治疗等均有重要科学意义和临床价值。IQGAP3基因位于染色体1q21.3,分子量大小约为180KD,含1631个氨基酸,主要在肠道、肝脏及脑、肺等器官中表达。研究发现,IQGAP3能够通过特定的结构区结合相关蛋白质,影响细胞增殖、分化、细胞粘附、细胞骨架以及细胞运动等。目前对IQGAP3的研究主要集中在神经突增生、细胞骨架形成等方面,研究还发现IQGAP3表达紊乱还参与肝癌、肺癌等恶性肿瘤的发生和发展等过程;然而,其表达失调在CRC发生和发展过程中的作用及分子机制目前尚不清楚。因此,本研究拟深入探讨IQGAP3在CRC发生和演进中的功能和分子机制。方法1.利用TCGA公共数据库、免疫组化(Immunohistochemistry,IHC)法分析、检测CRC组织及相应癌旁正常粘膜组织中IQGAP3的表达情况;2.利用荧光实时定量PCR、蛋白免疫印迹法检测IQGAP3在肠癌组织和细胞株中的表达,并构建IQGAP3稳定过表达和干扰细胞株;3.采用MTT、平板克隆形成、软琼脂克隆形成实验和裸鼠皮下成瘤实验,划痕愈合实验、Transwell小室实验,检测IQGAP3对肿瘤细胞增殖和迁移能力的影响;4.通过GSEA基因富集分析预测IQGAP3参与的生物过程,利用流式细胞术及Western blot的方法检测IQGAP3过表达或被干扰后相关信号通路靶基因的变化情况,探讨IQGAP3参与CRC发生和迁移的分子机制。结果1.IQGAP3在结直肠癌组织中的表达水平高于相配对的癌旁正常组织;2.IQGAP3在结直肠癌细胞株中的表达高于正常肠粘膜细胞;3.IQGAP3过表达能够显著促进CRC细胞的增殖及迁移能力,而IQGAP3被干扰后能够显著抑制CRC细胞的增殖及迁移能力;4.IQGAP3影响KRAS信号通路的活性及其下游靶基因的表达。结论1.IQGAP3在CRC组织中的表达水平显著高于配对的正常肠黏膜组织;2.IQGAP3过表达能够显著促进CRC细胞的增殖及迁移能力,而IQGAP3被干扰后能够显著抑制CRC细胞的增殖及迁移能力;3.IQGAP3通过调控KRAS/AKT信号通路、KRAS/Rac1、CDC42信号通路,促进CRC细胞的增殖和迁移。
[Abstract]:Background and objective: colorectal cancer (Colorectal, Cancer, CRC) is one of the most common malignant tumors in the world, the incidence rate of third among all malignant tumors, the mortality rate ranks fourth only to lung cancer, liver cancer and gastric cancer. In China, the incidence and mortality of CRC since 2010 has continued to grow, become the main cause of death in cancer patients, and there is a trend of young.CRC not only reduces the patient's quality of life, also brought a heavy economic burden; however, the molecular mechanism of the occurrence and development of CRC is not entirely clear to the occurrence of.CRC is a multi-stage, multi-step complex process, follow the "development changes in normal hyperplasia adenoma carcinoma" process; including the main mechanism of chromosomal instability (CNI) and microsatellite instability (MSI), CpG island methylator phenotype (CIMP), zigzag way, and a The occurrence of CRC involved in the development of two and above. The clinical tumor metastasis is the leading cause of death in patients, which mainly includes local infiltration, infiltration of blood flow, the blood circulation, through blood vessels, eventually in distant organs colonization and the formation of new metastases in this process, a series of process. Involves many complicated factors. Therefore, in-depth study of the molecular mechanism of CRC occurrence, development and metastasis and find valuable molecular markers for early diagnosis, CRC, prognosis and targeted therapy has important scientific significance and clinical value of.IQGAP3 gene is located on chromosome 1q21.3, the molecular weight is about 180KD, including 1631 amino acids, mainly in the intestine, liver and brain, the expression of lung and other organs. The study found that IQGAP3 was able to structure specific binding proteins, adhesion affect cell proliferation, differentiation, cells, bone cells Frame and cell movement. The current research on IQGAP3 mainly focus on the proliferation of neurites, cytoskeleton formation and other aspects, the study also found that IQGAP3 expression disorder is also involved in the occurrence and development of liver cancer, lung cancer and other malignant tumors; however, its expression is dysregulated in CRC incidence and effects and the molecular mechanisms in the development process is still not clear. Therefore, this study intends to explore the function of IQGAP3 and CRC in the molecular mechanism of carcinogenesis and progression. Methods 1. using the TCGA public database, immunohistochemistry (Immunohistochemistry, IHC) analysis to detect expression of IQGAP3 CRC tissues and adjacent normal mucosa tissues; 2. by real-time fluorescent quantitative PCR the expression of IQGAP3 protein was detected by Western blot in colorectal cancer tissues and cell lines, and the construction of stable IQGAP3 overexpression and RNAi cells; 3. by MTT, clone formation, soft agar solid Check and subcutaneous tumor formation experiment, wound healing assay, Transwell assay, to study the effect of IQGAP3 on proliferation and migration of tumor cells; 4. by GSEA gene enrichment analysis and prediction of biological processes involved in IQGAP3, using the method of flow cytometry and Western blot detection IQGAP3 changes related signal pathway or target gene expression after the interference of IQGAP3 in CRC, the occurrence and migration mechanism. Results 1.IQGAP3 expression in colorectal cancer tissues was higher than that of adjacent normal tissues; the expression of 2.IQGAP3 in colorectal cancer cell lines than that in normal intestinal mucosa cells; overexpression of 3.IQGAP3 can significantly promote the proliferation and migration of CRC cells, and IQGAP3 interference can significantly inhibit the proliferation and migration of CRC cells; the expression of 4.IQGAP3 activity and its downstream target genes. The effect of KRAS signaling node The expression level of 1.IQGAP3 in CRC tissues was significantly higher than that in normal mucosa pairing; overexpression of 2.IQGAP3 could significantly promote the proliferation and migration of CRC cells, and IQGAP3 interference can significantly inhibit the proliferation and migration of CRC cells through KRAS/AKT signaling pathway; 3.IQGAP3, KRAS/Rac1, CDC42 signaling pathway, and promote the proliferation of the migration of CRC cells.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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