發(fā)布時間：2018-03-02 10:53

  本文關鍵詞： 結直腸癌 IQGAP3 增殖 遷移 KRAS　出處：《南方醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究背景和目的:結直腸癌(Colorectal Cancer,CRC)是世界范圍內最常見的惡性腫瘤之一,其發(fā)病率位居所有惡性腫瘤第三位,死亡率位居第四位,僅次于肺癌、肝癌和胃癌。在中國,CRC的發(fā)病率和死亡率自2010年以來一直在持續(xù)增長,成為癌癥患者死亡的主要原因,并呈現年輕化趨勢。CRC不僅降低了患者的生活質量,也給社會帶來了沉重的經濟負擔;然而,CRC發(fā)生和發(fā)展的分子機制目前尚不完全清楚。CRC的發(fā)生是一個多階段、多步驟的復雜過程,遵循著“正常-增生-腺瘤-癌”等發(fā)展變化過程;主要機制有包括染色體不穩(wěn)定(CNI)、微衛(wèi)星不穩(wěn)定(MSI)、CpG島甲基化表型(CIMP)、鋸齒狀等發(fā)生途徑,還有一部分CRC的發(fā)生發(fā)展涉及兩條及以上方式。腫瘤轉移是臨床上導致患者死亡的主要原因,其過程主要包括局部浸潤、滲入血管、隨血循環(huán)流動、穿出血管,最終在遠處器官定植并形成新的轉移灶等過程,在這一系列過程中,涉及了許多復雜的因素。所以,深入研究CRC發(fā)生、發(fā)展和侵襲轉移的分子機制,尋找有價值的分子標志物,對CRC的早期診斷、預后評估以及靶向治療等均有重要科學意義和臨床價值。IQGAP3基因位于染色體1q21.3,分子量大小約為180KD,含1631個氨基酸,主要在腸道、肝臟及腦、肺等器官中表達。研究發(fā)現,IQGAP3能夠通過特定的結構區(qū)結合相關蛋白質,影響細胞增殖、分化、細胞粘附、細胞骨架以及細胞運動等。目前對IQGAP3的研究主要集中在神經突增生、細胞骨架形成等方面,研究還發(fā)現IQGAP3表達紊亂還參與肝癌、肺癌等惡性腫瘤的發(fā)生和發(fā)展等過程;然而,其表達失調在CRC發(fā)生和發(fā)展過程中的作用及分子機制目前尚不清楚。因此,本研究擬深入探討IQGAP3在CRC發(fā)生和演進中的功能和分子機制。方法1.利用TCGA公共數據庫、免疫組化(Immunohistochemistry,IHC)法分析、檢測CRC組織及相應癌旁正常粘膜組織中IQGAP3的表達情況;2.利用熒光實時定量PCR、蛋白免疫印跡法檢測IQGAP3在腸癌組織和細胞株中的表達,并構建IQGAP3穩(wěn)定過表達和干擾細胞株;3.采用MTT、平板克隆形成、軟瓊脂克隆形成實驗和裸鼠皮下成瘤實驗,劃痕愈合實驗、Transwell小室實驗,檢測IQGAP3對腫瘤細胞增殖和遷移能力的影響;4.通過GSEA基因富集分析預測IQGAP3參與的生物過程,利用流式細胞術及Western blot的方法檢測IQGAP3過表達或被干擾后相關信號通路靶基因的變化情況,探討IQGAP3參與CRC發(fā)生和遷移的分子機制。結果1.IQGAP3在結直腸癌組織中的表達水平高于相配對的癌旁正常組織;2.IQGAP3在結直腸癌細胞株中的表達高于正常腸粘膜細胞;3.IQGAP3過表達能夠顯著促進CRC細胞的增殖及遷移能力,而IQGAP3被干擾后能夠顯著抑制CRC細胞的增殖及遷移能力;4.IQGAP3影響KRAS信號通路的活性及其下游靶基因的表達。結論1.IQGAP3在CRC組織中的表達水平顯著高于配對的正常腸黏膜組織;2.IQGAP3過表達能夠顯著促進CRC細胞的增殖及遷移能力,而IQGAP3被干擾后能夠顯著抑制CRC細胞的增殖及遷移能力;3.IQGAP3通過調控KRAS/AKT信號通路、KRAS/Rac1、CDC42信號通路,促進CRC細胞的增殖和遷移。
[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.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.34

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