FOXK1协同Rufy3促进大肠癌侵袭转移机制研究

发布时间：2018-05-31 07:40

本文选题：Rufy3 + FOXK1　；参考：《南方医科大学》2017年硕士论文

【摘要】：目的:Rufy3蛋白参与细胞迁移、肌动蛋白细胞骨架的运动、脂质修饰、膜转运和细胞信号转导等多种细胞活动过程的调控。有研究表明,p21活化激酶(PAK1)可以促进Rufy3的表达且协同Rufy3促进胃癌细胞的侵袭和迁移。另外,转录因子FOXK1参与胚胎发育、细胞代谢和分化、组织修复等活动的调节,且在多种肿瘤发生发展中扮演癌基因的角色。本课题旨在明确FOXK1和Rufy3在大肠癌增殖、侵袭转移过程中发挥的作用及可能的作用机制。方法:1.免疫组织化学法检测Rufy3和FOXK1在大肠癌组织和正常大肠黏膜组织中的表达,分析Rufy3和FOXK1的表达量之间的关系、与大肠癌病人的病例特征、预后的关系;2.流式细胞周期技术、Western blot技术检测Rufy3对大肠癌细胞周期的影响;3.构建Rufy3稳定表达株、EdU染色、裸鼠皮下移植瘤模型检测Rufy3对大肠癌增殖的影响;4.Transwell侵袭实验、细胞划痕、免疫荧光、罗丹明鬼笔环肽染色等实验检测FOXK1和Rufy3对于大肠癌细胞上皮细胞间充质转化作用(EMT)、侵袭转移功能的影响;5.运用Smad2/3 siRNA和Smad4 siRNA、转化生长因子-β1(TGF-β1)相关实验研究Rufy3作用于大肠癌细胞可能的TGF-β/Smad通路机制;6.构建裸鼠原位肝转移模型,免疫组化、qRT-PCR方法检测E-cadherin、Vimentin的表达情况,研究在体内,FOXK1和Rufy3对大肠癌的作用。结果:1.Rufy3在大肠癌组织中的表达高于正常大肠粘膜组织,Rufy3高表达的大肠癌手术病人预后较低表达者差,Rufy3的表达与大肠癌的TNM分期、分化程度、AJCC分期、是否发生淋巴结转移具有显著的相关性;2.敲低Rufy3,处于G0/G1期的大肠癌细胞增多,G2/M细胞减少,相关周期蛋白表达发生改变;3.过表达Rufy3组的裸鼠皮下瘤较对照组生长快,Ki67表达量高;4.过表达Rufy3促进Vimentin的表达,抑制E-cadherin的表达,加快划痕愈合速度,增强大肠癌细胞穿透Transwell膜的能力,敲低Rufy3减弱TGF-β1对于大肠癌细胞的EMT作用;5.过表达Rufy3组的裸鼠较Vector组更多发生肝转移,且E-cadherin表达降低;6.Rufy3和FOXK1在大肠癌细胞中存在相互作用,并在大肠癌组织中高表达,表达量高者较低者平均生存时间短;7.Rufy3稳定表达株,敲低FOXK1后,Vimentin表达下降,E-cadherin表达上升,划痕愈合速度减慢,穿透 Transwell 膜的能力减弱;8.Rufy3 组较 Vector 组、Rufy3-FOXK1-siRNA组裸鼠形成的转移结节多而大,Vimentin的表达量最高。结论:1.Rufy3在大肠癌组织中的表达高于正常大肠粘膜组织,Rufy3高表达负性影响大肠癌手术病人的预后;3.Rufy3过表达促进大肠癌的增殖、侵袭转移能力;4.Rufy3过表达在大肠癌细胞中可能通过TGF-β/Smad信号通路促进EMT作用;5.Rufy3和FOXK1在大肠癌细胞内存在相互作用,两者在大肠癌组织中的表达呈正相关,协同负性影响预后;6.Rufy3对大肠癌EMT、侵袭转移能力的促进作用能被FOXK1-siRNA 抑制。
[Abstract]:Aim to regulate the cellular activity of cell migration, actin cytoskeleton movement, lipid modification, membrane transport and cell signal transduction. Some studies have shown that p21 activated kinase PAK1) can promote the expression of Rufy3 and promote the invasion and migration of gastric cancer cells in combination with Rufy3. In addition, transcription factor FOXK1 is involved in the regulation of embryonic development, cell metabolism and differentiation, tissue repair, and plays the role of oncogene in the development of many kinds of tumors. The purpose of this study was to clarify the role of FOXK1 and Rufy3 in the proliferation, invasion and metastasis of colorectal cancer. Method 1: 1. Immunohistochemical method was used to detect the expression of Rufy3 and FOXK1 in colorectal carcinoma and normal colorectal mucosa. The relationship between the expression of Rufy3 and FOXK1 was analyzed. Flow cytometry and Western blot were used to detect the effect of Rufy3 on the cell cycle of colorectal cancer. The effect of Rufy3 on the proliferation of colorectal carcinoma in nude mice was detected by using Rufy3 stable expression strain Edu staining. 4. Transwell invasion assay, cell scratch, immunofluorescence. The effects of FOXK1 and Rufy3 on epithelial mesenchymal transformation and invasion and metastasis of colorectal cancer cells were detected by rhodamine penicyclopeptide staining. Smad2/3 siRNA and Smad4 siRNAs were used to study the mechanism of TGF- 尾 / Smad pathway induced by Rufy3 in colorectal cancer cells. An in situ liver metastasis model was established in nude mice. The expression of E-cadherinus vimentin was detected by immunohistochemical qRT-PCR. The effect of FOXK1 and Rufy3 on colorectal carcinoma was studied in vivo. Results 1. The expression of Rufy3 in colorectal carcinoma tissues was higher than that in normal colorectal mucosa tissues. The prognosis of patients with lower expression of Rufy3 was lower than that of patients with lower expression of Rufy3. The expression of Rufy3 in colorectal carcinoma was lower than that in patients with colorectal cancer. The expression of Rufy3 in colorectal carcinoma was different from that in TNM stage. There is a significant correlation between lymph node metastasis and lymph node metastasis. With low Rufy3 knockout, the number of G2 / M cells in G0/G1 phase increased and the expression of cyclin was changed. The expression of Ki67 in subcutaneous tumor of nude mice with overexpression of Rufy3 was higher than that in control group. Overexpression of Rufy3 promoted the expression of Vimentin, inhibited the expression of E-cadherin, accelerated the healing rate of scratches, enhanced the ability of colorectal cancer cells to penetrate Transwell membrane, and knocked down Rufy3 to attenuate the EMT effect of TGF- 尾 1 on colorectal cancer cells. In nude mice with overexpression of Rufy3, liver metastasis was more than that in Vector, and the expression of E-cadherin decreased. 6. There was interaction between Rufy3 and FOXK1 in colorectal cancer cells, and high expression was found in colorectal carcinoma. The average survival time of those with higher expression was shorter than that of Vector, and the stable expression of Rufy3 was higher than that of Vector. The expression of Vimentin increased, the rate of scratch healing slowed down, and the ability to penetrate the Transwell membrane decreased. The expression of Vimentin in group Rufy3 was higher than that in group Vector (Rufy3-FOXK1-siRNA), but the expression of Vimentin was the highest. Conclusion: 1. The high expression of Rufy3 in colorectal carcinoma is higher than that in normal colorectal mucosa. The high expression of Rufy3 negatively affects the prognosis of colorectal cancer patients. 3. The overexpression of Rufy3 promotes the proliferation of colorectal cancer. 4. The overexpression of Rufy3 in colorectal cancer cells may promote the interaction between EMT and FOXK1 through TGF- 尾 / Smad signaling pathway. The expression of Rufy3 and FOXK1 in colorectal cancer cells is positively correlated. 6. Rufy3 can promote the invasion and metastasis of colorectal carcinoma and the ability of invasion and metastasis can be inhibited by FOXK1-siRNA.
【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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