FOXK1与FHL2在大肠癌的增殖、EMT、侵袭和转移中的相互作用

发布时间：2018-12-06 20:58

【摘要】：目的与意义转录因子FOXK1作为FOX家族的一个成员,参与细胞的生长代谢,在多种肿瘤的发生发展中可能发挥重要作用。有研究表明,FOXK1的高表达与结直肠癌的发生发展密切相关,能促进结直肠癌细胞的增值侵袭与转移,影响结直肠癌患者预后。FHL2(Four and a half LIM Protein 2)作为癌基因,在胃肠道肿瘤的发生发展过程中起着重要的作用。有文献报道,FHL2蛋白能与多种靶蛋白结合并调控靶蛋白的活性,是诱导上皮间质转化(EMT)和侵袭的一个关键的蛋白,在胃肠道肿瘤中高表达,正常组织中不表达。目前有研究FOXK1与FHL2在肌源性祖细胞中的相互作用,但在胃肠道肿瘤中的相互作用机制研究甚少。本课题主要研究FOXK1与FHL2在结直肠癌中的相互作用,评估FOXK1与FHL2的表达对结直肠预后的影响,为结直肠癌探索新的治疗靶点提供依据。材料和方法主要材料SW480、Caco2、SW620细胞,FOXK1抗兔单克隆抗体、FHL2抗兔/鼠单克隆抗体,E-cadherin、GAPDH 抗兔单克隆抗体、Vimentin、MMP2、MMP9、Snail抗鼠单克隆抗体,抗鼠荧光二抗、抗兔荧光二抗,罗丹明标记鬼笔环肽,EdU试剂盒,结晶紫。主要方法1.收集结直肠癌组织,用qRT-PCR和免疫组化检测并比较FOXK1在结直肠癌组织与正常组织中的表达。2.用免疫荧光的方法证实FOXK1与FHL2在结直肠癌细胞中的表达与定位。3.利用免疫共沉淀验证FOXK1与FHL2间的相互作用,探讨两者的关系。4.评估FOXK1和FHL2对结直肠癌预后的影响。87例结直肠癌组织芯片分别FOXK1和FHL2染色,相关性分析验证FOXK1与FHL2的相互作用,生存分析评估其对结直肠癌预后的影响。5.下调FHL2验证FOXK1与FHL2在结直肠癌中的相互作用。5.1建立稳定表达株Vector,及FOXK1,并在此基础上瞬转FHL2 siRNA(FOXK1-FHL2 siRNA)。5.2 EdU细胞增殖实验、划痕实验、Transwell细胞侵袭实验探究下调FHL2对FOXK1诱导的结直肠癌细胞增殖、侵袭、转移能力的影响。5.3观察三组细胞形态变化、罗丹明鬼笔环肽染色观察细胞骨架变化、Western blot 检测三组细胞 E-cadherin、Vimentin、Snai1、MMP2、MMP9 的表达。探究下调FHL2对FOXK1诱导的结直肠癌细胞的EMT的影响。6.收集结直肠癌淋巴结转移患者的淋巴结,免疫组化验证FOXK1和FHL2在转移瘤中的表达。7.建立裸鼠皮下成瘤模型、脾被膜下肝转移模型和尾静脉肺转移模型,体内验证压低FHL2表达对FOXK1诱导的侵袭转移作用的影响。结果1.FOXK1在结直肠癌组织中的表达高于正常组织;2.FOXK1和FHL2在结直肠癌组织细胞的表达呈正相关;3.FOXK1与FHL2在结直肠癌中存在相互协同作用;4.FHL2siRNA抑制体内外FOXK1诱导的结直肠癌细胞的增殖、EMT、侵袭和转移。结论FOXK1与FHL2在结直肠癌的增殖、EMT、侵袭和转移中具有协同作用,因此,FOXK1和FHL2可能作为结直肠癌的综合治疗的靶基因。
[Abstract]:Aim and significance transcription factor FOXK1, as a member of FOX family, is involved in cell growth and metabolism, and may play an important role in the occurrence and development of many kinds of tumors. Studies have shown that the high expression of FOXK1 is closely related to the occurrence and development of colorectal cancer, can promote the proliferation of colorectal cancer cell invasion and metastasis, affecting the prognosis of colorectal cancer patients. FHL2 (Four and a half LIM Protein 2) as a oncogene, It plays an important role in the occurrence and development of gastrointestinal cancer. It has been reported that FHL2 protein can bind to many target proteins and regulate the activity of target proteins. It is a key protein to induce the transformation of (EMT) and invasion in epithelial stroma. It is highly expressed in gastrointestinal tumors but not in normal tissues. At present, the interaction of FOXK1 and FHL2 in myogenic progenitor cells has been studied, but the mechanism of interaction in gastrointestinal neoplasms is rare. The purpose of this study was to investigate the interaction of FOXK1 and FHL2 in colorectal cancer, to evaluate the effect of FOXK1 and FHL2 expression on the prognosis of colorectal cancer, and to provide evidence for exploring new therapeutic targets for colorectal cancer. Materials and methods SW480,Caco2,SW620 cells, FOXK1 anti rabbit monoclonal antibody, FHL2 anti rabbit / mouse monoclonal antibody, E cadherin GAPDH anti rabbit monoclonal antibody, Vimentin,MMP2,MMP9,Snail anti mouse monoclonal antibody, anti mouse fluorescence second antibody were used. Anti-rabbit fluorescent second antibody, Rhodamine labeled Gypophorin, EdU kit, crystal violet. Main method 1. QRT-PCR and immunohistochemistry were used to detect and compare the expression of FOXK1 in colorectal cancer tissues and normal tissues. 2. 2. Immunofluorescence assay was used to confirm the expression and localization of FOXK1 and FHL2 in colorectal cancer cells. The interaction between FOXK1 and FHL2 was verified by immunoprecipitation, and the relationship between them was discussed. 4. To evaluate the influence of FOXK1 and FHL2 on the prognosis of colorectal cancer. 87 cases of colorectal cancer were stained with FOXK1 and FHL2 respectively. The correlation analysis verified the interaction between FOXK1 and FHL2, and the survival analysis evaluated the influence of FOXK1 on prognosis of colorectal cancer. FHL2 down-regulation was used to verify the interaction between FOXK1 and FHL2 in colorectal cancer. 5.1 the stable expression lines Vector, and FOXK1, were established and FHL2 siRNA (FOXK1-FHL2 siRNA). 5.2 EdU cell proliferation test, scratch test) were transiently transformed. The effect of down-regulation of FHL2 on the proliferation, invasion and metastasis of colorectal cancer cells induced by FOXK1 was investigated by Transwell cell invasion assay. 5.3 the morphologic changes of the three groups were observed, and the cytoskeleton changes were observed by Rhodamine phloropeptide staining. Western blot was used to detect the expression of E-cadherin and Vimentinon Snai1 MMP2 and MMP9 in three groups. To explore the effect of down-regulation of FHL2 on EMT induced by FOXK1 in colorectal cancer cells. 6. 6. The lymph nodes of patients with lymph node metastasis from colorectal cancer were collected, and the expressions of FOXK1 and FHL2 in metastatic tumors were confirmed by immunohistochemistry. 7. 7%. Subcutaneous tumorigenesis model, splenic submembrane liver metastasis model and caudal vein lung metastasis model were established in nude mice to verify the effect of low expression of FHL2 on invasion and metastasis induced by FOXK1 in vivo. Results the expression of 1.FOXK1 in colorectal cancer tissues was higher than that in normal tissues, the expression of 2.FOXK1 and FHL2 was positively correlated with the expression of FHL2 in colorectal cancer tissues, and there was a synergistic effect between 3.FOXK1 and FHL2 in colorectal cancer. 4.FHL2siRNA inhibits the proliferation, EMT, invasion and metastasis of colorectal cancer cells induced by FOXK1 in vivo and in vitro. Conclusion FOXK1 and FHL2 have synergistic effects on the proliferation, EMT, invasion and metastasis of colorectal cancer. Therefore, FOXK1 and FHL2 may be the target genes for comprehensive therapy of colorectal cancer.
【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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