FOXK1與FHL2在大腸癌的增殖、EMT、侵襲和轉(zhuǎn)移中的相互作用
發(fā)布時間：2018-12-06 20:58
【摘要】：目的與意義轉(zhuǎn)錄因子FOXK1作為FOX家族的一個成員,參與細胞的生長代謝,在多種腫瘤的發(fā)生發(fā)展中可能發(fā)揮重要作用。有研究表明,FOXK1的高表達與結(jié)直腸癌的發(fā)生發(fā)展密切相關,能促進結(jié)直腸癌細胞的增值侵襲與轉(zhuǎn)移,影響結(jié)直腸癌患者預后。FHL2(Four and a half LIM Protein 2)作為癌基因,在胃腸道腫瘤的發(fā)生發(fā)展過程中起著重要的作用。有文獻報道,FHL2蛋白能與多種靶蛋白結(jié)合并調(diào)控靶蛋白的活性,是誘導上皮間質(zhì)轉(zhuǎn)化(EMT)和侵襲的一個關鍵的蛋白,在胃腸道腫瘤中高表達,正常組織中不表達。目前有研究FOXK1與FHL2在肌源性祖細胞中的相互作用,但在胃腸道腫瘤中的相互作用機制研究甚少。本課題主要研究FOXK1與FHL2在結(jié)直腸癌中的相互作用,評估FOXK1與FHL2的表達對結(jié)直腸預后的影響,為結(jié)直腸癌探索新的治療靶點提供依據(jù)。材料和方法主要材料SW480、Caco2、SW620細胞,FOXK1抗兔單克隆抗體、FHL2抗兔/鼠單克隆抗體,E-cadherin、GAPDH 抗兔單克隆抗體、Vimentin、MMP2、MMP9、Snail抗鼠單克隆抗體,抗鼠熒光二抗、抗兔熒光二抗,羅丹明標記鬼筆環(huán)肽,EdU試劑盒,結(jié)晶紫。主要方法1.收集結(jié)直腸癌組織,用qRT-PCR和免疫組化檢測并比較FOXK1在結(jié)直腸癌組織與正常組織中的表達。2.用免疫熒光的方法證實FOXK1與FHL2在結(jié)直腸癌細胞中的表達與定位。3.利用免疫共沉淀驗證FOXK1與FHL2間的相互作用,探討兩者的關系。4.評估FOXK1和FHL2對結(jié)直腸癌預后的影響。87例結(jié)直腸癌組織芯片分別FOXK1和FHL2染色,相關性分析驗證FOXK1與FHL2的相互作用,生存分析評估其對結(jié)直腸癌預后的影響。5.下調(diào)FHL2驗證FOXK1與FHL2在結(jié)直腸癌中的相互作用。5.1建立穩(wěn)定表達株Vector,及FOXK1,并在此基礎上瞬轉(zhuǎn)FHL2 siRNA(FOXK1-FHL2 siRNA)。5.2 EdU細胞增殖實驗、劃痕實驗、Transwell細胞侵襲實驗探究下調(diào)FHL2對FOXK1誘導的結(jié)直腸癌細胞增殖、侵襲、轉(zhuǎn)移能力的影響。5.3觀察三組細胞形態(tài)變化、羅丹明鬼筆環(huán)肽染色觀察細胞骨架變化、Western blot 檢測三組細胞 E-cadherin、Vimentin、Snai1、MMP2、MMP9 的表達。探究下調(diào)FHL2對FOXK1誘導的結(jié)直腸癌細胞的EMT的影響。6.收集結(jié)直腸癌淋巴結(jié)轉(zhuǎn)移患者的淋巴結(jié),免疫組化驗證FOXK1和FHL2在轉(zhuǎn)移瘤中的表達。7.建立裸鼠皮下成瘤模型、脾被膜下肝轉(zhuǎn)移模型和尾靜脈肺轉(zhuǎn)移模型,體內(nèi)驗證壓低FHL2表達對FOXK1誘導的侵襲轉(zhuǎn)移作用的影響。結(jié)果1.FOXK1在結(jié)直腸癌組織中的表達高于正常組織;2.FOXK1和FHL2在結(jié)直腸癌組織細胞的表達呈正相關;3.FOXK1與FHL2在結(jié)直腸癌中存在相互協(xié)同作用;4.FHL2siRNA抑制體內(nèi)外FOXK1誘導的結(jié)直腸癌細胞的增殖、EMT、侵襲和轉(zhuǎn)移。結(jié)論FOXK1與FHL2在結(jié)直腸癌的增殖、EMT、侵襲和轉(zhuǎn)移中具有協(xié)同作用,因此,FOXK1和FHL2可能作為結(jié)直腸癌的綜合治療的靶基因。
[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.
【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
本文編號：2366650
[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.
【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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