【摘要】：第一章FOXKl在結(jié)腸癌中的表達與臨床相關性的研究目的和意義1、 FOXKl的結(jié)構特點FOX(叉頭框)基因家族為一個轉(zhuǎn)錄因子家族,共包含43個成員。FOX蛋白家族1類包括FOXA-G, I-L以及Q亞家族,其在碳端有一個基本結(jié)構域；而FOXH以及FOXM-P則歸屬于FOX蛋白家族2類。在人的胚胎干細胞(ES)中檢測到了FOXH1以及FOXO1的mRNA表達。在一些人類先天性疾病中也檢測到了FOXC1、FOXC2、 FOXE1、 FOXE3、 FOXL2、 FOXN1、FOXP2和FOXP3等該家族基因的突變。本研究的研究對象為叉頭框基因家族中的一員,FOXK1,其編碼的蛋白與鼠肌細胞核因子(MNF)/FOXK1有高度的同源性。人FOXK1基因位于染色體7p22,包含13個外顯子。研究發(fā)現(xiàn)FOXK1a和FOXK1b分別為FORK1的兩個轉(zhuǎn)錄子,它們的5’端包含一個叉頭結(jié)構。人FOXK1蛋白包括733個氨基酸,約75.5 kDa,其中88.7%的氨基酸與鼠FOXK1(719個氨基酸)相同,48.7%的氨基酸與人FOXK2相同,47.5%氨基酸與非洲蟾蜍FOXK2相同。在人FOXK1、 FOXK2、鼠FOXK1以及非洲蟾蜍FOXK2中FHA元件(叉頭框相關元件)和FOX元件(叉頭框元件)均為保守元件。2、 FOXK1與腫瘤的關系已有許多報道認為FOX轉(zhuǎn)錄因子家族與腫瘤以及胚胎形成有著密切的關系。FOXO3和FOXO4基因在血液系統(tǒng)惡性腫瘤中與MLL基因融合；FOXO1基因在橫紋肌肉瘤中則與PAX3或PAX7基因融合。FOXA1基因在食管癌、胰腺癌以及肺癌中均過表達;而在胃腸道腫瘤中FOXP1基因表達缺失。這些FOX蛋白在細胞生長,增殖以及分化過程中都起了雙相的調(diào)節(jié)作用。在結(jié)直腸癌、淋巴瘤和膠質(zhì)母細胞瘤細胞系中,Akt作為P13K的下游調(diào)節(jié)子,可以通過磷酸化FOXO1、FOXO3以及FOXO4來激活細胞周期,從而有利于腫瘤細胞轉(zhuǎn)化的過程；而去磷酸化的(活化的)FOXO1、FOXO3和FOXO4則轉(zhuǎn)入細胞核中誘導細胞休眠或細胞凋亡。In silico檢測分析253個胚胎及成人cDNA文庫(每個文庫包括5000個序列),有85個文庫檢測到FOXK1轉(zhuǎn)錄子表達。在胚胎及幼兒文庫里,FOXK1主要表達于胎兒腦部,眼睛,心臟,肺,胸腺,幼兒腦部以及胚胎中。在55個成人組織文庫里(33個腫瘤文庫,18個正常組織文庫),正常腦,結(jié)直腸,腎和肺組織未檢測到FOXK1的表達,正常淋巴,眼睛以及皮膚中可表達；而這些器官對應的腫瘤組織中FOXK1均有表達,尤其在腦(95／0),結(jié)直腸(124／0)和淋巴結(jié)組織(120／24),腫瘤中FOXK1的表達遠高于對應的正常組織,提示FOXK1為癌基因。FOXK1基因在腫瘤發(fā)病機制中的研究較少,在乳腺癌細胞的血管生成及轉(zhuǎn)移過程中,FOXK1是以抑癌基因的角色存在。Freddie等發(fā)現(xiàn)在人骨肉瘤細胞中,FOXK1通過作用于SRF抑制平滑肌a-肌動蛋白的表達。Wang等報道了FOXK1、 FOXK2在結(jié)直腸癌組織、結(jié)直腸癌細胞(HT29、 DLD1)中高表達,通過抬高FOXK1、 FOXK2的表達,證實了FOXK1通過定位DVL蛋白入核從而激活Wnt信號通路。綜合來看,FOXK1的在腫瘤中的研究限于乳腺癌、骨肉瘤、結(jié)直腸癌；在人體多器官腫瘤的表達情況多見于In silico中的生物信息分析。而在腸癌的研究中,對FOXK1的研究數(shù)據(jù)提示其為癌基因,是通過激活DVL蛋白激活Wnt通路而發(fā)揮癌基因的作用,對于FOXK1表達狀況與臨床患者的預后相關性、生存率的關系、腫瘤患者各類臨床特點(如年齡、性別、腫瘤大小、腫瘤部位、是否有淋巴結(jié)轉(zhuǎn)移)的關系的研究幾乎沒有報道,這就成為本課題研究的第一個切入點,我們將分析FOXK1蛋白的表達情況對患者臨床特點的關系,找出其作為癌基因與腸癌患者生存率是否存在相關性的可能。本課題此章節(jié),以組織芯片、免疫組化、Western、qRT-PCR、穩(wěn)定表達株構建技術、雙熒光素酶基因報告檢測系統(tǒng)、生存分析等實驗手段。對FOXK1的人體腫瘤組織(包括結(jié)直腸癌)中表達情況進行具體檢測,進一步證實FOXK1在結(jié)直腸癌中癌基因的作用。圍繞FOXK1在結(jié)腸腫瘤中的表達強弱及其與臨床特點、生存率的關系進行探討,繼而為結(jié)腸癌的基因治療提供幫助。主要材料組織與細胞：組織芯片(15種人體正常與腫瘤組織),93例結(jié)直腸癌及癌旁組織,FHC、SW480、LOVO、SW1116、Colo205、 SW620、 HT29和DLD1細胞。試劑：RNA提取試劑盒、逆轉(zhuǎn)錄試劑盒、qRT-PCR試劑盒、雙熒光素酶報告基因檢測試劑盒、免疫組織化學相關試劑盒、兔抗人FOXK1單克隆抗體,辣根過氧化物酶標記的山羊抗兔多克隆抗體。主要方法1、 FOXK1在15類正常及腫瘤組織中的表達情況將嵌有15類(每類3例)正常組織的芯片及15類(每類3例)對應腫瘤組織的芯片(皮膚、睪丸、腎臟、卵巢、前列腺、肺、食管、胰腺、宮頸、大腦、胃、小腸、乳腺、結(jié)腸、直腸)進行免疫組織化學染色,探討FOXK1在正常組織及腫瘤組織中的表達情況。2、 FOXKl在結(jié)直腸癌組織及細胞中的表達情況2.1 FOXK1在原發(fā)性結(jié)腸癌組織中的表達情況收集9例來自不同結(jié)直腸癌患者手術標本,提取組織蛋白,進行western blotting,比較FOXK1在腫瘤組織及配對癌旁組織中表達的差異。2.2 FOXK1在7個結(jié)直腸癌細胞系中的表達情況提取7個結(jié)直腸癌細胞系(HT-29, SW480, LoVo, SW1116, SW620, Colo205和DLD1)和人正常結(jié)直腸上皮細胞FHC細胞的的細胞總蛋白,利用Western blotting技術檢測FOXK1在結(jié)直腸癌細胞內(nèi)的表達情況。3、過表達FOXK1對結(jié)直腸癌細胞內(nèi)癌基因表達的影響3.1構建過表達FOXK1的穩(wěn)定細胞株將質(zhì)粒pcDNA3.1-FOXK1和對照pcDNA3.1質(zhì)粒瞬時轉(zhuǎn)染到SW480細胞,48小時后用含有1000μg/ml的G418培養(yǎng)基連續(xù)培養(yǎng)2周,挑選單克隆細胞株,消化至單個細胞并接種至96孔板內(nèi),繼續(xù)以1000μg/ml的G418培養(yǎng)基連續(xù)培養(yǎng)兩周,得到單克隆穩(wěn)定表達株,并以Western blotting及qRT-PCR進行驗證單克隆株是否高表達FOXK1基因。3.2過表達FOXK1后對癌基因在mRNA表達水平的影響提取兩組細胞：pcDNA3.1-FOXK1-SW480和對照pcDNA3.1-SW480的細胞總RNA,逆轉(zhuǎn)錄獲得mRNA后進行qRT-PCR,比較FOXK1表達變化對癌基因Survivin、Cyclin D1和AP-1在mRNA表達水平的影響。3.3過表達FOXK1后對癌基因的轉(zhuǎn)錄活性的影響分別構建以PGL3為載體的,含有Survivin, Cyclin D1和AP-1啟動子片段的雙熒光素酶報告基因質(zhì)粒,pluc340、pluc416和pluc450,瞬時轉(zhuǎn)染入pcDNA3.1-FOXK1-SW480和對照pcDNA3.1-SW480兩組結(jié)直腸癌細胞,評價FOXK1過表達對上述3個癌基因轉(zhuǎn)錄活性的影響。4、FOXK1在結(jié)直腸癌組織中的表達與臨床特點及預后的關系收集93例腸癌患者手術標本經(jīng)福爾馬林固定、石蠟包埋并切片,進行免疫組織化學染色,根據(jù)FOXK1表達情況分為高表達組及低表達組,分析FOXK1的表達情況與年齡、性別、腫瘤部位、TNM分期、病理分化程度、淋巴結(jié)轉(zhuǎn)移情況的相關性,通過繪制Kaplan-Meier生存曲線來表現(xiàn)FOXK1表達情況對總體生存率的影響。5、數(shù)據(jù)的統(tǒng)計學處理雙熒光素酶報告基因檢測、qRT-PCR結(jié)果用三或兩次實驗結(jié)果先進行方差齊性檢驗,方差齊(P0.05)則進行兩個處理組間獨立樣本t檢驗或多個處理組間one way ANOVA統(tǒng)計分析,整體比較有顯著性差異后多重比較采用LSD法；方差不齊(P0.05)則應用Satterthwaite近似t檢驗或Welch檢驗分析,整體比較有顯著性差異后多重比較采用Duunett's T3檢驗。通過免疫組織化學的得分判斷FOXK1的表達高低與臨床病理(多個處理組)特點運用兩樣本率比較采用四格表資料的χ2檢驗,多樣本率的比較采用行列表資料的x2檢驗,如果某一個分組的樣本特別小,相關性分析則采用Fisher's精確檢驗,生存分析方法采用log-rank檢驗。分析與生存相關的指標有：性別(男/女),年齡(
[Abstract]:Chapter One: The expression of FOXKl in colon cancer and its clinical significance 1. The structural characteristics of FOXKl gene family is a family of transcription factors, including 43 members. The expression of FOXH1 and FOXO1 mRNA was detected in human embryonic stem cells (ES). Mutations in FOXC1, FOXC2, FOXE1, FOXE3, FOXL2, FOXN1, FOXP 2 and FOXP3 genes were also detected in some human congenital diseases. The human FOXK1 gene is located on chromosome 7p22 and contains 13 exons. Two FORK1 transcripts, FOXK1a and FOXK1b, respectively, have a fork structure at their 5'ends. The human FOXK1 protein contains 733 amino acids, about 75.5 kDa, of which 88.7% are related to the amino acids. Mouse FOXK1 (719 amino acids) is the same, 48.7% of the amino acids are the same as human FOXK2, and 47.5% of the amino acids are the same as African toad FOXK2. In human FOXK1, FOXK2, mouse FOXK1 and African toad FOXK2, FHA and FOX elements (fork frame related elements) are conservative elements. FOXO3 and FOXO4 genes fuse with MLL genes in hematological malignancies; FOXO1 gene fuses with PAX3 or PAX7 genes in rhabdomyosarcoma; FOXA1 gene is overexpressed in esophageal, pancreatic and lung cancer; and FOXP1 gene is overexpressed in gastrointestinal tumors. Deletion. These FOX proteins play biphasic roles in cell growth, proliferation, and differentiation. In colorectal cancer, lymphoma and glioblastoma cell lines, Akt acts as the downstream regulator of P13K and can activate cell cycle by phosphorylating FOXO1, FOXO3, and FOXO4, thus facilitating the process of tumor cell transformation. Dephosphorylated (activated) FOXO1, FOXO3, and FOXO4 were transfected into the nucleus to induce cell dormancy or apoptosis. In silico assay, 253 embryonic and adult cDNA libraries (each containing 5000 sequences) were analyzed, and 85 libraries detected FOXK1 transcription. In embryonic and infant libraries, FOXK1 was mainly expressed in the fetal brain, eyes. FOXK1 was not detected in the eyes, heart, lungs, thymus glands, infant brains and embryos of 55 adult tissue libraries (33 tumor libraries, 18 normal tissue libraries), normal brain, colorectal, kidney and lung tissues, but in normal lymph, eyes and skin; FOXK1 was expressed in the corresponding tumor tissues of these organs, especially in the skin. The expression of FOXK1 in brain (95/0), colorectal (124/0) and lymph nodes (120/24) was much higher than that in normal tissues, suggesting that FOXK1 is an oncogene. The study of FOXK1 gene in the pathogenesis of cancer is less. In the process of angiogenesis and metastasis of breast cancer cells, FOXK1 is a tumor suppressor gene. In human osteosarcoma cells, FOXK1 inhibits the expression of smooth muscle a-actin by acting on SRF. Wang et al reported that FOXK1 and FOXK2 are highly expressed in colorectal cancer tissues and colorectal cancer cells (HT29, DLD1). By elevating the expression of FOXK1 and FOXK2, FOXK1 activates Wnt signaling pathway by localizing the entry of DVL protein into the nucleus. The study of FOXK1 in cancer is limited to breast cancer, osteosarcoma, colorectal cancer, and the expression of FOXK1 in multiple organ tumors is more common in In silico bioinformatics analysis. The relationship between reaching status and prognosis, survival rate, clinical characteristics of tumor patients (such as age, sex, tumor size, tumor location, lymph node metastasis) has not been reported. This is the first entry point of this study. We will analyze the expression of FOXK1 protein in patients. In this chapter, tissue microarray, immunohistochemistry, Western, qRT-PCR, construction of stable expression strains, double luciferase gene reporting and detection system, survival analysis and other experimental means were used to detect the correlation between FOXK1 and survival rate of patients with colorectal cancer. Specific detection of FOXK1 expression in colorectal cancer further confirmed the role of oncogene in colorectal cancer. Focus on the expression of FOXK1 in colorectal cancer and its relationship with clinical characteristics, survival rate, and then provide help for gene therapy of colorectal cancer. Normal and tumor tissues), 93 colorectal cancer and adjacent tissues, FHC, SW480, LOVO, SW1116, Colo205, SW620, HT29 and DLD1 cells. Reagents: RNA extraction kit, reverse transcription kit, qRT-PCR kit, double luciferase reporter gene detection kit, immunohistochemical related kit, rabbit anti-human FOXK1 monoclonal antibody, horseradish radish peroxide Major Methods 1. FOXK1 expression in 15 normal and tumor tissues will be embedded with 15 types of normal tissue chips and 15 types of corresponding tumor tissue chips (skin, testis, kidney, ovary, prostate, lung, esophagus, pancreas, cervix, brain, stomach, small intestine, breast, nodule). Immunohistochemical staining was used to investigate the expression of FOXK1 in normal tissues and tumor tissues. 2. The expression of FOXKl in colorectal cancer tissues and cells 2. 1. The expression of FOXK1 in primary colorectal cancer tissues was detected by immunohistochemical staining. Loting, compared the expression of FOXK1 in tumor tissues and adjacent tissues. 2.2 The expression of FOXK1 in seven colorectal cancer cell lines (HT-29, SW480, LoVo, SW1116, SW620, Colo205 and DLD1) and human normal colorectal epithelial FHC cells was detected by Western blotting. The expression of FOXK1 in colorectal cancer cells was detected. 3. Overexpression of FOXK1 affected oncogene expression in colorectal cancer cells. 3. 1 A stable cell line was constructed to overexpress FOXK1. Plasmid pcDNA3.1-FOXK1 and control pcDNA3.1 were transfected into SW480 cells. After 48 hours, the plasmid was cultured in G418 medium containing 1000 ug/ml for 2 weeks. Monoclonal cell lines were selected and digested into a single cell and inoculated into 96-well plate. The stable expression of FOXK1 was obtained by continuous culture in G418 medium of 1000 ug/ml for two weeks. Western blotting and qRT-PCR were used to verify the effect of FOXK1 gene overexpression on oncogene mRNA expression. Total RNA was extracted from pcDNA3.1-FOXK1-SW480 and control pcDNA3.1-SW480 cells. After reverse transcription, the mRNA was obtained and qRT-PCR was performed. The effects of FOXK1 expression on the mRNA expression levels of Survivin, Cyclin D 1 and AP-1 were compared. Survivin, Cyclin D 1 and AP-1 promoter fragments of double luciferase reporter gene plasmid pluc340, pluc416 and pluc450 were transfected into two groups of colorectal cancer cells, pcDNA3.1-FOXK1-SW480 and control pcDNA3.1-SW480. The effects of FOXK1 overexpression on the transcriptional activity of the three oncogenes were evaluated. 4. FOXK1 expression and its clinical significance in colorectal cancer tissues The clinical features and prognosis of 93 patients with colorectal cancer were studied. The specimens were fixed with formalin, embedded in paraffin and sectioned for immunohistochemical staining. According to FOXK1 expression, they were divided into high expression group and low expression group. The expression of FOXK1 was analyzed with age, sex, tumor location, TNM stage, degree of pathological differentiation, lymph node metastasis. The correlation between the two conditions was analyzed by mapping the Kaplan-Meier survival curve to show the effect of FOXK1 expression on overall survival rate. 5. The data were statistically processed by double luciferase reporter gene detection. The results of qRT-PCR were tested by homogeneity of variance first with three or two experimental results, while those of homogeneity of variance (P 0.05) were tested by independent sample t test or non-homogeneity of variance between the two treatment groups. One way ANOVA statistical analysis showed that there was significant difference between the two groups and LSD was used for multiple comparisons. Satterthwaite approximation t test or Welch test was used for multiple comparisons with significant difference in variance (P 0.05). Duunett's T3 test was used for multiple comparisons. The FOXK1 table was judged by the score of immunohistochemistry. The_2 test of four-grid data was used to compare the two sample rates. The x2 test of row list data was used to compare the multiple sample rates. If the sample size of a certain group was very small, the Fisher's exact test was used for correlation analysis. The log-rank test was used for survival analysis. Relevant indicators are: sex (male / female), age (
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.34

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