致癌基因FOXK1通过诱导EMT促进结直肠癌细胞的侵袭转移

发布时间：2018-08-28 17:46

【摘要】：第一章FOXKl在结肠癌中的表达与临床相关性的研究目的和意义1、 FOXKl的结构特点FOX(叉头框)基因家族为一个转录因子家族,共包含43个成员。FOX蛋白家族1类包括FOXA-G, I-L以及Q亚家族,其在碳端有一个基本结构域；而FOXH以及FOXM-P则归属于FOX蛋白家族2类。在人的胚胎干细胞(ES)中检测到了FOXH1以及FOXO1的mRNA表达。在一些人类先天性疾病中也检测到了FOXC1、FOXC2、 FOXE1、 FOXE3、 FOXL2、 FOXN1、FOXP2和FOXP3等该家族基因的突变。本研究的研究对象为叉头框基因家族中的一员,FOXK1,其编码的蛋白与鼠肌细胞核因子(MNF)/FOXK1有高度的同源性。人FOXK1基因位于染色体7p22,包含13个外显子。研究发现FOXK1a和FOXK1b分别为FORK1的两个转录子,它们的5’端包含一个叉头结构。人FOXK1蛋白包括733个氨基酸,约75.5 kDa,其中88.7%的氨基酸与鼠FOXK1(719个氨基酸)相同,48.7%的氨基酸与人FOXK2相同,47.5%氨基酸与非洲蟾蜍FOXK2相同。在人FOXK1、 FOXK2、鼠FOXK1以及非洲蟾蜍FOXK2中FHA元件(叉头框相关元件)和FOX元件(叉头框元件)均为保守元件。2、 FOXK1与肿瘤的关系已有许多报道认为FOX转录因子家族与肿瘤以及胚胎形成有着密切的关系。FOXO3和FOXO4基因在血液系统恶性肿瘤中与MLL基因融合；FOXO1基因在横纹肌肉瘤中则与PAX3或PAX7基因融合。FOXA1基因在食管癌、胰腺癌以及肺癌中均过表达;而在胃肠道肿瘤中FOXP1基因表达缺失。这些FOX蛋白在细胞生长,增殖以及分化过程中都起了双相的调节作用。在结直肠癌、淋巴瘤和胶质母细胞瘤细胞系中,Akt作为P13K的下游调节子,可以通过磷酸化FOXO1、FOXO3以及FOXO4来激活细胞周期,从而有利于肿瘤细胞转化的过程；而去磷酸化的(活化的)FOXO1、FOXO3和FOXO4则转入细胞核中诱导细胞休眠或细胞凋亡。In silico检测分析253个胚胎及成人cDNA文库(每个文库包括5000个序列),有85个文库检测到FOXK1转录子表达。在胚胎及幼儿文库里,FOXK1主要表达于胎儿脑部,眼睛,心脏,肺,胸腺,幼儿脑部以及胚胎中。在55个成人组织文库里(33个肿瘤文库,18个正常组织文库),正常脑,结直肠,肾和肺组织未检测到FOXK1的表达,正常淋巴,眼睛以及皮肤中可表达；而这些器官对应的肿瘤组织中FOXK1均有表达,尤其在脑(95／0),结直肠(124／0)和淋巴结组织(120／24),肿瘤中FOXK1的表达远高于对应的正常组织,提示FOXK1为癌基因。FOXK1基因在肿瘤发病机制中的研究较少,在乳腺癌细胞的血管生成及转移过程中,FOXK1是以抑癌基因的角色存在。Freddie等发现在人骨肉瘤细胞中,FOXK1通过作用于SRF抑制平滑肌a-肌动蛋白的表达。Wang等报道了FOXK1、 FOXK2在结直肠癌组织、结直肠癌细胞(HT29、 DLD1)中高表达,通过抬高FOXK1、 FOXK2的表达,证实了FOXK1通过定位DVL蛋白入核从而激活Wnt信号通路。综合来看,FOXK1的在肿瘤中的研究限于乳腺癌、骨肉瘤、结直肠癌；在人体多器官肿瘤的表达情况多见于In silico中的生物信息分析。而在肠癌的研究中,对FOXK1的研究数据提示其为癌基因,是通过激活DVL蛋白激活Wnt通路而发挥癌基因的作用,对于FOXK1表达状况与临床患者的预后相关性、生存率的关系、肿瘤患者各类临床特点(如年龄、性别、肿瘤大小、肿瘤部位、是否有淋巴结转移)的关系的研究几乎没有报道,这就成为本课题研究的第一个切入点,我们将分析FOXK1蛋白的表达情况对患者临床特点的关系,找出其作为癌基因与肠癌患者生存率是否存在相关性的可能。本课题此章节,以组织芯片、免疫组化、Western、qRT-PCR、稳定表达株构建技术、双荧光素酶基因报告检测系统、生存分析等实验手段。对FOXK1的人体肿瘤组织(包括结直肠癌)中表达情况进行具体检测,进一步证实FOXK1在结直肠癌中癌基因的作用。围绕FOXK1在结肠肿瘤中的表达强弱及其与临床特点、生存率的关系进行探讨,继而为结肠癌的基因治疗提供帮助。主要材料组织与细胞：组织芯片(15种人体正常与肿瘤组织),93例结直肠癌及癌旁组织,FHC、SW480、LOVO、SW1116、Colo205、 SW620、 HT29和DLD1细胞。试剂：RNA提取试剂盒、逆转录试剂盒、qRT-PCR试剂盒、双荧光素酶报告基因检测试剂盒、免疫组织化学相关试剂盒、兔抗人FOXK1单克隆抗体,辣根过氧化物酶标记的山羊抗兔多克隆抗体。主要方法1、 FOXK1在15类正常及肿瘤组织中的表达情况将嵌有15类(每类3例)正常组织的芯片及15类(每类3例)对应肿瘤组织的芯片(皮肤、睾丸、肾脏、卵巢、前列腺、肺、食管、胰腺、宫颈、大脑、胃、小肠、乳腺、结肠、直肠)进行免疫组织化学染色,探讨FOXK1在正常组织及肿瘤组织中的表达情况。2、 FOXKl在结直肠癌组织及细胞中的表达情况2.1 FOXK1在原发性结肠癌组织中的表达情况收集9例来自不同结直肠癌患者手术标本,提取组织蛋白,进行western blotting,比较FOXK1在肿瘤组织及配对癌旁组织中表达的差异。2.2 FOXK1在7个结直肠癌细胞系中的表达情况提取7个结直肠癌细胞系(HT-29, SW480, LoVo, SW1116, SW620, Colo205和DLD1)和人正常结直肠上皮细胞FHC细胞的的细胞总蛋白,利用Western blotting技术检测FOXK1在结直肠癌细胞内的表达情况。3、过表达FOXK1对结直肠癌细胞内癌基因表达的影响3.1构建过表达FOXK1的稳定细胞株将质粒pcDNA3.1-FOXK1和对照pcDNA3.1质粒瞬时转染到SW480细胞,48小时后用含有1000μg/ml的G418培养基连续培养2周,挑选单克隆细胞株,消化至单个细胞并接种至96孔板内,继续以1000μg/ml的G418培养基连续培养两周,得到单克隆稳定表达株,并以Western blotting及qRT-PCR进行验证单克隆株是否高表达FOXK1基因。3.2过表达FOXK1后对癌基因在mRNA表达水平的影响提取两组细胞：pcDNA3.1-FOXK1-SW480和对照pcDNA3.1-SW480的细胞总RNA,逆转录获得mRNA后进行qRT-PCR,比较FOXK1表达变化对癌基因Survivin、Cyclin D1和AP-1在mRNA表达水平的影响。3.3过表达FOXK1后对癌基因的转录活性的影响分别构建以PGL3为载体的,含有Survivin, Cyclin D1和AP-1启动子片段的双荧光素酶报告基因质粒,pluc340、pluc416和pluc450,瞬时转染入pcDNA3.1-FOXK1-SW480和对照pcDNA3.1-SW480两组结直肠癌细胞,评价FOXK1过表达对上述3个癌基因转录活性的影响。4、FOXK1在结直肠癌组织中的表达与临床特点及预后的关系收集93例肠癌患者手术标本经福尔马林固定、石蜡包埋并切片,进行免疫组织化学染色,根据FOXK1表达情况分为高表达组及低表达组,分析FOXK1的表达情况与年龄、性别、肿瘤部位、TNM分期、病理分化程度、淋巴结转移情况的相关性,通过绘制Kaplan-Meier生存曲线来表现FOXK1表达情况对总体生存率的影响。5、数据的统计学处理双荧光素酶报告基因检测、qRT-PCR结果用三或两次实验结果先进行方差齐性检验,方差齐(P0.05)则进行两个处理组间独立样本t检验或多个处理组间one way ANOVA统计分析,整体比较有显著性差异后多重比较采用LSD法；方差不齐(P0.05)则应用Satterthwaite近似t检验或Welch检验分析,整体比较有显著性差异后多重比较采用Duunett's T3检验。通过免疫组织化学的得分判断FOXK1的表达高低与临床病理(多个处理组)特点运用两样本率比较采用四格表资料的χ2检验,多样本率的比较采用行列表资料的x2检验,如果某一个分组的样本特别小,相关性分析则采用Fisher's精确检验,生存分析方法采用log-rank检验。分析与生存相关的指标有：性别(男/女),年龄(
[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 (
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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