【作者】 王磊；

【导师】 朱虹光； 吴莹； 郑莉；

【作者基本信息】 复旦大学 ， 病理学， 2010， 博士

【摘要】 DIXDC1是新近克隆的人类Wnt通路的新基因,即ccdl基因(Coiled-coil-DIX1)人类的异构体,ccdl基因在斑马鱼神经管发育过程中被证实是一个Wnt通路的正调控因子。25%左右的人微卫星不稳定性(microsatellite instability)MSI-H的结直肠癌中检测到Wnt通路重要组分Axin2基因突变。上述突变集中于外显子7上的4个单核苷酸连续序列,导致移码和Axin2蛋白C-末端(包括DIX结构域)的缺失,提示Axin2蛋白的C-末端可能参与抑制结直肠癌的发生和发展,然而机制不甚清楚。DIXDC1蛋白就是以Axin2的C-末端为诱饵,通过酵母双杂交实验,筛选出的与Axin2 C-末端存在相互作用的一个蛋白。目前DIXDC1基因全长已经成功地被克隆并制备了相应的抗体,但关于DIXDC1生物化学及细胞生物学的研究报道较少。有研究显示DIXDC1属于actin和tubulin结合蛋白家族的新成员,提示其可能参与对细胞形态和运动的调节。DIXDC1作为Wnt通路的新成员分子,其具体功能怎样,如何通过Wnt通路对于结直肠癌的发生、发展进行调节；其本身是否受到Wnt通路的调节,其调节方式如何；nicroRNA是否也参与调控DIXDC1的表达,与其相关的miRNA与结直肠癌的发生发展关系如何,目前未见国内外报道。本课题从研究DIXDC1的功能为切入点,将首次阐明其在结直肠癌发生发展过程中的生物学作用及机制；并进一步探讨Wnt通路对DIXDC1蛋白表达水平的影响及机制；利用miRNA芯片及生物信息数据库筛选出与DIXDC1存在相互作用的miRNA,进一步验证相关miRNA对DIXDC1表达的调控并探讨相关miRNA对结直肠癌发生发展的影响。为结直肠癌发生发展机制研究提供新的实验依据,为临床结直肠癌的诊断和治疗提供一定的理论基础。目的阐明DIXDC1对大肠癌细胞生物学特性的影响并进一步深入研究其机制。方法脂质体介导的稳定转染方法建立DIXDC1过表达的DLD1细胞；MTT法和裸鼠成瘤实验检测细胞增殖能力；免疫组织化学方法检测DIXDC1在结直肠癌组织中的表达情况及其与增殖指数(Ki-67)的相关性；流式细胞仪检测细胞周期分布和凋亡情况；Western blot检测细胞周期相关蛋白表达水平和PI3K、MAPK信号通路激活状态；双荧光素酶报告系统检测DIXDC1对TCF/LEF转录活性的影响；免疫共沉淀技术验证DIXDC1与Phospho-AKT之间的相互作用；采用PI3K信号通路抑制剂LY294002处理,观察其对DIXDC1过表达细胞生长情况、细胞周期及周期相关蛋白表达的影响；转染DIXDC1 siRNA干扰DIXDC1表达后,观察其对DIXDC1过表达细胞生长情况、细胞周期及周期相关蛋白表达和PI3K信号通路的影响。结果DIXDC1的过表达于体外水平促进大肠癌细胞的增殖,于体内水平促进裸鼠移植瘤形成及生长。与正常肠粘膜相比,结直肠癌组织中DIXDC1蛋白表达水平升高,并且与增殖指数Ki-67的表达具有密切的相关性。DIXDC1通过上调cyclin D1、下调p21蛋白表达进而促进细胞周期G0／G1到S期的转化,且激活PI3K信号通路。同时DIXDC1可以上调β-catenin蛋白表达,并且具有增强TCF/LEF转录活性的作用。通过PI3K信号通路抑制剂作用或者干扰DIXDC1蛋白的表达后,β-catenin和cyclin Dl蛋白表达下调而p21蛋白表达升高,同时阻滞了细胞周期G0／G1到S期的转化。结论DIXDC1具有促进大肠癌细胞增殖和肿瘤形成的作用,其过表达后可以通过上调cyclin D1和下调p21蛋白表达进而促进细胞周期G0／G1到S期的转化,DIXDC1促大肠癌细胞增殖效应可能与PI3K信号通路的激活相关。目的阐明Wnt通路对DIXDC1蛋白表达的调控及其相关机制。方法免疫组织化学方法检测DIXDC1与P-catenin在结直肠癌中共表达及共定位情况；Real-time RT-PCR检测正常肠粘膜、结直肠癌组织及Wnt-3a刺激前后大肠癌细胞中DIXDC1 mRNA表达水平的改变；Western blot检测Wnt-3a刺激后大肠癌细胞中DIXDC1蛋白表达水平的改变；CHX chase assay检测DIXDC1蛋白的半衰期,并观察Wnt-3a对DIXDC1蛋白稳定性的影响；采用MG132处理细胞后应用Western blot检测DIXDC1蛋白表达的改变；免疫沉淀技术检测DIXDC1与泛素蛋白的结合情况,并观察Wnt-3a对DIXDC1蛋白泛素化水平的影响；免疫共沉淀技术检测Wnt-3a对DIXDC1蛋白磷酸化水平的影响。结果结直肠癌组织中DIXDC1与β-catenin存在共表达及共定位情况。与正常组织相比,结直肠癌组织中DIXDC1蛋白表达水平升高但mRNA表达水平却下降。体外实验中Wnt-3a可以诱导DIXDC1蛋白表达水平的升高,但mRNA表达水平却未见升高。Wnt-3a增加DIXDC1蛋白的稳定性,蛋白酶体抑制剂MG132处理细胞后,DIXDC1蛋白表达水平升高。Wnt-3a抑制了DIXDC1与泛素蛋白的结合从而增加了DIXDC1蛋白稳定性,同时抑制了DIXDC1蛋白磷酸化水平。结论DIXDC1蛋白是通过蛋白酶体途径降解的,经典Wnt通路的激活可以通过抑制其泛素化而增加DIXDC1蛋白的稳定性；Wnt通路可能通过抑制DIXDC1蛋白的磷酸化水平而影响其泛素化降解。目的筛选并验证与DIXDC1存在相互作用的niRNA,并阐明其对结直肠癌发生发展的影响。方法首先通过一系列重复实验对Agilent公司nicroRNA芯片的稳定性和可靠性进行评估；芯片筛选正常肠粘膜、炎性息肉、肠腺瘤和结直肠癌中差异表达的miRNA;选取芯片检测结果中差异表达的17个miRNA,对14对样本RNA进行Real-tmie RT-PCR检测；运用Significance Analysis of Microarray (SAM)芯片数据分析技术对结直肠癌发生发展各阶段中差异表达的miRNA进行分析；通过TargetScan4.2数据库预测,筛选与DIXDC1存在较为保守的相互作用,且芯片结果中变化较为显著的miRNA;通过双荧光素酶报告系统检测miRNA与DIXDC1 3’UTR区域的结合情况；miRNA寡核苷酸转染实验和Western-blot实验检验miRNA对DIXDC1蛋白表达水平的影响。通过Corrected t test:Bonferroni correction统计分析进一步探讨与DIXDC1相关miRNA对结直肠癌发生发展的影响。结果Agilent公司的microRNA芯片(chip)含有8个独立的阵列(array),阵列内重复检测探针的平均变异系数仅为6.3%,同一芯片内阵列之间的相关性到达0.999,不同芯片之间的相关性也到达0.999,Real-time RT-PCR检测的17个miRNA的差异变化与芯片检测结果的相关性到达0.90。经过SAM分析显示,在正常肠粘膜-炎性息肉与肠腺瘤-结直肠癌中表达存在明显差异的有63个miRNA,其中40个表达升高,23个表达下降；在肠腺瘤与结直肠癌中表达存在明显差异的有38个miRNA,其中11个表达升高,27个表达下降。选择下调表达最明显的miR-195和miR-497进行深入研究,发现miR-195和miR-497均能作用于DIXDC1 3’UTR,进而抑制DIXDC1 mRNA的转录,且均可以抑制DIXDC1蛋白的表达。通过Corrected t test:Bonferroni correction统计分析发现一部分miRNA只在从正常肠粘膜-炎性息肉到肠腺瘤病变过程中存在差异表达；另外一部分miRNA却只在从肠腺瘤到结直肠癌(Dukes’A/B)的病变过程中存在差异表达；而还有一部分miRNA在肠癌发生发展的整个过程中均存在差异表达,即从正常肠粘膜-炎性息肉到肠腺瘤到结直肠癌(Dukes’A/B),miR-195和miR-497在肠癌发生发展的整个过程中均存在差异表达,参与了结直肠癌的发生发展的整个过程。结论在结直肠癌发生发展过程中存在一系列差异表达的microRNA。miR-195和miR-497在肠腺瘤和结直肠癌组织中的表达均显著下调,且与DIXDC1存在相互作用,参与DIXDC1蛋白表达的调节,我们认为DIXDC1、niR-195和miR-497均参与了肠癌的发生发展过程,miR-195和miR-497通过与DIXDC1之间的相互作用间接影响了结直肠癌的发生发展过程。更多还原

【Abstract】 DIXDC1 is the human homolog of Ccdl (Coiled-coil-DIX1), a third type of DIX (Dishevelled-Axin) domain-possessing protein and a positive regulator in the Wnt signaling pathway during zebrafish neural patterning. DIX domains of Dvl and Axin, two major Wnt downstream mediators, are involved in homomeric or heteromeric protein interactions and are essential for the signal transduction. Frameshift mutations of Axin2 gene resulting in complete deletion of C-terminal DIX domain of the Axin2 protein were detected in more than 25% of colorectal cancers (CRC) with micro-satellite instability (MSI). Human DIXDC1 was recently isolated as an Axin2 C-terminus binding protein. DIXDC1 was postulated to be associated with CRC tumorigenesis.Full-length DIXDC1 was cloned and rabbit polyclonal antibody was generated. However, there has been very little research on human DIXDC1 gene since it was firstly identified as an actin-binding protein in 2006. The biological role of DIXDC1 remained largely unknown.As a positive regulator in the Wnt pathway, what’s the biological role of DIXDC1 in the formation and progression of human colorectal cancer? Is DIXDC1 also regulated by Wnt signaling pathway? Is microRNA also involved in regulation of DIXDC1 expression? What’s the relationship between its related miRNA and colorectal cancer tumorigensis? All of these remain unknown. In our study, we explored the biological roles of DIXDC1 and its related microRNA in the progression of human colorectal cancer tumorigensis. DIXDC1 might be used as a potential target for colon cancer prevention and therapy. Our study also shed some new light on the mechanism of Wnt pathway gene activation in human colon carcinogenesis. Purpose To explore the biological role of DIXDC1 in the progression of human colon carcinogenesis and its mechanisms.Methods Lipofectin-mediated gene transfection method was used to establish DIXDC1 overexpression DLD1 cells. Cells proliferation viability was tested by MTT assay in vitro and Tumorigenicity assay in nude mice in vivo. Immunohistochemical staining was used to detect the expression of DIXDC1 in human colorectal cancer tissues and its correlation with high proliferation index. Flow cytometry was used to assay the cell cycle and apoptosis. Western blot was used to detect the cell cycle-related protein and the activity of PI3K and MAPK signaling pathway. Luciferase assay was performed to observe the effect of DIXDC1 on the TCF binding site reporter gene activity. The interaction between DIXDC1 and Phospho-AKT was demonstrated by immunoprecipitation assay. PI3K pathway inhibitor LY294002 was used to detect whether blocking this pathwasy can affect the cell proliferation, cell cycle and cell cycle-related protein expression in DIXDC1 overexpression cells. DIXDC1 siRNA was transfected to knock down DIXDC1 preotein expression, and its effect on cell proliferation, cell cycle, cell cycle-related protein expression and the activity of PI3K signaling pathway was also detected.Results In the current study, ectopic over-expression of DIXDC1 resulted in increased cell proliferation in vitro and accelerated tumorigenesis on nude mice in vivo. Up-regulation of DIXDC1 protein was detected in human colorectal adenocarcinoma tissues and was found to be correlated well with high cell proliferation index. We also showed that DIXDC1 promoted G0/G1 to S phase transition concomitantly with up-regulation of cyclinDl and down-regulation of p21 protein. DIXDC1 over-expression cells showed activation of PI3K/AKT pathway.β-catenin was also found to be upregulated in DIXDC1 overexpression cells. The TCF binding site reporter gene activity was higher in DIXDC 1 transfection cells compared with the control group. DIXDC1 might up-regulateβ-catenin and the Wnt pathway target gene cyclin D1. Both siRNA knockdown of DIXDCl and blocking PI3K pathway using specific inhibitor caused G1-S phase arrest, as well as down-regulation of CyclinD1 and up-regulation of p21 in DIXDC1 over-expression colon cancer cells. Conclusion Our study demonstrates that over-expression of DIXDC1 might target p21 and CyclinDl to promote colon cancer cell proliferation and tumorigenesis at least partially through activation of PI3K/Akt pathway.Purpose To clarify whether DIXDC1 protein expression is regulated by Wnt signaling pathway and elucidate the mechanisms involved in this process.Methods Immunohistoshemical staining was used to examine whether DIXDC1 andβ-catenin protein co-localized in human colorectal cancer tissue. DIXDC1 mRNA expression in the human colon tumoral and matched normal tissues was evaluated by Realtmie RT-PCR. In vitro experiment was performed to detect if Wnt-3a affected DIXDC1 mRNA and protein expression using Realtmie RT-PCR and Western blot. The decay rate of DIXDC1 protein in the presence or absence of Wnt-3a was measured by CHX chase assay. Western blot was used to detect the effect of MG132 on DIXDC1 protein expression. To determine whether DIXDC1 protein was targeted for ubiquitination, co-immunoprecipitation was performed to detect the association of DIXDC1 and ubiquitin with and without Wnt-3a treatment. Anti-phosphoserine, anti-phosphothreonine and anti-phosphotyrosine antibodies were used to detect the phosphorylation of endogenous DIXDC 1 after immunoprecipitation by anti-DIXDC1 antibody.Results Positive DIXDC1 staining was detected in colon cancer cells and was co-localized well withβ-catenin staining. However, DIXDC1 mRNA expression was decreased in human colon cancer cells comparing to the matched normal colon epithelial cells. We also found that DIXDC1 protein was induced upon Wnt-3a stimulation, whereas DIXDC1 mRNA level was not significantly increased after Wnt-3a treatment. Our further investigation showed that DIXDC1 protein was degraded through the proteasome pathway, and activation of canonical Wnt signaling decreased the ubiquitin-dependent degradation of both ectopic and endogenous DIXDC 1 protein. In order to explore the possible mechanism of ubiquitination of DIXDC 1, we found that the phosphorylation of DIXDC1 was inhibited by Wnt-3a. Conclution Canonical Wnt/β-catenin pathway activation might up-regulate DIXDC1 through post-translational mechanism by inhibiting the ubiquitin-mediated degradation of DIXDC1 protein. It is possible that Wnt-3a affected the phosphorylation of DIXDC1, resulting in the accumulation of DIXDC1.Purpose To detect the differential expression of DIXDC1 interaction miRNAs in the progression of colon carcinogenesis and evaluate their effects on the expression of DIXDC1 and colon tumorigenesis.Methods Human miRNA microarrays from Agilent Technologies were used in our study. Replicate experiments were performed to determine the variability of array hybridization. The microarray was used to analyze the miRNA expression pattern of normal colon mucosa, inflammatory polyps, colorectal adenoma and carcinoma. Real-time RT-PCR was performed to validate the data from array using 17 miRNAs probes. miRNAs that were differentially expressed between groups were identified using significance analysis of microarray (SAM). miRNAs that predicted to target DIXDC1 and conserved across various species were selected according to TargetScan 4.2. Luciferase assay was perfomed to detect the interaction between miRNAs and the 3’UTR of DIXDC1. miRNAs were transfected into colon cancer cells, the effect of miRNAs on the DIXDC1 protein expression was evaluated by Western blot. Corrected t test:Bonferroni correction was used to estimate differential expression of miRNAs contained the miRNAs related to DIXDC1 during the disease state progression.Results Each miRNA chip from Agilent Technologies contains 8 identical arrays, the average intra-array variation across 112 arrays was 6.3%; Inter-array correlation was 0.999, while inter-chip correlation with reference RNA was also 0.999. The quantitative correlation of fold change between Agilent miRNA microarrays and Real-time RT-PCR was 0.90. The SAM application identified 63 miRNAs that were significantly differentially expressed (40 over-expressed,23 under-expressed) in adenoma-carcinoma compared with normal-polyps tissue, while there were 38 miRNAs significantly differentially expressed (11 over-expressed,27 under-expressed) in adenoma compared with carcinoma. miR-195 and miR-497 were chosen for further investigation, because they were the most two significantly under-expressed. We found that miR-195 and miR-497 can interact with the 3’UTR of DIXDC1 and inhibit DIXDC1 protein expression. Differential expression of miRNAs among different stages of colorectal cancer was deteted. Dynamically, these miRNAs gave us a vision of the transformation and progression of colorectal cancer. Some of them differentially expressed from normal to adenoma transition, some of them differentially expressed from adenoma to carcinoma transition, while others differentially expressed from normal to adenoma to carcinoma transition. miR-195 and miR-497 were progressively downregulated from normal to adenoma to carcinoma, which indicated that miR-195 and miR-497 related to DIXDC1 might have some impacts on the transformation and progression of colorectal cancer.Conclution Series of miRNAs were significantly differentially expressed in the progression of colon carcinogenesis. miR-195 and miR-497 were progressively downregulated from normal to adenoma to carcinoma, while also interacted with the 3’UTR of DIXDC1 and inhibit DIXDC1 protein expression. miR-195 and miR-497 may have some impacts on the progression of colon carcinogenesis through interaction with DIXDC1.更多还原