结直肠癌中EMT标志物的表达对预后的意义以及IGFBP-rP1在EMT和转移中的作用及机制研究
发布时间：2018-07-13 20:50
【摘要】：结直肠癌是当今社会主要恶性肿瘤之一,其发病率和死亡率在全世界范围内的恶性肿瘤中分别位于第三位和第四位。全球每年大约有1,400,000名新确诊的结直肠癌病例,并且约有693,900名患者最终死于结直肠癌。随着经济的发展,所谓的西方式生活使国内结直肠癌的发病率飞速增长。据《2015中国癌症统计》报道,我国在2015年新确诊376,300名结直肠癌病例,有191,000名患者死于结直肠癌。结直肠癌已严重威胁全世界人民健康,造成了严重的社会和经济负担。TNM分期虽然可以从整体上评估预后和指导规范化治疗,但是它已经无法满足个体化治疗的需求,有大部分Ⅱ期和Ⅲ期的患者并没有从TNM分期指导的治疗中获益。因此,挖掘有效的与预后和治疗有关的分子标志物来辅助筛选有转移或者复发风险的早期结直肠癌患者,使他们在后续的辅助治疗中受益具有重要的临床价值。并且,积极开展结直肠癌病理机制的研究对于结直肠癌的防治势在必行。临床上90%以上的恶性肿瘤患者最终死于肿瘤转移或复发。大约20%的患者在诊断出结直肠癌时就已经发生了远处转移,40-50%的患者在确诊时未发现转移,治疗后最终也会出现远处转移。肿瘤的转移是一个多步骤、多阶段、多途径、涉及多基因变化的一系列复杂过程。近年来,越来越多的研究表明上皮间质转化(Epithelial mesenchymal transition, EMT)在肿瘤的侵袭转移中发挥着重要的功能。EMT是指上皮细胞受到外界信号刺激后,细胞形态转变为间质表型的生物学过程。在这一过程中,上皮细胞失去极性,失去细胞间的紧密黏附连接,上皮标志物表达下调或缺失,而逐渐获得间质细胞形态特征以及间质标志物表达,同时增强细胞的运动能力和迁移能力。EMT过程中最显著的分子事件是E-cadherin表达降低,而N-cadherin, Vimentin和Fibronectin的表达增强,一些转录因子如Snail, Slug, Twist和Zeb1/2等通过直接或间接调控E-cadherin等EMT相关基因的表达,诱导EMT过程。因此,我们检测这些与EMT相关的经典标志物在结直肠癌中的表达,明确其在结直肠癌预后中的作用,并试图从中遴选出关键的分子,以筛选具有高转移风险的患者群体,为患者的预后判断、转移预测、个体化治疗等方面提供有效的辅助支撑。EMT过程受众多重要的信号通路调控,包括TGF-β, RTK, Wnt, Notch, Hedgehog, Matrix和缺氧等。其中TGF-β1是体外刺激肿瘤细胞构建EMT模型的经典因子,目前关于TGF-P信号通路参与调控EMT的报道颇为详尽,TGF-β与其受体结合后激活Smad2/3使其磷酸化,接着磷酸化的Smad2/3与Smad4结合入核,调节一系列与转移以及EMT相关靶基因的功能。另外TGF-β诱导EMT的过程还涉及与细胞运动和凋亡相关的一些其他信号通路,如PI3K/Akt通路、MAPK通路和Rho-like GTPase通路等。除了经典的EMT标志物之外,越来越多的报道揭示存在许多新分子在EMT过程中扮演重要的角色,而胰岛素样生长因子结合蛋白相关蛋白1 (insulin-like growth factor binding protein-related protein 1, IGFBP-rP1)是我们实验室通过抑制性差减杂交法从结肠腺癌-正常粘膜的差减cDNA文库中筛选出并在结肠腺癌中高表达的一个基因。前期一系列研究发现IGFBP-rP1抑制结直肠癌细胞的生长,诱导细胞凋亡和衰老,其表达越高患者预后越好,我们认为它在结直肠癌中发挥着潜在的抑癌作用。另外,实验室前期体外细胞系研究发现,IGFBP-rP1可在EMT过程中发挥抑制作用。因此,我们欲在体内和体外双水平明确IGFBP-rP1在结直肠癌中与EMT和转移的关系。目前虽也有一些关于IGFBP-rP1抑制肿瘤细胞迁移能力的报道,但是具体机制尚不清楚。IGFBP-rP1虽与IGFs以及insulin都可以结合,但是结合能力较弱,这提示IGFBP-rP1在除了通过经典的IGF系统发挥其生物学效应之外,还具有独立于IGF的作用。我们实验室前期芯片数据发现IGFBP-rP1过表达引起TGF-β通路的下游效应分子Smad3 mRNA表达水平降低。因此,IGFBP-rP1是否通过TGF-β/Smad信号通路发挥抑制EMT的功能,值得我们深入研究。首先我们选取了2004-2008年间在邵逸夫医院接受治疗的346名结直肠癌患者的癌组织cDNA标本,采用实时荧光定量PCR(qPCR)检测八个EMT标志物的表达,包括E-cadherin, Vimentin, Fibronectin, Snail, Slug, Twist, Zeb1和Zeb2。这组病例中的167例有患者预后信息,随访信息由电话随访获得,随访时间截止于2013年9月30日,随访时间最短3个月,最长115个月,中位随访时间72个月,平均随访时间68个月。接着我们在另一组实验室保存的组织芯片标本中对mRNA的结果进行了后续的验证,采用免疫组织化学方法检测了E-cadherin, Fibronectin, Snail, Slug和Twist的表达。这组病例来自1991-2006年间在杭州萧山第一人民医院接受治疗的415名结直肠癌患者的癌组织标本,全部有患者预后信息,随访信息由萧山疾控中心提供,随访时间截止于2006年12月31日,随访时间最短1个月,最长186个月,中位随访时间33个月,平均随访时间48个月。SPSS 20.0 for windows统计学软件用于以下统计学处理。采用Mann-Whitney秩和检验计算基因在不同临床表型的表达差异；采用非配对t检验计算转录因子在两种表型中的表达差异；蛋白表达分组结果与临床表现的关联分析采用卡方检验或者Fisher精确检验；两因子间相关性分析采用Spearman双变量相关性分析；单因素Cox比例风险模型对连续性变量进行单因素生存分析；用Kaplan-Meier法对分类变量进行单因素生存分析(log-rank法进行显著性检验),并绘制生存曲线；将单因素分析对预后有统计学意义的因素再纳入多因素生存分析。以上分析均以P值≤0.05作为显著性阈值。在两组结直肠癌样本中在nRNA和蛋白两个水平对EMT标志物与临床病理参数和预后的关系分别分析。首先在mRNA水平发现：EMT标志物分别在不同的临床表型间表达存在差异；各转录因子在间质表型(Vim/E-cad或者Fibro/E-cad第75百分位数)的患者中的表达常高于上皮表型(Vim/E-cad或者Fibro/E-cad第75百分位数)患者；五个转录因子两两之间存在正相关；在众多EMT标志物中只有Snail的高表达对患者预后是一个不利因素,并且是一个独立预后指标；进一步在将TNM分期分层后的生存分析中发现,Snail的高表达在Ⅰ-Ⅱ期患者中是一个不利的独立预后指标,另外在Slug或Twist高表达时,Ⅰ-Ⅱ期患者预后也较差,但这二者不是独立的预后因素。基于mRNA水平的结果,我们选取了五个EMT标志物(E-cadherin, Fibronectin, Snail, Slug和Twist)进行后续蛋白水平的验证。蛋白水平的结果与mRNA水平的结果总体上具有一致性,但也有一些新的发现。Snail或Twist核阳性表达时患者预后差；将Snail或Twist与E-cadherin分别联合分析表明,E-cadherin低表达且Snail阳性或E-cadherin低表达且Twist阳性患者较其他分组来说生存率最低；并且Snail核阳性表达是一个不利的独立预后指标；进一步更细致的分析发现,Snail或Twist核阳性的预后意义只存在于Ⅰ-Ⅱ期患者,在Ⅲ-Ⅳ期患者中,是否存在Snail或Twist核阳性表达在生存上并无显著性差异；同样地,Snail核阳性表达也在Ⅰ-Ⅱ期患者中具有独立预后意义；另外我们还发现Snail对结直肠癌患者预后的影响依赖于p53的状态,只有在p53野生型的患者中,Snail阳性表达不利于生存。前期我们在体外结直肠癌细胞系中研究发现,IGFBP-rP1在诱导上皮标记物的表达的同时可以下调间质标记物的表达,降低细胞的迁移侵袭能力,在EMT过程中发挥抑制作用。因此我们欲在体内水平明确IGFBP-rP1与EMT和转移的关系。首先我们采用免疫组织化学的方法检测了217例结直肠癌组织中IGFBP-rP1的表达,通过卡方检验发现IGFBP-rP1的高表达与组织学分级、局部淋巴结累犯及病理分期呈负相关,并且与EMT标志物E-cadherin及P-catenin的膜表达呈正相关,与间质标志物Fibronectin的表达呈负相关。在组织水平上进一步明确了在结直肠癌中IGFBP-rP1发挥抑制EMT的作用。接着采用裸鼠皮下成瘤实验,揭示了过表达IGFBP-rP1后抑制结直肠癌细胞SW620在动物体内的生长,并且在移植瘤中同样检测到过表达IGFBP-rP1后促进上皮标志物的表达,同时抑制间质标志物的表达,而在IGFBP-rP1敲除的SW480中则存在相反的作用,这进一步说明IGFBP-rP1可抑制结直肠癌发生EMT过程。为了明确IGFBP-rP1在体内对于转移的影响,我们通过尾静脉注射过表达和敲除IGFBP-rP1的两组细胞株所构建的肺转移模型,从正反两方面揭示了IGFBP-rP1的存在会抑制结直肠癌细胞的肺转移。随后我们进一步探讨了IGFBP-rP1是通过何种机制来调控结直肠癌中EMT的发生。首先,在体外细胞系中发现内源性的IGFBP-rP1可以抑制TGF-β受体以及其下游分子Smad的表达,抑制TGF-β/Smad信号通路的活化。接着通过在SW480敲除IGFBP-rP1的细胞株中加入TGF-β受体抑制剂SB431542,发现由IGFBP-rP1敲除所诱导的EMT现象会被逆转,这进一步证明TGF-β/Smad信号通路是IGFBP-rP1参与EMT调控所必需的。最后我们在TGF-β1刺激HT29细胞后所诱导的EMT模型中,加入外源性重组蛋白IGFBP-rP1,不仅可扭转所诱导的EMT现象,还可阻碍并抑制TGF-β受体激活,下调Smad2/3的磷酸化水平,这也再次验证了IGFBP-rP1是通过负性调控TGF-β/Smad信号通路而抑制EMT过程的。综上所述,我们可以得出以下结论：(1) Snail对结直肠癌患者预后的影响依赖于p53的状态,只有在p53野生型患者中,Snail表达具有不良预后意义,并且在结直肠癌Ⅰ-Ⅱ期患者中Snail高表达是一个不利的独立预后因素或者Snail阳性是一个独立危险因素。(2) IGFBP-rP1通过TGF-β/Smad信号通路抑制结直肠癌EMT和转移。
[Abstract]:Colorectal cancer is one of the major malignant tumors in today's society. Its incidence and mortality rate are located in third and fourth of the world's malignant tumors. There are about 1400000 newly diagnosed cases of colorectal cancer in the world each year, and about 693900 patients die of colorectal cancer. Western life has caused a rapid increase in the incidence of colorectal cancer in China. According to <2015 China cancer statistics, 376300 cases of colorectal cancer were newly confirmed in China in 2015, and 191000 patients died of colorectal cancer. Colorectal cancer has seriously threatened the health of the people all over the world, causing serious social and economic burdens in.TNM staging. To assess the overall prognosis and guide standardized treatment, but it has not been able to meet the needs of individualized treatment. Most patients in phase II and stage III have not benefited from the treatment guided by TNM staging. Therefore, effective molecular markers related to prognosis and treatment are excavated to assist in the screening of early stages of the risk of metastasis or recurrence. Colorectal cancer patients have an important clinical value to benefit from subsequent adjuvant therapy. Moreover, it is imperative to actively carry out the study of the pathological mechanism of colorectal cancer for the prevention and treatment of colorectal cancer. More than 90% of the patients with malignant tumors eventually died of tumor metastasis or relapse. About 20% of the patients were diagnosed with colorectal cancer. Distant metastasis has occurred. Patients with 40-50% have not found metastasis at the time of diagnosis, and distant metastasis will eventually occur. Tumor metastasis is a multistep, multi stage, multi pathway, and a series of complex processes involving multiple gene changes. In recent years, more and more studies have shown that epithelial mesenchymal transition (Epithelial mesenchymal transi) Tion, EMT) plays an important role in the invasion and metastasis of tumor, which refers to the biological process of epithelial cells transformed into interstitial phenotypes after external stimuli are stimulated by external signals. In this process, the epithelial cells lose their polarity, lose the close adhesion of the cells, and the expression of epithelial markers is downregulated or missing, and gradually acquired between the epithelial cells. The most significant molecular event in.EMT process is the decrease of E-cadherin expression, while the expression of N-cadherin, Vimentin and Fibronectin is enhanced, and some transcription factors such as Snail, Slug, Twist and Zeb1/2 are directly or indirectly regulated by the E-cadher. The expression of EMT related genes, such as in, induces the EMT process. Therefore, we detect the expression of these classical EMT related markers in colorectal cancer, and make clear the role of these markers in the prognosis of colorectal cancer, and try to select the key molecules to select the group with high metastasis risk, to judge the prognosis of the patients, to transfer the prediction, and to predict the prognosis of the patients. There are many important signaling pathways in the.EMT process, including TGF- beta, RTK, Wnt, Notch, Hedgehog, Matrix and anoxia, including TGF- beta 1, which is a classic factor to stimulate tumor cells to construct EMT models in vitro. The present report on TGF-P signaling pathway involved in EMT is quite detailed, TGF- beta After binding to its receptor, it activates Smad2/3 to phosphorylate it, then the phosphorylated Smad2/3 combines with Smad4 into the nucleus to regulate a series of functions associated with metastasis and EMT related target genes. In addition, the process of TGF- beta induced EMT involves some other signaling pathways related to cell movement and apoptosis, such as the PI3K/Akt pathway, MAPK pathway and Rho-like GTPase. In addition to the classic EMT markers, more and more reports reveal that many new molecules play an important role in the EMT process, and the insulin like growth factor binding protein related protein 1 (insulin-like growth factor binding protein-related protein 1, IGFBP-rP1) is our laboratory through inhibitory subtraction A gene expressed in colon adenocarcinoma cDNA library from colon adenocarcinoma normal mucosa and in colorectal adenocarcinoma. A series of previous studies found that IGFBP-rP1 inhibits the growth of colorectal cancer cells, induces apoptosis and senescence, and the higher the expression, the better the prognosis of the patients. We believe that it plays a potential inhibition in colorectal cancer. In addition, in the early laboratory in vitro cell line study found that IGFBP-rP1 could play an inhibitory role in the EMT process. Therefore, we would like to identify the relationship between IGFBP-rP1 and EMT and metastasis in colorectal cancer both in vivo and in vitro. Although there are some reports on the ability of IGFBP-rP1 to inhibit tumor cell migration, the specific machine It is not clear that.IGFBP-rP1 can be combined with IGFs and insulin, but the ability to bind is weak, which suggests that IGFBP-rP1 is independent of IGF in addition to the classical IGF system. Our early lab chip data found that IGFBP-rP1 overexpression causes the downstream effector of TGF- beta pathway S The expression level of Mad3 mRNA is reduced. Therefore, whether or not IGFBP-rP1 plays the function of inhibiting EMT through the TGF- beta /Smad signaling pathway is worthy of our in-depth study. First, we selected the cDNA specimens of the cancer tissues of 346 colorectal cancer patients who were treated in Sir Run Run Shaw Hospital for 2004-2008 years, and used real time fluorescent quantitative PCR (qPCR) to detect eight EMT markers. The expression of substance, including E-cadherin, Vimentin, Fibronectin, Snail, Slug, Twist, Zeb1 and Zeb2., had 167 patients' prognosis information. Follow-up information was obtained by telephone follow-up. The follow-up time was up to September 30, 2013, the shortest follow-up time was 3 months, the longest period was 115 months, the median follow-up time was 72 months, and the average follow-up time was 68 months. Then we followed up the results of mRNA in the tissue microarray specimens preserved in another group of laboratory tests, using immunohistochemical methods to detect the expression of E-cadherin, Fibronectin, Snail, Slug and Twist. This group of cases came from 415 colorectal cancer, which was treated at the first people's Hospital in Hangzhou, Hangzhou. All the patients' cancer tissue specimens were provided with the patient's prognosis information. The follow-up information was provided by the Xiaoshan CDC. The follow-up time was up to December 31, 2006, the shortest 1 months, the longest 186 months, the median follow-up time of 33 months, the average follow-up time of 48 months.SPSS 20 for windows statistics software for the following statistical processing. M Ann-Whitney rank sum test calculated the difference in the expression of gene in different clinical phenotypes; the expression difference of transcription factors in two phenotypes was calculated by non paired t test; the correlation analysis of protein expression grouping results and clinical manifestations was determined by chi square test or Fisher accurate test; two the correlation analysis of the subgroups used Spearman bivariate analysis. Correlation analysis; single factor Cox proportional risk model for single factor survival analysis of continuous variables; Kaplan-Meier method for single factor survival analysis (log-rank method for significant test) and mapping of survival curves; single factor analysis was used for multiple factor survival analysis for prognostic factors. The above analysis took P value less than 0.05 as a significant threshold. In the two groups of colorectal cancer samples, the relationship between the EMT markers and the clinicopathological parameters and prognosis was analyzed at the level of nRNA and protein at two levels. First, at the level of mRNA, the difference between the EMT markers in different clinical phenotypes was found, and the transcription factors were in the stromal table. The expression in patients with type (Vim/E-cad or Fibro/E-cad seventy-fifth percentile) was often higher than the epithelial phenotype (Vim/E-cad or Fibro/E-cad seventy-fifth percentile); five transcription factor 22 had a positive correlation; in a number of EMT markers, the high expression of Snail was an adverse factor for the patient and was an independent factor. Prognostic indicators; further in the subsistence analysis after stratified TNM staging, the high expression of Snail was an unfavorable independent prognostic indicator in stage I - II patients. In addition to the high expression of Slug or Twist, the prognosis of stage I - II patients was also poor, but these two were not independent preconditioning factors. Based on the mRNA level, we selected five EMT markers (E-cadherin, Fibronectin, Snail, Slug and Twist) were tested for subsequent protein levels. The results of protein levels were generally consistent with the results of mRNA levels, but some new findings were found to be poor in the positive expression of.Snail or Twist nuclei; the joint analysis of Snail or Twist and E-cadherin showed that, Herin low expression and Snail positive or E-cadherin low expression and Twist positive patients have the lowest survival rate; and Snail nuclear positive expression is an unfavorable independent prognostic indicator. Further more detailed analysis found that the prognostic significance of Snail or Twist nuclear positive only lies in stage I - II patients, in stage III - IV patients, There is no significant difference in survival between the presence of Snail or Twist nuclear positive; similarly, the positive expression of Snail nuclear positive is also of independent prognostic significance in stage I - II patients; furthermore, we also found that the effect of Snail on the prognosis of colorectal cancer patients depends on the state of p53, and that the positive expression of Snail is negative only in the p53 wild type patients. In the early stage of survival. In our early colorectal cancer cell line, we found that IGFBP-rP1 can downregulate the expression of epithelial markers, reduce the cell migration and invasion, and play an inhibitory role in the EMT process. Therefore, we would like to define the relationship between IGFBP-rP1 and EMT and metastasis in the body level. First, we detected the expression of IGFBP-rP1 in 217 cases of colorectal cancer by immunohistochemical method. Through chi square test, we found that the high expression of IGFBP-rP1 was negatively correlated with the histological grade, local lymph node recidivism and pathological stage, and was positively correlated with the expression of the EMT marker E-cadherin and P-catenin membrane, and the interstitial marker Fib. The expression of ronectin was negatively correlated. At the tissue level, it was further clarified that IGFBP-rP1 played a role in inhibiting EMT in colorectal cancer. Then, the subcutaneous tumor test in nude mice revealed the inhibition of the growth of SW620 in the colorectal cancer cells after overexpression of IGFBP-rP1, and the expression of IGFBP-rP1 was also detected in the transplanted tumor. The expression of the epithelial markers and the inhibition of the expression of the interstitial markers and the opposite effect in the IGFBP-rP1 knockout SW480 further indicate that IGFBP-rP1 inhibits the EMT process in colorectal cancer. In order to clarify the effect of IGFBP-rP1 on metastasis in the body, we pass the tail vein over expression and knockout IGFBP-rP1 two The model of lung metastasis constructed by the group cell line reveals that the presence of IGFBP-rP1 inhibits the lung metastasis of colorectal cancer cells from positive and negative two aspects. Then we further explore the mechanism of IGFBP-rP1 to regulate the occurrence of EMT in colorectal cancer. First, it is found that endogenous IGFBP-rP1 can inhibit the TGF- beta in the cell line in vitro. The expression of the body and its downstream molecule Smad inhibits the activation of the TGF- beta /Smad signaling pathway. Then the EMT phenomenon induced by the knockout of IGFBP-rP1 is reversed by adding the TGF- beta receptor inhibitor SB431542 in the SW480 knockout IGFBP-rP1 cell line. This further proves that the TGF- beta / Smad signaling pathway is necessary for IGFBP-rP1 to participate in the regulation and control of the IGFBP-rP1. Finally, in the EMT model induced by TGF- beta 1 stimulated HT29 cells, the addition of exogenous recombinant protein IGFBP-rP1 not only reverses the induced EMT phenomenon, but also inhibits and inhibits the activation of TGF- beta receptor and downregulates the phosphorylation level of Smad2/3, which is again verified that IGFBP-rP1 is suppressed by the negative regulation of TGF- beta /Smad signaling pathway. In the EMT process, we can conclude that (1) the effect of Snail on the prognosis of colorectal cancer patients depends on the state of p53. Only in the p53 wild type patients, Snail expression has a bad prognostic significance, and the high expression of Snail in patients with stage I and II of colorectal cancer is an unfavorable independent prognostic factor or Snail Yang. Sex is an independent risk factor. (2) IGFBP-rP1 inhibits EMT and metastasis of colorectal cancer through TGF- beta /Smad signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34
本文编号：2120699
[Abstract]:Colorectal cancer is one of the major malignant tumors in today's society. Its incidence and mortality rate are located in third and fourth of the world's malignant tumors. There are about 1400000 newly diagnosed cases of colorectal cancer in the world each year, and about 693900 patients die of colorectal cancer. Western life has caused a rapid increase in the incidence of colorectal cancer in China. According to <2015 China cancer statistics, 376300 cases of colorectal cancer were newly confirmed in China in 2015, and 191000 patients died of colorectal cancer. Colorectal cancer has seriously threatened the health of the people all over the world, causing serious social and economic burdens in.TNM staging. To assess the overall prognosis and guide standardized treatment, but it has not been able to meet the needs of individualized treatment. Most patients in phase II and stage III have not benefited from the treatment guided by TNM staging. Therefore, effective molecular markers related to prognosis and treatment are excavated to assist in the screening of early stages of the risk of metastasis or recurrence. Colorectal cancer patients have an important clinical value to benefit from subsequent adjuvant therapy. Moreover, it is imperative to actively carry out the study of the pathological mechanism of colorectal cancer for the prevention and treatment of colorectal cancer. More than 90% of the patients with malignant tumors eventually died of tumor metastasis or relapse. About 20% of the patients were diagnosed with colorectal cancer. Distant metastasis has occurred. Patients with 40-50% have not found metastasis at the time of diagnosis, and distant metastasis will eventually occur. Tumor metastasis is a multistep, multi stage, multi pathway, and a series of complex processes involving multiple gene changes. In recent years, more and more studies have shown that epithelial mesenchymal transition (Epithelial mesenchymal transi) Tion, EMT) plays an important role in the invasion and metastasis of tumor, which refers to the biological process of epithelial cells transformed into interstitial phenotypes after external stimuli are stimulated by external signals. In this process, the epithelial cells lose their polarity, lose the close adhesion of the cells, and the expression of epithelial markers is downregulated or missing, and gradually acquired between the epithelial cells. The most significant molecular event in.EMT process is the decrease of E-cadherin expression, while the expression of N-cadherin, Vimentin and Fibronectin is enhanced, and some transcription factors such as Snail, Slug, Twist and Zeb1/2 are directly or indirectly regulated by the E-cadher. The expression of EMT related genes, such as in, induces the EMT process. Therefore, we detect the expression of these classical EMT related markers in colorectal cancer, and make clear the role of these markers in the prognosis of colorectal cancer, and try to select the key molecules to select the group with high metastasis risk, to judge the prognosis of the patients, to transfer the prediction, and to predict the prognosis of the patients. There are many important signaling pathways in the.EMT process, including TGF- beta, RTK, Wnt, Notch, Hedgehog, Matrix and anoxia, including TGF- beta 1, which is a classic factor to stimulate tumor cells to construct EMT models in vitro. The present report on TGF-P signaling pathway involved in EMT is quite detailed, TGF- beta After binding to its receptor, it activates Smad2/3 to phosphorylate it, then the phosphorylated Smad2/3 combines with Smad4 into the nucleus to regulate a series of functions associated with metastasis and EMT related target genes. In addition, the process of TGF- beta induced EMT involves some other signaling pathways related to cell movement and apoptosis, such as the PI3K/Akt pathway, MAPK pathway and Rho-like GTPase. In addition to the classic EMT markers, more and more reports reveal that many new molecules play an important role in the EMT process, and the insulin like growth factor binding protein related protein 1 (insulin-like growth factor binding protein-related protein 1, IGFBP-rP1) is our laboratory through inhibitory subtraction A gene expressed in colon adenocarcinoma cDNA library from colon adenocarcinoma normal mucosa and in colorectal adenocarcinoma. A series of previous studies found that IGFBP-rP1 inhibits the growth of colorectal cancer cells, induces apoptosis and senescence, and the higher the expression, the better the prognosis of the patients. We believe that it plays a potential inhibition in colorectal cancer. In addition, in the early laboratory in vitro cell line study found that IGFBP-rP1 could play an inhibitory role in the EMT process. Therefore, we would like to identify the relationship between IGFBP-rP1 and EMT and metastasis in colorectal cancer both in vivo and in vitro. Although there are some reports on the ability of IGFBP-rP1 to inhibit tumor cell migration, the specific machine It is not clear that.IGFBP-rP1 can be combined with IGFs and insulin, but the ability to bind is weak, which suggests that IGFBP-rP1 is independent of IGF in addition to the classical IGF system. Our early lab chip data found that IGFBP-rP1 overexpression causes the downstream effector of TGF- beta pathway S The expression level of Mad3 mRNA is reduced. Therefore, whether or not IGFBP-rP1 plays the function of inhibiting EMT through the TGF- beta /Smad signaling pathway is worthy of our in-depth study. First, we selected the cDNA specimens of the cancer tissues of 346 colorectal cancer patients who were treated in Sir Run Run Shaw Hospital for 2004-2008 years, and used real time fluorescent quantitative PCR (qPCR) to detect eight EMT markers. The expression of substance, including E-cadherin, Vimentin, Fibronectin, Snail, Slug, Twist, Zeb1 and Zeb2., had 167 patients' prognosis information. Follow-up information was obtained by telephone follow-up. The follow-up time was up to September 30, 2013, the shortest follow-up time was 3 months, the longest period was 115 months, the median follow-up time was 72 months, and the average follow-up time was 68 months. Then we followed up the results of mRNA in the tissue microarray specimens preserved in another group of laboratory tests, using immunohistochemical methods to detect the expression of E-cadherin, Fibronectin, Snail, Slug and Twist. This group of cases came from 415 colorectal cancer, which was treated at the first people's Hospital in Hangzhou, Hangzhou. All the patients' cancer tissue specimens were provided with the patient's prognosis information. The follow-up information was provided by the Xiaoshan CDC. The follow-up time was up to December 31, 2006, the shortest 1 months, the longest 186 months, the median follow-up time of 33 months, the average follow-up time of 48 months.SPSS 20 for windows statistics software for the following statistical processing. M Ann-Whitney rank sum test calculated the difference in the expression of gene in different clinical phenotypes; the expression difference of transcription factors in two phenotypes was calculated by non paired t test; the correlation analysis of protein expression grouping results and clinical manifestations was determined by chi square test or Fisher accurate test; two the correlation analysis of the subgroups used Spearman bivariate analysis. Correlation analysis; single factor Cox proportional risk model for single factor survival analysis of continuous variables; Kaplan-Meier method for single factor survival analysis (log-rank method for significant test) and mapping of survival curves; single factor analysis was used for multiple factor survival analysis for prognostic factors. The above analysis took P value less than 0.05 as a significant threshold. In the two groups of colorectal cancer samples, the relationship between the EMT markers and the clinicopathological parameters and prognosis was analyzed at the level of nRNA and protein at two levels. First, at the level of mRNA, the difference between the EMT markers in different clinical phenotypes was found, and the transcription factors were in the stromal table. The expression in patients with type (Vim/E-cad or Fibro/E-cad seventy-fifth percentile) was often higher than the epithelial phenotype (Vim/E-cad or Fibro/E-cad seventy-fifth percentile); five transcription factor 22 had a positive correlation; in a number of EMT markers, the high expression of Snail was an adverse factor for the patient and was an independent factor. Prognostic indicators; further in the subsistence analysis after stratified TNM staging, the high expression of Snail was an unfavorable independent prognostic indicator in stage I - II patients. In addition to the high expression of Slug or Twist, the prognosis of stage I - II patients was also poor, but these two were not independent preconditioning factors. Based on the mRNA level, we selected five EMT markers (E-cadherin, Fibronectin, Snail, Slug and Twist) were tested for subsequent protein levels. The results of protein levels were generally consistent with the results of mRNA levels, but some new findings were found to be poor in the positive expression of.Snail or Twist nuclei; the joint analysis of Snail or Twist and E-cadherin showed that, Herin low expression and Snail positive or E-cadherin low expression and Twist positive patients have the lowest survival rate; and Snail nuclear positive expression is an unfavorable independent prognostic indicator. Further more detailed analysis found that the prognostic significance of Snail or Twist nuclear positive only lies in stage I - II patients, in stage III - IV patients, There is no significant difference in survival between the presence of Snail or Twist nuclear positive; similarly, the positive expression of Snail nuclear positive is also of independent prognostic significance in stage I - II patients; furthermore, we also found that the effect of Snail on the prognosis of colorectal cancer patients depends on the state of p53, and that the positive expression of Snail is negative only in the p53 wild type patients. In the early stage of survival. In our early colorectal cancer cell line, we found that IGFBP-rP1 can downregulate the expression of epithelial markers, reduce the cell migration and invasion, and play an inhibitory role in the EMT process. Therefore, we would like to define the relationship between IGFBP-rP1 and EMT and metastasis in the body level. First, we detected the expression of IGFBP-rP1 in 217 cases of colorectal cancer by immunohistochemical method. Through chi square test, we found that the high expression of IGFBP-rP1 was negatively correlated with the histological grade, local lymph node recidivism and pathological stage, and was positively correlated with the expression of the EMT marker E-cadherin and P-catenin membrane, and the interstitial marker Fib. The expression of ronectin was negatively correlated. At the tissue level, it was further clarified that IGFBP-rP1 played a role in inhibiting EMT in colorectal cancer. Then, the subcutaneous tumor test in nude mice revealed the inhibition of the growth of SW620 in the colorectal cancer cells after overexpression of IGFBP-rP1, and the expression of IGFBP-rP1 was also detected in the transplanted tumor. The expression of the epithelial markers and the inhibition of the expression of the interstitial markers and the opposite effect in the IGFBP-rP1 knockout SW480 further indicate that IGFBP-rP1 inhibits the EMT process in colorectal cancer. In order to clarify the effect of IGFBP-rP1 on metastasis in the body, we pass the tail vein over expression and knockout IGFBP-rP1 two The model of lung metastasis constructed by the group cell line reveals that the presence of IGFBP-rP1 inhibits the lung metastasis of colorectal cancer cells from positive and negative two aspects. Then we further explore the mechanism of IGFBP-rP1 to regulate the occurrence of EMT in colorectal cancer. First, it is found that endogenous IGFBP-rP1 can inhibit the TGF- beta in the cell line in vitro. The expression of the body and its downstream molecule Smad inhibits the activation of the TGF- beta /Smad signaling pathway. Then the EMT phenomenon induced by the knockout of IGFBP-rP1 is reversed by adding the TGF- beta receptor inhibitor SB431542 in the SW480 knockout IGFBP-rP1 cell line. This further proves that the TGF- beta / Smad signaling pathway is necessary for IGFBP-rP1 to participate in the regulation and control of the IGFBP-rP1. Finally, in the EMT model induced by TGF- beta 1 stimulated HT29 cells, the addition of exogenous recombinant protein IGFBP-rP1 not only reverses the induced EMT phenomenon, but also inhibits and inhibits the activation of TGF- beta receptor and downregulates the phosphorylation level of Smad2/3, which is again verified that IGFBP-rP1 is suppressed by the negative regulation of TGF- beta /Smad signaling pathway. In the EMT process, we can conclude that (1) the effect of Snail on the prognosis of colorectal cancer patients depends on the state of p53. Only in the p53 wild type patients, Snail expression has a bad prognostic significance, and the high expression of Snail in patients with stage I and II of colorectal cancer is an unfavorable independent prognostic factor or Snail Yang. Sex is an independent risk factor. (2) IGFBP-rP1 inhibits EMT and metastasis of colorectal cancer through TGF- beta /Smad signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34
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