【摘要】：結(jié)直腸癌是當(dāng)今社會(huì)主要惡性腫瘤之一,其發(fā)病率和死亡率在全世界范圍內(nèi)的惡性腫瘤中分別位于第三位和第四位。全球每年大約有1,400,000名新確診的結(jié)直腸癌病例,并且約有693,900名患者最終死于結(jié)直腸癌。隨著經(jīng)濟(jì)的發(fā)展,所謂的西方式生活使國(guó)內(nèi)結(jié)直腸癌的發(fā)病率飛速增長(zhǎng)。據(jù)《2015中國(guó)癌癥統(tǒng)計(jì)》報(bào)道,我國(guó)在2015年新確診376,300名結(jié)直腸癌病例,有191,000名患者死于結(jié)直腸癌。結(jié)直腸癌已嚴(yán)重威脅全世界人民健康,造成了嚴(yán)重的社會(huì)和經(jīng)濟(jì)負(fù)擔(dān)。TNM分期雖然可以從整體上評(píng)估預(yù)后和指導(dǎo)規(guī)范化治療,但是它已經(jīng)無(wú)法滿足個(gè)體化治療的需求,有大部分Ⅱ期和Ⅲ期的患者并沒(méi)有從TNM分期指導(dǎo)的治療中獲益。因此,挖掘有效的與預(yù)后和治療有關(guān)的分子標(biāo)志物來(lái)輔助篩選有轉(zhuǎn)移或者復(fù)發(fā)風(fēng)險(xiǎn)的早期結(jié)直腸癌患者,使他們?cè)诤罄m(xù)的輔助治療中受益具有重要的臨床價(jià)值。并且,積極開(kāi)展結(jié)直腸癌病理機(jī)制的研究對(duì)于結(jié)直腸癌的防治勢(shì)在必行。臨床上90%以上的惡性腫瘤患者最終死于腫瘤轉(zhuǎn)移或復(fù)發(fā)。大約20%的患者在診斷出結(jié)直腸癌時(shí)就已經(jīng)發(fā)生了遠(yuǎn)處轉(zhuǎn)移,40-50%的患者在確診時(shí)未發(fā)現(xiàn)轉(zhuǎn)移,治療后最終也會(huì)出現(xiàn)遠(yuǎn)處轉(zhuǎn)移。腫瘤的轉(zhuǎn)移是一個(gè)多步驟、多階段、多途徑、涉及多基因變化的一系列復(fù)雜過(guò)程。近年來(lái),越來(lái)越多的研究表明上皮間質(zhì)轉(zhuǎn)化(Epithelial mesenchymal transition, EMT)在腫瘤的侵襲轉(zhuǎn)移中發(fā)揮著重要的功能。EMT是指上皮細(xì)胞受到外界信號(hào)刺激后,細(xì)胞形態(tài)轉(zhuǎn)變?yōu)殚g質(zhì)表型的生物學(xué)過(guò)程。在這一過(guò)程中,上皮細(xì)胞失去極性,失去細(xì)胞間的緊密黏附連接,上皮標(biāo)志物表達(dá)下調(diào)或缺失,而逐漸獲得間質(zhì)細(xì)胞形態(tài)特征以及間質(zhì)標(biāo)志物表達(dá),同時(shí)增強(qiáng)細(xì)胞的運(yùn)動(dòng)能力和遷移能力。EMT過(guò)程中最顯著的分子事件是E-cadherin表達(dá)降低,而N-cadherin, Vimentin和Fibronectin的表達(dá)增強(qiáng),一些轉(zhuǎn)錄因子如Snail, Slug, Twist和Zeb1/2等通過(guò)直接或間接調(diào)控E-cadherin等EMT相關(guān)基因的表達(dá),誘導(dǎo)EMT過(guò)程。因此,我們檢測(cè)這些與EMT相關(guān)的經(jīng)典標(biāo)志物在結(jié)直腸癌中的表達(dá),明確其在結(jié)直腸癌預(yù)后中的作用,并試圖從中遴選出關(guān)鍵的分子,以篩選具有高轉(zhuǎn)移風(fēng)險(xiǎn)的患者群體,為患者的預(yù)后判斷、轉(zhuǎn)移預(yù)測(cè)、個(gè)體化治療等方面提供有效的輔助支撐。EMT過(guò)程受眾多重要的信號(hào)通路調(diào)控,包括TGF-β, RTK, Wnt, Notch, Hedgehog, Matrix和缺氧等。其中TGF-β1是體外刺激腫瘤細(xì)胞構(gòu)建EMT模型的經(jīng)典因子,目前關(guān)于TGF-P信號(hào)通路參與調(diào)控EMT的報(bào)道頗為詳盡,TGF-β與其受體結(jié)合后激活Smad2/3使其磷酸化,接著磷酸化的Smad2/3與Smad4結(jié)合入核,調(diào)節(jié)一系列與轉(zhuǎn)移以及EMT相關(guān)靶基因的功能。另外TGF-β誘導(dǎo)EMT的過(guò)程還涉及與細(xì)胞運(yùn)動(dòng)和凋亡相關(guān)的一些其他信號(hào)通路,如PI3K/Akt通路、MAPK通路和Rho-like GTPase通路等。除了經(jīng)典的EMT標(biāo)志物之外,越來(lái)越多的報(bào)道揭示存在許多新分子在EMT過(guò)程中扮演重要的角色,而胰島素樣生長(zhǎng)因子結(jié)合蛋白相關(guān)蛋白1 (insulin-like growth factor binding protein-related protein 1, IGFBP-rP1)是我們實(shí)驗(yàn)室通過(guò)抑制性差減雜交法從結(jié)腸腺癌-正常粘膜的差減cDNA文庫(kù)中篩選出并在結(jié)腸腺癌中高表達(dá)的一個(gè)基因。前期一系列研究發(fā)現(xiàn)IGFBP-rP1抑制結(jié)直腸癌細(xì)胞的生長(zhǎng),誘導(dǎo)細(xì)胞凋亡和衰老,其表達(dá)越高患者預(yù)后越好,我們認(rèn)為它在結(jié)直腸癌中發(fā)揮著潛在的抑癌作用。另外,實(shí)驗(yàn)室前期體外細(xì)胞系研究發(fā)現(xiàn),IGFBP-rP1可在EMT過(guò)程中發(fā)揮抑制作用。因此,我們欲在體內(nèi)和體外雙水平明確IGFBP-rP1在結(jié)直腸癌中與EMT和轉(zhuǎn)移的關(guān)系。目前雖也有一些關(guān)于IGFBP-rP1抑制腫瘤細(xì)胞遷移能力的報(bào)道,但是具體機(jī)制尚不清楚。IGFBP-rP1雖與IGFs以及insulin都可以結(jié)合,但是結(jié)合能力較弱,這提示IGFBP-rP1在除了通過(guò)經(jīng)典的IGF系統(tǒng)發(fā)揮其生物學(xué)效應(yīng)之外,還具有獨(dú)立于IGF的作用。我們實(shí)驗(yàn)室前期芯片數(shù)據(jù)發(fā)現(xiàn)IGFBP-rP1過(guò)表達(dá)引起TGF-β通路的下游效應(yīng)分子Smad3 mRNA表達(dá)水平降低。因此,IGFBP-rP1是否通過(guò)TGF-β/Smad信號(hào)通路發(fā)揮抑制EMT的功能,值得我們深入研究。首先我們選取了2004-2008年間在邵逸夫醫(yī)院接受治療的346名結(jié)直腸癌患者的癌組織cDNA標(biāo)本,采用實(shí)時(shí)熒光定量PCR(qPCR)檢測(cè)八個(gè)EMT標(biāo)志物的表達(dá),包括E-cadherin, Vimentin, Fibronectin, Snail, Slug, Twist, Zeb1和Zeb2。這組病例中的167例有患者預(yù)后信息,隨訪信息由電話隨訪獲得,隨訪時(shí)間截止于2013年9月30日,隨訪時(shí)間最短3個(gè)月,最長(zhǎng)115個(gè)月,中位隨訪時(shí)間72個(gè)月,平均隨訪時(shí)間68個(gè)月。接著我們?cè)诹硪唤M實(shí)驗(yàn)室保存的組織芯片標(biāo)本中對(duì)mRNA的結(jié)果進(jìn)行了后續(xù)的驗(yàn)證,采用免疫組織化學(xué)方法檢測(cè)了E-cadherin, Fibronectin, Snail, Slug和Twist的表達(dá)。這組病例來(lái)自1991-2006年間在杭州蕭山第一人民醫(yī)院接受治療的415名結(jié)直腸癌患者的癌組織標(biāo)本,全部有患者預(yù)后信息,隨訪信息由蕭山疾控中心提供,隨訪時(shí)間截止于2006年12月31日,隨訪時(shí)間最短1個(gè)月,最長(zhǎng)186個(gè)月,中位隨訪時(shí)間33個(gè)月,平均隨訪時(shí)間48個(gè)月。SPSS 20.0 for windows統(tǒng)計(jì)學(xué)軟件用于以下統(tǒng)計(jì)學(xué)處理。采用Mann-Whitney秩和檢驗(yàn)計(jì)算基因在不同臨床表型的表達(dá)差異；采用非配對(duì)t檢驗(yàn)計(jì)算轉(zhuǎn)錄因子在兩種表型中的表達(dá)差異；蛋白表達(dá)分組結(jié)果與臨床表現(xiàn)的關(guān)聯(lián)分析采用卡方檢驗(yàn)或者Fisher精確檢驗(yàn)；兩因子間相關(guān)性分析采用Spearman雙變量相關(guān)性分析；單因素Cox比例風(fēng)險(xiǎn)模型對(duì)連續(xù)性變量進(jìn)行單因素生存分析；用Kaplan-Meier法對(duì)分類變量進(jìn)行單因素生存分析(log-rank法進(jìn)行顯著性檢驗(yàn)),并繪制生存曲線；將單因素分析對(duì)預(yù)后有統(tǒng)計(jì)學(xué)意義的因素再納入多因素生存分析。以上分析均以P值≤0.05作為顯著性閾值。在兩組結(jié)直腸癌樣本中在nRNA和蛋白兩個(gè)水平對(duì)EMT標(biāo)志物與臨床病理參數(shù)和預(yù)后的關(guān)系分別分析。首先在mRNA水平發(fā)現(xiàn)：EMT標(biāo)志物分別在不同的臨床表型間表達(dá)存在差異；各轉(zhuǎn)錄因子在間質(zhì)表型(Vim/E-cad或者Fibro/E-cad第75百分位數(shù))的患者中的表達(dá)常高于上皮表型(Vim/E-cad或者Fibro/E-cad第75百分位數(shù))患者；五個(gè)轉(zhuǎn)錄因子兩兩之間存在正相關(guān)；在眾多EMT標(biāo)志物中只有Snail的高表達(dá)對(duì)患者預(yù)后是一個(gè)不利因素,并且是一個(gè)獨(dú)立預(yù)后指標(biāo)；進(jìn)一步在將TNM分期分層后的生存分析中發(fā)現(xiàn),Snail的高表達(dá)在Ⅰ-Ⅱ期患者中是一個(gè)不利的獨(dú)立預(yù)后指標(biāo),另外在Slug或Twist高表達(dá)時(shí),Ⅰ-Ⅱ期患者預(yù)后也較差,但這二者不是獨(dú)立的預(yù)后因素。基于mRNA水平的結(jié)果,我們選取了五個(gè)EMT標(biāo)志物(E-cadherin, Fibronectin, Snail, Slug和Twist)進(jìn)行后續(xù)蛋白水平的驗(yàn)證。蛋白水平的結(jié)果與mRNA水平的結(jié)果總體上具有一致性,但也有一些新的發(fā)現(xiàn)。Snail或Twist核陽(yáng)性表達(dá)時(shí)患者預(yù)后差；將Snail或Twist與E-cadherin分別聯(lián)合分析表明,E-cadherin低表達(dá)且Snail陽(yáng)性或E-cadherin低表達(dá)且Twist陽(yáng)性患者較其他分組來(lái)說(shuō)生存率最低；并且Snail核陽(yáng)性表達(dá)是一個(gè)不利的獨(dú)立預(yù)后指標(biāo)；進(jìn)一步更細(xì)致的分析發(fā)現(xiàn),Snail或Twist核陽(yáng)性的預(yù)后意義只存在于Ⅰ-Ⅱ期患者,在Ⅲ-Ⅳ期患者中,是否存在Snail或Twist核陽(yáng)性表達(dá)在生存上并無(wú)顯著性差異；同樣地,Snail核陽(yáng)性表達(dá)也在Ⅰ-Ⅱ期患者中具有獨(dú)立預(yù)后意義；另外我們還發(fā)現(xiàn)Snail對(duì)結(jié)直腸癌患者預(yù)后的影響依賴于p53的狀態(tài),只有在p53野生型的患者中,Snail陽(yáng)性表達(dá)不利于生存。前期我們?cè)隗w外結(jié)直腸癌細(xì)胞系中研究發(fā)現(xiàn),IGFBP-rP1在誘導(dǎo)上皮標(biāo)記物的表達(dá)的同時(shí)可以下調(diào)間質(zhì)標(biāo)記物的表達(dá),降低細(xì)胞的遷移侵襲能力,在EMT過(guò)程中發(fā)揮抑制作用。因此我們欲在體內(nèi)水平明確IGFBP-rP1與EMT和轉(zhuǎn)移的關(guān)系。首先我們采用免疫組織化學(xué)的方法檢測(cè)了217例結(jié)直腸癌組織中IGFBP-rP1的表達(dá),通過(guò)卡方檢驗(yàn)發(fā)現(xiàn)IGFBP-rP1的高表達(dá)與組織學(xué)分級(jí)、局部淋巴結(jié)累犯及病理分期呈負(fù)相關(guān),并且與EMT標(biāo)志物E-cadherin及P-catenin的膜表達(dá)呈正相關(guān),與間質(zhì)標(biāo)志物Fibronectin的表達(dá)呈負(fù)相關(guān)。在組織水平上進(jìn)一步明確了在結(jié)直腸癌中IGFBP-rP1發(fā)揮抑制EMT的作用。接著采用裸鼠皮下成瘤實(shí)驗(yàn),揭示了過(guò)表達(dá)IGFBP-rP1后抑制結(jié)直腸癌細(xì)胞SW620在動(dòng)物體內(nèi)的生長(zhǎng),并且在移植瘤中同樣檢測(cè)到過(guò)表達(dá)IGFBP-rP1后促進(jìn)上皮標(biāo)志物的表達(dá),同時(shí)抑制間質(zhì)標(biāo)志物的表達(dá),而在IGFBP-rP1敲除的SW480中則存在相反的作用,這進(jìn)一步說(shuō)明IGFBP-rP1可抑制結(jié)直腸癌發(fā)生EMT過(guò)程。為了明確IGFBP-rP1在體內(nèi)對(duì)于轉(zhuǎn)移的影響,我們通過(guò)尾靜脈注射過(guò)表達(dá)和敲除IGFBP-rP1的兩組細(xì)胞株所構(gòu)建的肺轉(zhuǎn)移模型,從正反兩方面揭示了IGFBP-rP1的存在會(huì)抑制結(jié)直腸癌細(xì)胞的肺轉(zhuǎn)移。隨后我們進(jìn)一步探討了IGFBP-rP1是通過(guò)何種機(jī)制來(lái)調(diào)控結(jié)直腸癌中EMT的發(fā)生。首先,在體外細(xì)胞系中發(fā)現(xiàn)內(nèi)源性的IGFBP-rP1可以抑制TGF-β受體以及其下游分子Smad的表達(dá),抑制TGF-β/Smad信號(hào)通路的活化。接著通過(guò)在SW480敲除IGFBP-rP1的細(xì)胞株中加入TGF-β受體抑制劑SB431542,發(fā)現(xiàn)由IGFBP-rP1敲除所誘導(dǎo)的EMT現(xiàn)象會(huì)被逆轉(zhuǎn),這進(jìn)一步證明TGF-β/Smad信號(hào)通路是IGFBP-rP1參與EMT調(diào)控所必需的。最后我們?cè)赥GF-β1刺激HT29細(xì)胞后所誘導(dǎo)的EMT模型中,加入外源性重組蛋白IGFBP-rP1,不僅可扭轉(zhuǎn)所誘導(dǎo)的EMT現(xiàn)象,還可阻礙并抑制TGF-β受體激活,下調(diào)Smad2/3的磷酸化水平,這也再次驗(yàn)證了IGFBP-rP1是通過(guò)負(fù)性調(diào)控TGF-β/Smad信號(hào)通路而抑制EMT過(guò)程的。綜上所述,我們可以得出以下結(jié)論：(1) Snail對(duì)結(jié)直腸癌患者預(yù)后的影響依賴于p53的狀態(tài),只有在p53野生型患者中,Snail表達(dá)具有不良預(yù)后意義,并且在結(jié)直腸癌Ⅰ-Ⅱ期患者中Snail高表達(dá)是一個(gè)不利的獨(dú)立預(yù)后因素或者Snail陽(yáng)性是一個(gè)獨(dú)立危險(xiǎn)因素。(2) IGFBP-rP1通過(guò)TGF-β/Smad信號(hào)通路抑制結(jié)直腸癌EMT和轉(zhuǎn)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.34

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