本文選題：食管鱗癌 + NRP1�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：食管癌發(fā)生發(fā)展過(guò)程中存在大量分子異常改變。實(shí)驗(yàn)室前期工作中獲得一個(gè)食管癌組織高表達(dá)蛋白Calreticulin(CRT),功能研究發(fā)現(xiàn)CRT可促進(jìn)食管癌細(xì)胞侵襲遷移,表達(dá)譜芯片分析顯示包括Neuropilin-1(NRP1)在內(nèi)的多個(gè)基因在食管癌細(xì)胞中受CRT調(diào)控。本工作進(jìn)一步研究了NRP1介導(dǎo)CRT影響食管癌細(xì)胞惡性表型的分子機(jī)制。Real-time PCR與Western blot驗(yàn)證結(jié)果表明CRT可以促進(jìn)NRP1mRNA口蛋白表達(dá)。體外細(xì)胞侵襲與遷移實(shí)驗(yàn)顯示,敲降NRP1食管癌細(xì)胞侵襲遷移能力明顯降低；敲降CRT回復(fù)NRP1表達(dá),食管癌細(xì)胞侵襲遷移能力得到回復(fù)。裸鼠肺轉(zhuǎn)移實(shí)驗(yàn)顯示,抑制NRP1表達(dá),食管癌細(xì)胞肺轉(zhuǎn)移結(jié)節(jié)數(shù)量顯著降低。敲降CRT或NRP1均能抑制MMP2、MMP9及FAK的表達(dá),提示CRT-NRP1通路異�？赡苁鞘彻馨┘�(xì)胞侵襲遷移的重要機(jī)制。染色質(zhì)免疫沉淀(ChIP)與雙熒光素酶報(bào)告基因?qū)嶒?yàn)結(jié)果顯示,轉(zhuǎn)錄因子STAT5A能夠調(diào)控NRP1的轉(zhuǎn)錄。檢測(cè)發(fā)現(xiàn)CRT增強(qiáng)STAT5A的轉(zhuǎn)錄活性。在食管癌組織中驗(yàn)證發(fā)現(xiàn),CRT與NRP1表達(dá)顯著正相關(guān)。這些結(jié)果說(shuō)明,我們發(fā)現(xiàn)了一個(gè)新的促進(jìn)食管癌細(xì)胞侵襲遷移信號(hào)通路,即CRT-STAT5A-NRP1。食管癌組織分析顯示,NRP1高表達(dá)與原發(fā)性腫瘤浸潤(rùn)深度和患者不良預(yù)后顯著正相關(guān)。細(xì)胞表型研究結(jié)果顯示,NRP1可以增強(qiáng)食管癌細(xì)胞增殖及裸鼠成瘤能力。進(jìn)一步研究發(fā)現(xiàn),NRP1可以上調(diào)N F-κB P65亞基的mRNA和蛋白表達(dá),回復(fù)實(shí)驗(yàn)結(jié)果提示NRP1可調(diào)控P65表達(dá)進(jìn)而影響食管癌細(xì)胞的增殖。由于NRP1不具備轉(zhuǎn)錄因子功能,我們進(jìn)一步研究了介導(dǎo)NRP1促進(jìn)P65轉(zhuǎn)錄的機(jī)制。通過(guò)生物信息學(xué)分析、ChIP實(shí)驗(yàn)和雙熒光素酶報(bào)告基因?qū)嶒?yàn)獲得了P65的候選轉(zhuǎn)錄因子cAMP反應(yīng)元件結(jié)合蛋白CREB。我們發(fā)現(xiàn)NRP1可與表皮生長(zhǎng)因子受體EGFR相互作用并激活EGFR,NRP1的b1/b2結(jié)構(gòu)域?qū)GFR的活化起到關(guān)鍵作用；EGFR通過(guò)AKT通路調(diào)控CREB的轉(zhuǎn)錄因子活性,進(jìn)而影響P65表達(dá)。在食管癌組織中利用連續(xù)切片檢測(cè)發(fā)現(xiàn),NRP1與P65表達(dá)水平顯著正相關(guān)。由此我們揭示了NRP1調(diào)控食管癌增殖的信號(hào)通路,即NRP1-EGFR-AKT-CREB-P65。利用NRP1靶向多肽處理腫瘤細(xì)胞,結(jié)果顯示多肽終濃度為5μmol/L時(shí),大多數(shù)NRP1高表達(dá)的腫瘤細(xì)胞系生長(zhǎng)顯著受到抑制；按照250μg/只的劑量,每周腹腔注射給藥2次,能夠明顯抑制食管癌細(xì)胞裸鼠皮下瘤的生長(zhǎng),提示NRP1可能作為潛在的腫瘤治療靶點(diǎn)。
[Abstract]:There are a lot of abnormal molecular changes during the development of esophageal carcinoma. A high expression protein Calreticulinine (CRT) was obtained in our laboratory. Functional studies showed that CRT could promote invasion and migration of esophageal cancer cells. Expression microarray analysis showed that many genes, including Neuropilin-1 NRP1, were regulated by CRT in esophageal cancer cells. In this work, we further studied the molecular mechanism of CRT mediated by NRP1 on the malignant phenotype of esophageal cancer cells. Real-time PCR and Western blot verification showed that CRT could promote the expression of NRP1mRNA protein. In vitro cell invasion and migration assay showed that knockdown of NRP1 esophageal carcinoma cells significantly decreased the ability of invasion and migration, knock down CRT returned to NRP1 expression, esophageal cancer cell invasion and migration ability was restored. Lung metastasis test in nude mice showed that inhibition of NRP1 expression significantly decreased the number of metastatic nodules in esophageal cancer cells. Knockdown of CRT or NRP1 can inhibit the expression of MMP2, MMP9 and FAK, suggesting that abnormal CRT-NRP1 pathway may be an important mechanism of invasion and migration of esophageal cancer cells. The results of chromatin immunoprecipitation and double luciferase reporter gene experiments showed that the transcription factor STAT5A could regulate the transcription of NRP1. CRT was found to enhance the transcriptional activity of STAT5A. A positive correlation between CRT and NRP1 expression was found in esophageal carcinoma tissues. These results suggest that we have discovered a new signaling pathway that promotes invasion and migration of esophageal cancer cells, namely CRT-STAT5A-NRP1. Tissue analysis showed that the high expression of NRP1 was positively correlated with the depth of primary tumor invasion and poor prognosis. The results of phenotypic study showed that NRP1 could enhance the proliferation of esophageal cancer cells and the tumorigenic ability of nude mice. Furthermore, it was found that NRP1 could up-regulate the expression of mRNA and protein of NF- 魏 B p65 subunit, and the response experiment suggested that NRP1 could regulate the expression of p65 and affect the proliferation of esophageal carcinoma cells. Since NRP1 does not have the function of transcription factor, we have further studied the mechanism of mediating NRP1 to promote the transcription of p65. The candidate transcription factor cAMP response element binding protein CREB for p65 was obtained by bioinformatics analysis and double luciferase reporter gene experiment. We found that NRP1 interacts with epidermal growth factor receptor EGFR and activates the b1/b2 domain of EGFR NRP1, which plays a key role in the activation of EGFR. EGFR regulates the activity of CREB transcription factors through the AKT pathway, thus affecting the expression of p65. A positive correlation between the expression of NRP1 and p65 was found in esophageal carcinoma by serial section detection. Therefore, we reveal the signal pathway of NRP1 regulating the proliferation of esophageal carcinoma, namely NRP1-EGFR-AKT-CREB-P65. Tumor cells were treated with NRP1 targeted polypeptides. The results showed that the growth of most tumor cell lines with high NRP1 expression was significantly inhibited when the final concentration of polypeptide was 5 渭 mol/L, and was injected twice a week at the dose of 250 渭 g / mouse. It can obviously inhibit the growth of subcutaneous tumor of esophageal cancer cells in nude mice, suggesting that NRP1 may be a potential target for tumor therapy.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.1

【共引文獻(xiàn)】

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1 ;Modulating autophagy:a strategy for cancer therapy[J];癌癥;2011年10期

2 熊輝強(qiáng);李黎;張少容;;鈣網(wǎng)蛋白與抗腫瘤免疫的相關(guān)性研究新進(jìn)展[J];重慶醫(yī)學(xué);2012年13期

3 郭春香;宋靜慧;;宮頸病變與陰道局部細(xì)胞免疫研究新進(jìn)展[J];中國(guó)婦產(chǎn)科臨床雜志;2013年05期

4 侯麗穎;孫燕佩;張旭光;趙棟;吳坤;;維生素E琥珀酸酯通過(guò)Akt/mTOR信號(hào)通路誘導(dǎo)人胃癌細(xì)胞SGC-7901發(fā)生保護(hù)性自噬[J];癌變.畸變.突變;2013年05期

5 陳松峰;邵增務(wù);;自噬相關(guān)信號(hào)轉(zhuǎn)導(dǎo)通路研究進(jìn)展[J];國(guó)際骨科學(xué)雜志;2013年04期

6 王峗;柯飛;趙安芳;陳蘭英;;自噬在癌癥發(fā)生中的雙重作用[J];癌變.畸變.突變;2013年06期

7 Yu-Shan Chen;Xiao-Bo Qiu;;Ubiquitin at the crossroad of cell death and survival[J];Chinese Journal of Cancer;2013年12期

8 肖玲;舒暢;唐記華;王曉萍;王高華;;不同時(shí)程的慢性不可預(yù)計(jì)溫和應(yīng)激對(duì)大鼠海馬神經(jīng)可塑性相關(guān)蛋白水平的影響[J];神經(jīng)損傷與功能重建;2013年06期

9 張松;鄒曉平;;HSP27與消化系統(tǒng)腫瘤耐藥相關(guān)性的研究進(jìn)展[J];國(guó)際消化病雜志;2013年05期

10 吳同申;孟彥;彭圣智;;肝細(xì)胞生長(zhǎng)因子及其受體與神經(jīng)纖毛蛋白1在骨肉瘤中的表達(dá)及意義[J];重慶醫(yī)學(xué);2013年27期

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1 Chong Zhang;Lin-yi Feng;Xiao-dan Shuai;Ya-si Xu;Dan Zhang;Da-yong Zhang;Bao-ming Wang;Li-huang Zhang;Jun-bo Wang;Jian-ping Pan;Hua Zhou;Si-cong Wang;Neng-ming Lin;;Aspirin enhanced ABT-737-induced apoptosis via regulation of PI3K pathway in human cancer cells[A];抗腫瘤藥物研究新進(jìn)展與腫瘤個(gè)性化藥物治療論壇論文集[C];2013年

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1 劉飛;食管癌差異表達(dá)蛋白的分離鑒定及功能研究[D];北京協(xié)和醫(yī)學(xué)院;2011年

2 高紅軍;食管癌自身抗體和血清小分子蛋白的鑒定[D];中國(guó)協(xié)和醫(yī)科大學(xué);2008年

3 張廣平;河南食管癌高發(fā)區(qū)同卵雙胞胎食管癌和癌前病變患者血清蛋白組學(xué)研究[D];鄭州大學(xué);2009年

4 韓冰;內(nèi)質(zhì)網(wǎng)蛋白質(zhì)折疊及其相關(guān)通路在耐藥絨癌中的作用[D];中國(guó)協(xié)和醫(yī)科大學(xué);2010年

5 慕容;TRB3在腫瘤進(jìn)展中的作用及其機(jī)制探討[D];北京協(xié)和醫(yī)學(xué)院;2012年

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7 汪曉敏;Calreticulin蛋白在食管癌轉(zhuǎn)移中作用機(jī)制研究[D];北京協(xié)和醫(yī)學(xué)院;2011年

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2 劉贊;新疆三個(gè)民族食管癌與癌旁組織比較蛋白質(zhì)組的研究[D];新疆醫(yī)科大學(xué);2009年

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