發(fā)布時(shí)間：2018-02-12 05:31

  本文關(guān)鍵詞： 直腸癌 基因表達(dá)譜 放化療敏感性 MicroRNA 種子區(qū) 遺傳變異 毒副反應(yīng) 生存　出處：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：世界范圍內(nèi)直腸癌的發(fā)病率呈持續(xù)上升的趨勢(shì)。進(jìn)展期直腸癌患者出現(xiàn)復(fù)發(fā)和轉(zhuǎn)移的風(fēng)險(xiǎn)較高,同步放化療聯(lián)合根治性切除手術(shù)是進(jìn)展期直腸癌患者的主要臨床治療方式。由于個(gè)體遺傳差異和腫瘤異質(zhì)性,患者對(duì)同步放化療的療效反應(yīng)和毒副反應(yīng)存在較大差異,本研究旨在通過(guò)全基因組表達(dá)譜芯片檢測(cè)探討直腸癌術(shù)前同步放化療療效相關(guān)的基因表達(dá)特征和分子生物學(xué)機(jī)制,通過(guò)候選基因策略鑒定直腸癌術(shù)后同步放化療毒副反應(yīng)和生存相關(guān)的遺傳變異。本研究首先入組81例術(shù)前同步化療聯(lián)合根治性手術(shù)的直腸癌患者,進(jìn)行術(shù)前腫瘤組織的全基因組表達(dá)檢測(cè),通過(guò)t檢驗(yàn)比較30例治療敏感組和14例不敏感組基因表達(dá)的差異,采用P0.05且Fold Change≥2.0的標(biāo)準(zhǔn)共篩選出179個(gè)差異表達(dá)的探針,通過(guò)模型篩選構(gòu)建了由差異最顯著的20個(gè)基因組成的療效預(yù)測(cè)模型,在發(fā)現(xiàn)階段的44例樣本中該模型的療效預(yù)測(cè)正確率為100%。根據(jù)該模型可以將37例中度敏感的患者分成30例治療敏感組和7例治療不敏感組,這兩組在無(wú)病生存時(shí)間和殘余癌細(xì)胞比例兩個(gè)方面均具有顯著的差異(P=0.0004；P=0.0377)。通過(guò)定量PCR的方法在45例驗(yàn)證樣本中檢測(cè)20個(gè)基因的表達(dá)水平,采用受試者工作曲線的方法發(fā)現(xiàn)20個(gè)基因組成的表達(dá)特征能夠較好地區(qū)分不同治療反應(yīng)的病人(AUC=0.815；95% CI=0.662-0.968；P=0.0173)。通過(guò)進(jìn)一步的數(shù)據(jù)分析,發(fā)現(xiàn)ZNF37A高表達(dá)的直腸癌患者對(duì)同步放化療更敏感,而且無(wú)病生存期延長(zhǎng)。通過(guò)體內(nèi)和體外表型實(shí)驗(yàn)發(fā)現(xiàn)高表達(dá)ZNF37A后腸癌細(xì)胞和皮下移植瘤對(duì)放化療敏感性增加,低表達(dá)ZNF37A后其對(duì)放化療敏感性降低。ZNF37A是已知的轉(zhuǎn)錄抑制因子,通過(guò)共表達(dá)分析發(fā)現(xiàn)與ZNF37A表達(dá)負(fù)相關(guān)的372個(gè)基因中有8個(gè)顯著富集在凋亡信號(hào)通路中,通過(guò)在細(xì)胞系中進(jìn)行檢測(cè)發(fā)現(xiàn)高表達(dá)ZNF37A后TNFRSF6B的表達(dá)顯著下降,而低表達(dá)ZNF37A后TNFRSF6B的表達(dá)顯著增高。通過(guò)ChIP-qPCR和報(bào)告基因?qū)嶒?yàn)證實(shí)ZNF37A可以和TNFRSF6B的啟動(dòng)子區(qū)結(jié)合并抑制其轉(zhuǎn)錄。而且在放化療處理組,高表達(dá)ZNF37A后促進(jìn)細(xì)胞凋亡的發(fā)生；而低表達(dá)ZNF37A后抑制細(xì)胞凋亡的發(fā)生。進(jìn)一步降低細(xì)胞系中TNFRSF6B的表達(dá)水平后,細(xì)胞凋亡增加,恢復(fù)對(duì)放化療的敏感性。臨床特征和治療方式相似的直腸癌患者,接受同步放化療后的毒副反應(yīng)和生存時(shí)間相差較大,而個(gè)體遺傳因素是導(dǎo)致毒副反應(yīng)和生存時(shí)間差異的重要因素。MicroRNAs是一類重要的小分子非編碼RNA,主要通過(guò)5’端的2-8個(gè)堿基(種子區(qū))與目的基因的3’UTR結(jié)合,通過(guò)對(duì)目的基因的轉(zhuǎn)錄后調(diào)控,參與多種細(xì)胞生物學(xué)過(guò)程。因此我們候選位于miRNA種子區(qū)且在中國(guó)北方漢族人群中MAF大于0.1的18個(gè)遺傳變異,在365例直腸癌術(shù)后同步放化療的患者中進(jìn)行遺傳變異與患者毒副作用的發(fā)生和生存時(shí)間的關(guān)聯(lián)研究。通過(guò)Logistic回歸模型計(jì)算各遺傳變異基因型與毒副反應(yīng)發(fā)生風(fēng)險(xiǎn)的OR和95%可信區(qū)間;Cox比例風(fēng)險(xiǎn)模型計(jì)算各遺傳變異基因型與患者生存時(shí)間的HR和95%可信區(qū)間。通過(guò)數(shù)據(jù)分析發(fā)現(xiàn),位于hsa-miR-4707-3p種子區(qū)的rs2273626與直腸癌患者中重度白細(xì)胞減少的發(fā)生相關(guān),加性模型中多因素校正后的OR值為0.48(95% CI=0.31-0.74；P=0.0009)；位于hsa-miR-4274種子區(qū)的rs202195689與直腸癌患者總生存時(shí)間和無(wú)病生存時(shí)間相關(guān),加性模型中多因素校正后的HR值分別為1.94(95%CI=1.19-3.14；P=0.0074)和1.83(95% CI=1.24-2.72；P=0.0026)。本研究構(gòu)建了直腸癌術(shù)前同步放化療的療效預(yù)測(cè)模型,并且發(fā)現(xiàn)療效差異表達(dá)基因ZNF37A能夠通過(guò)轉(zhuǎn)錄抑制TNFRSF6B的表達(dá),促進(jìn)放化療后細(xì)胞凋亡的發(fā)生,增加細(xì)胞的放化療敏感性。直腸癌放化療毒副反應(yīng)和預(yù)后相關(guān)的遺傳變異研究顯示,位于miRNA種子區(qū)的遺傳變異能夠降低中重度白細(xì)胞減少的發(fā)生或與患者的預(yù)后不良相關(guān)。這些研究對(duì)于深入理解直腸癌患者臨床治療反應(yīng)的差異和指導(dǎo)個(gè)體化治療具有重要的理論意義和潛在的應(yīng)用價(jià)值。
[Abstract]:In this study , we studied the gene expression profiles and molecular biological mechanisms of the patients with advanced rectal cancer . The results of this study were used to detect the gene expression profiles and molecular biological mechanisms of the patients with advanced rectal cancer . The expression level of 20 genes was detected in 45 validation samples by quantitative PCR , and the expression profiles of 20 genes were identified by the method of working curve of subjects ( AUC = 0.815 ; 95 % CI = 0.662 - 0.968 ; P = 0.0173 ) . The results showed that the expression of TNFRSF6B was significantly decreased after the expression of ZNF37A . The results showed that the expression of TNFRSF6B was significantly decreased after the expression of ZNF37A . The results showed that the expression of TNFRSF6B was significantly decreased after the expression of ZNF37A . It was found that the gene ZNF37A can inhibit the expression of TNFRSF6B by transcription , promote the occurrence of apoptosis after chemotherapy , and increase the sensitivity of radiotherapy and chemotherapy . The study of genetic variation associated with chemotherapy toxicity and prognosis of rectal cancer shows that genetic variation in the miRNA seed region can reduce the occurrence of moderate or severe white blood cell loss or the poor prognosis of patients . These studies have important theoretical significance and potential application value for the deep understanding of the differences in clinical treatment response in rectal cancer patients and the guidance of individualized treatment .

【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】：2016
【分類號(hào)】：R735.37
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本文編號(hào)：1504887