本文選題：MPDZ + 肺癌��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：背景:長(zhǎng)期以來,惡性腫瘤的發(fā)病率和死亡率一直居高不下,嚴(yán)重威脅著人類的生命健康。越來越多的研究證實(shí),遺傳學(xué)和表觀遺傳學(xué)的異常改變?cè)诎┗蚝鸵职┗虻谋磉_(dá)調(diào)控中起著重要的作用,抑癌基因拷貝數(shù)變異和(或)啟動(dòng)子CpG島甲基化導(dǎo)致的表達(dá)失活與腫瘤的發(fā)生密切相關(guān)。因此,通過尋找腫瘤發(fā)生過程中抑癌基因拷貝數(shù)變異和DNA甲基化變化的規(guī)律,將為惡性腫瘤的臨床診斷和靶向治療方式提供新的思路。MPDZ基因是我們前期在肺癌研究中篩選出的一個(gè)新的甲基化調(diào)控候選抑癌基因,但是其在肺癌以及其他腫瘤中的作用和機(jī)制還不清楚,因此本課題擬通過對(duì)MPDZ基因在腫瘤中的臨床特征分析,初步評(píng)價(jià)其在腫瘤發(fā)生中的臨床意義;同時(shí),全面分析MPDZ基因在腫瘤中的功能和分子機(jī)制,為后續(xù)深入研究該基因在腫瘤發(fā)生中的生物學(xué)意義和有效的腫瘤治療提供資料。目的:1.明確MPDZ基因在腫瘤中的突變、拷貝數(shù)變異和甲基化的發(fā)生情況及其臨床意義;解析MPDZ基因甲基化和表達(dá)對(duì)腫瘤患者生存預(yù)后的影響。2.明確MPDZ基因在肺癌組織、痰液和血漿中的甲基化發(fā)生情況,評(píng)價(jià)MPDZ甲基化檢測(cè)在肺癌診斷中的價(jià)值。3.證實(shí)MPDZ基因?qū)δ[瘤細(xì)胞增殖、周期和凋亡、侵襲和轉(zhuǎn)移的作用;闡明MPDZ發(fā)揮抑癌作用的分子信號(hào)通路。方法:1.利用COSMIC和cBioPortal在線軟件分析MPDZ基因在正常組織中的表達(dá);同時(shí)分析MPDZ基因在腫瘤中突變的發(fā)生情況和突變的主要形式。2.利用TCGA數(shù)據(jù)庫,分析MPDZ基因在肝癌、腎透明細(xì)胞癌(ccRCC)和肺癌中的拷貝數(shù)變異和基因表達(dá)情況及其兩者之間的關(guān)系;Kaplan-Meier方法分析拷貝數(shù)變異和基因表達(dá)對(duì)腫瘤患者生存預(yù)后的影響。3.利用MSP檢測(cè)MPDZ基因在肺癌組織、痰液和血漿中的甲基化發(fā)生情況,同時(shí)結(jié)合TCGA數(shù)據(jù)庫評(píng)價(jià)MPDZ基因甲基化在肺癌診斷中的潛在生物學(xué)價(jià)值。4.利用qRT-PCR和WB檢測(cè)肺癌細(xì)胞系和HBE細(xì)胞中MPDZ基因的表達(dá),同時(shí)利用免疫組織化學(xué)方法分析MPDZ蛋白在腫瘤組織和癌旁組織中的表達(dá),利用Kaplan-Meier方法分析MPDZ蛋白表達(dá)與肺癌患者生存預(yù)后的關(guān)系。5.通過細(xì)胞模型和動(dòng)物模型分析MPDZ基因在腫瘤中的作用功能,利用MTS分析細(xì)胞增殖情況、流式細(xì)胞儀分析細(xì)胞周期的改變和細(xì)胞凋亡的發(fā)生、Transwell和劃痕愈合實(shí)驗(yàn)分析腫瘤細(xì)胞侵襲轉(zhuǎn)移能力、裸鼠移植瘤模型評(píng)價(jià)體內(nèi)腫瘤生長(zhǎng)情況。6.利用qRT-PCR、WB、基因轉(zhuǎn)染、RNAi技術(shù)和免疫熒光等多種分子生物學(xué)技術(shù)研究MPDZ在腫瘤中作用的分子機(jī)制,并確定在腫瘤中發(fā)揮作用的主要信號(hào)通路。結(jié)果:1.MPDZ基因在不同器官組織中的表達(dá)存在一定差異,其中在血液系統(tǒng)表達(dá)較低,在神經(jīng)系統(tǒng)表達(dá)較高;且該基因在多數(shù)腫瘤中發(fā)生突變,以彌漫性B細(xì)胞淋巴瘤中突變發(fā)生率最高(16%)。2.MPDZ基因在肝癌和ccRCC中發(fā)生明顯的拷貝數(shù)變異,主要的形式為拷貝數(shù)缺失,且拷貝數(shù)缺失影響該基因的表達(dá);拷貝數(shù)缺失的ccRCC患者的生存時(shí)間顯著短于拷貝數(shù)正常的患者(P0.01)。3.MPDZ基因在肺癌中發(fā)生了明顯的甲基化,在腫瘤組織的甲基化檢出率為69.4%,腫瘤組織發(fā)生甲基化的患者,其痰液和血漿的檢出率分別為46.5%和76.7%;MPDZ基因啟動(dòng)子CpG島中存在三個(gè)CG位點(diǎn):cg14655855、cg06202228和cg10899099,且肺癌組織中的甲基化水平顯著高于癌旁組織(P0.01);三個(gè)CG位點(diǎn)及其平均值ROC曲線下面積AUC在0.80~0.84之間,95%CI在0.74~0.88之間,是一個(gè)靈敏度和特異度較好的肺癌臨床診斷指標(biāo)。4.MPDZ基因在肺癌組織和多數(shù)肺癌細(xì)胞系中低表達(dá),且該基因表達(dá)受甲基化的調(diào)控;MPDZ基因表達(dá)與患者的生存期呈負(fù)相關(guān),且該蛋白表達(dá)是一個(gè)獨(dú)立的預(yù)后因素。5.MPDZ基因高表達(dá)可以通過促而進(jìn)細(xì)胞凋亡抑制肺癌細(xì)胞的增殖,同時(shí)可以抑制肺癌細(xì)胞的侵襲和轉(zhuǎn)移;而干擾MPDZ表達(dá)后可以促進(jìn)肺癌細(xì)胞的增殖、侵襲和轉(zhuǎn)移。6.MPDZ基因發(fā)揮抑癌作用依賴于hippo-YAP通路,干擾YAP后,肺癌細(xì)胞的遷移明顯抑制。MPDZ蛋白可以與Hippo通路中的多數(shù)蛋白相互作用;MPDZ基因高表達(dá)后可以抑制YAP基因及其下游靶分子CTGF和CYR61的表達(dá),而干擾MPDZ表達(dá)后則可以促進(jìn)YAP基因及其下游靶分子CTGF和CYR61的表達(dá);同時(shí)MPDZ基因差異表達(dá)可以影響YAP蛋白的總量。結(jié)論:MPDZ基因在多數(shù)腫瘤中發(fā)生突變,但突變率較低,單純基因突變并不影響腫瘤患者的生存預(yù)后;在肝癌和ccRCC中MPDZ基因拷貝數(shù)缺失影響該基因的表達(dá)以及患者的生存預(yù)后;MPDZ基因在肺癌中發(fā)生明顯的甲基化,且該基因甲基化是一個(gè)較好的肺癌臨床診斷生物標(biāo)志物;MPDZ基因在肺癌中低表達(dá),其表達(dá)與肺癌患者的生存時(shí)間呈正相關(guān),是肺癌患者的一個(gè)獨(dú)立預(yù)后因素;MPDZ基因抑制腫瘤細(xì)胞的增殖、侵襲和轉(zhuǎn)移;MPDZ基因在腫瘤中發(fā)揮抑癌作用依賴于Hippo-YAP通路。
[Abstract]:Background: for a long time, the incidence and mortality of malignant tumors remain high, which seriously threaten human life and health. More and more studies have confirmed that abnormal changes in genetics and epigenetics play an important role in the regulation of the expression and regulation of oncogene and tumor suppressor genes, the copy number variation of tumor suppressor genes and / or promoter CpG island. The expression inactivation induced by methylation is closely related to the occurrence of tumor. Therefore, by looking for the variation of the copy number of tumor suppressor genes and the change of DNA methylation in the process of tumor occurrence, the new thought of.MPDZ gene for the clinical diagnosis and targeting therapy of malignant tumor is a new one in the early stage of our study on lung cancer. Methylation regulates the candidate tumor suppressor gene, but its role and mechanism in lung cancer and other tumors is not clear. Therefore, this subject is intended to evaluate the clinical significance of MPDZ in tumor development by analyzing the clinical features of the MPDZ gene in the tumor. At the same time, the function and molecular mechanism of the gene in the tumor are analyzed in order to follow up. In-depth study of the biological significance of the gene in the carcinogenesis and the effective treatment of tumor treatment. Objective: 1. to clarify the mutation of the MPDZ gene in the tumor, the occurrence of copy number and methylation and its clinical significance, and to analyze the effect of MPDZ gene methylation and expression on the survival prognosis of the tumor patients.2. clear MPDZ gene in the lung Methylation in cancer tissue, sputum and plasma, evaluation of the value of MPDZ methylation in the diagnosis of lung cancer.3. confirms the role of MPDZ gene in tumor cell proliferation, cycle and apoptosis, invasion and metastasis, and clarifies the molecular signaling pathway for MPDZ to play the role of tumor suppressor. Method: 1. using COSMIC and cBioPortal online software to analyze MPDZ genes. The expression in normal tissues; the analysis of the mutation of the MPDZ gene in the tumor and the main form of the mutation.2. using the TCGA database to analyze the copy number and gene expression of the MPDZ gene in the liver cancer, the renal clear cell carcinoma (ccRCC) and the lung cancer and the relationship between the gene expression and their relationship; the Kaplan-Meier method analyses the copy number variation. The effect of gene expression on survival prognosis of tumor patients.3. using MSP to detect the methylation of MPDZ gene in lung cancer tissue, sputum and plasma, and to evaluate the potential biological value of MPDZ gene methylation in the diagnosis of lung cancer combined with TCGA database.4. using qRT-PCR and WB to detect the MPDZ gene in lung cancer cell lines and HBE cells. The expression of MPDZ protein in tumor tissues and adjacent tissues was analyzed by immunohistochemistry. The relationship between the expression of MPDZ protein and the survival prognosis of lung cancer patients was analyzed by Kaplan-Meier method. The function of the MPDZ gene in the tumor was analyzed by the cell model and animal model, and the proliferation of the cells was analyzed by MTS. The cell proliferation was analyzed by MTS. Flow cytometry analysis of cell cycle changes and apoptosis, Transwell and scratch healing test analysis of tumor cell invasion and metastasis ability, nude mouse model of transplanted tumor evaluation of tumor growth in vivo.6. using qRT-PCR, WB, gene transfection, RNAi technology and immunofluorescence and other molecular biology techniques, MPDZ in the tumor The molecular mechanism of action and the main signaling pathway to play a role in the tumor. Results: the expression of 1.MPDZ gene in different organ tissues is different, in which the expression of the blood system is low, the expression of the gene is higher in the nervous system, and the gene is mutated in most of the tumors, and the mutation rate in the diffuse B cell lymphoma The highest (16%).2.MPDZ gene has obvious copy number variation in liver cancer and ccRCC. The main form is the deletion of copy number, and the deletion of the copy number affects the expression of the gene. The survival time of the ccRCC patients with the missing copy number is significantly shorter than that of the patients with normal copy number (P0.01).3.MPDZ gene is obviously methylation in lung cancer. The detectable rate of methylation in tumor tissue was 69.4%. The detection rates of sputum and plasma were 46.5% and 76.7% in the patients with tumor tissue methylation, three CG loci in MPDZ gene promoter CpG Island: cg14655855, cg06202228 and cg10899099, and the level of methylation in lung cancer tissues was significantly higher than that of the para cancerous tissue (P0.01); three CG loci were found. The area AUC under the average ROC curve is between 0.80~0.84 and 95%CI in 0.74~0.88, which is a diagnostic index of sensitivity and specificity of lung cancer, the.4.MPDZ gene is low expressed in lung cancer tissues and most lung cancer cell lines, and the expression of the gene is regulated by methylation, and the expression of MPDZ gene is negatively correlated with the survival time of the patients. The expression of this protein is an independent prognostic factor, the high expression of.5.MPDZ gene can inhibit the proliferation of lung cancer cells by promoting apoptosis and inhibit the invasion and metastasis of lung cancer cells, and the interference of MPDZ expression can promote the proliferation of lung cancer cells, and the invasion and metastasis of.6.MPDZ genes play an important role in inhibiting the cancer of the lung cancer cells. After the interference of YAP, the migration of lung cancer cells obviously inhibits the interaction of.MPDZ protein with most of the proteins in the Hippo pathway, and the expression of CTGF and CYR61 in the YAP gene and its downstream target molecules can be inhibited after the high expression of the MPDZ gene, and the expression of CTGF and CYR61 in the YAP gene and its downstream target molecules can be promoted after the interference of MPDZ expression. The differential expression of Z gene can affect the total amount of YAP protein. Conclusion: the MPDZ gene is mutated in most tumors, but the mutation rate is low. The mutation of the simple gene does not affect the survival prognosis of the tumor patients. The deletion of the MPDZ gene in the hepatocellular carcinoma and ccRCC affects the expression of the gene and the survival prognosis of the patients; the MPDZ gene is in the middle of the lung cancer. The methylation of the gene is a good biomarker for the clinical diagnosis of lung cancer. The expression of MPDZ gene is low in lung cancer, and its expression is positively correlated with the survival time of the lung cancer patients. It is an independent prognostic factor for lung cancer patients; the MPDZ gene inhibits the proliferation, invasion and metastasis of the tumor cells; the MPDZ gene is in the tumor. The role of cancer suppressor is dependent on the Hippo-YAP pathway.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 封杰;劉文斌;陳洪強(qiáng);黃永勝;韓飛;曹佳;劉晉yN;;肺癌患者M(jìn)PDZ基因異常甲基化的研究[J];癌變·畸變·突變;2015年06期

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本文編號(hào)：1954320