MPDZ基因在肿瘤中作用及机制的初步研究

发布时间：2018-05-30 06:41

本文选题：MPDZ + 肺癌　；参考：《第三军医大学》2017年硕士论文

【摘要】：背景:长期以来,恶性肿瘤的发病率和死亡率一直居高不下,严重威胁着人类的生命健康。越来越多的研究证实,遗传学和表观遗传学的异常改变在癌基因和抑癌基因的表达调控中起着重要的作用,抑癌基因拷贝数变异和(或)启动子CpG岛甲基化导致的表达失活与肿瘤的发生密切相关。因此,通过寻找肿瘤发生过程中抑癌基因拷贝数变异和DNA甲基化变化的规律,将为恶性肿瘤的临床诊断和靶向治疗方式提供新的思路。MPDZ基因是我们前期在肺癌研究中筛选出的一个新的甲基化调控候选抑癌基因,但是其在肺癌以及其他肿瘤中的作用和机制还不清楚,因此本课题拟通过对MPDZ基因在肿瘤中的临床特征分析,初步评价其在肿瘤发生中的临床意义;同时,全面分析MPDZ基因在肿瘤中的功能和分子机制,为后续深入研究该基因在肿瘤发生中的生物学意义和有效的肿瘤治疗提供资料。目的:1.明确MPDZ基因在肿瘤中的突变、拷贝数变异和甲基化的发生情况及其临床意义;解析MPDZ基因甲基化和表达对肿瘤患者生存预后的影响。2.明确MPDZ基因在肺癌组织、痰液和血浆中的甲基化发生情况,评价MPDZ甲基化检测在肺癌诊断中的价值。3.证实MPDZ基因对肿瘤细胞增殖、周期和凋亡、侵袭和转移的作用;阐明MPDZ发挥抑癌作用的分子信号通路。方法:1.利用COSMIC和cBioPortal在线软件分析MPDZ基因在正常组织中的表达;同时分析MPDZ基因在肿瘤中突变的发生情况和突变的主要形式。2.利用TCGA数据库,分析MPDZ基因在肝癌、肾透明细胞癌(ccRCC)和肺癌中的拷贝数变异和基因表达情况及其两者之间的关系;Kaplan-Meier方法分析拷贝数变异和基因表达对肿瘤患者生存预后的影响。3.利用MSP检测MPDZ基因在肺癌组织、痰液和血浆中的甲基化发生情况,同时结合TCGA数据库评价MPDZ基因甲基化在肺癌诊断中的潜在生物学价值。4.利用qRT-PCR和WB检测肺癌细胞系和HBE细胞中MPDZ基因的表达,同时利用免疫组织化学方法分析MPDZ蛋白在肿瘤组织和癌旁组织中的表达,利用Kaplan-Meier方法分析MPDZ蛋白表达与肺癌患者生存预后的关系。5.通过细胞模型和动物模型分析MPDZ基因在肿瘤中的作用功能,利用MTS分析细胞增殖情况、流式细胞仪分析细胞周期的改变和细胞凋亡的发生、Transwell和划痕愈合实验分析肿瘤细胞侵袭转移能力、裸鼠移植瘤模型评价体内肿瘤生长情况。6.利用qRT-PCR、WB、基因转染、RNAi技术和免疫荧光等多种分子生物学技术研究MPDZ在肿瘤中作用的分子机制,并确定在肿瘤中发挥作用的主要信号通路。结果:1.MPDZ基因在不同器官组织中的表达存在一定差异,其中在血液系统表达较低,在神经系统表达较高;且该基因在多数肿瘤中发生突变,以弥漫性B细胞淋巴瘤中突变发生率最高(16%)。2.MPDZ基因在肝癌和ccRCC中发生明显的拷贝数变异,主要的形式为拷贝数缺失,且拷贝数缺失影响该基因的表达;拷贝数缺失的ccRCC患者的生存时间显著短于拷贝数正常的患者(P0.01)。3.MPDZ基因在肺癌中发生了明显的甲基化,在肿瘤组织的甲基化检出率为69.4%,肿瘤组织发生甲基化的患者,其痰液和血浆的检出率分别为46.5%和76.7%;MPDZ基因启动子CpG岛中存在三个CG位点:cg14655855、cg06202228和cg10899099,且肺癌组织中的甲基化水平显著高于癌旁组织(P0.01);三个CG位点及其平均值ROC曲线下面积AUC在0.80~0.84之间,95%CI在0.74~0.88之间,是一个灵敏度和特异度较好的肺癌临床诊断指标。4.MPDZ基因在肺癌组织和多数肺癌细胞系中低表达,且该基因表达受甲基化的调控;MPDZ基因表达与患者的生存期呈负相关,且该蛋白表达是一个独立的预后因素。5.MPDZ基因高表达可以通过促而进细胞凋亡抑制肺癌细胞的增殖,同时可以抑制肺癌细胞的侵袭和转移;而干扰MPDZ表达后可以促进肺癌细胞的增殖、侵袭和转移。6.MPDZ基因发挥抑癌作用依赖于hippo-YAP通路,干扰YAP后,肺癌细胞的迁移明显抑制。MPDZ蛋白可以与Hippo通路中的多数蛋白相互作用;MPDZ基因高表达后可以抑制YAP基因及其下游靶分子CTGF和CYR61的表达,而干扰MPDZ表达后则可以促进YAP基因及其下游靶分子CTGF和CYR61的表达;同时MPDZ基因差异表达可以影响YAP蛋白的总量。结论:MPDZ基因在多数肿瘤中发生突变,但突变率较低,单纯基因突变并不影响肿瘤患者的生存预后;在肝癌和ccRCC中MPDZ基因拷贝数缺失影响该基因的表达以及患者的生存预后;MPDZ基因在肺癌中发生明显的甲基化,且该基因甲基化是一个较好的肺癌临床诊断生物标志物;MPDZ基因在肺癌中低表达,其表达与肺癌患者的生存时间呈正相关,是肺癌患者的一个独立预后因素;MPDZ基因抑制肿瘤细胞的增殖、侵袭和转移;MPDZ基因在肿瘤中发挥抑癌作用依赖于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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.2

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