| 题名 | 新的肺癌相关基因MCRS1病理生理功能及其机制研究 |
| 作者 | 刘敏霞 |
| 学位类别 | 博士 |
| 答辩日期 | 2013-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 曹毅 |
| 关键词 | 非小细胞肺癌 MCRS1 异常高表达 细胞增生 上皮间质转化（EMT） 分子机制 |
| 其他题名 | Pathophysiological function of oncogenic MCRS1 and its regulation mechanism in non-small cell lung cancer |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 肺癌是一种常见的恶性肿瘤。随着吸烟和环境污染影响的增加，肺癌的发病率和病死率均迅速上升。肺癌已成为我国恶性肿瘤首位死亡原因。肿瘤细胞分子水平的改变， 如染色体、基因的改变，已被认为是恶性肿瘤发生的最主要机理。寻找新的肺癌相关基因——癌基因和抑癌基因，对深入研究肺癌发生、发展的病理生理机制，发现新的诊断标记物和治疗靶点有实际意义。在前期工作中，我们结合细胞遗传学和分子遗传学方法，对肺癌细胞系进行染色体分析，发现12q13区域异常。本研究中我们在12q13区域约 14M 范围内不同位点选择20个基因，分别在肺癌细胞系和组织中对其mRNA表达水平进行定量RT-PCR分析。经筛选后，我们发现2个基因mRNA在肺癌组织中表达降低；而9个基因mRNA表达升高。在9个上调基因中，RACGAP1，MCRS1，EIF4B，WNT1和PTGES3 5个基因在正常肺组织中不表达或痕量表达，在肺癌细胞系和肺癌肿瘤组织中高表达。微小球蛋白MCRS1 在肺癌组织中表达水平升高尤为明显，统计学分析有非常显著差异（p<0.001）。免疫组织化学染色和免疫印迹分析进一步证实， MCRS1蛋白水平在肺癌细胞和组织也确有升高。共聚焦荧光显微镜观察发现在正常的永生化支气管上皮细胞中，MCRS1清晰定位于中心体；而在培养的肺癌细胞，MCRS1的分布明显不同。MCRS1异常表达可能与肺癌发生发展相关。MCRS1定位于细胞核，参与基因转录调节、细胞转化和恶性肿瘤发生及发展等过程。基于MCRS1在非小细胞肺癌中异常表达，我们应用RNA interference技术降低MCRS1的表达，研究其对肿瘤的生长和转移两大主要特征的影响。（1）通过MTT检测细胞增殖、流式细胞术分析细胞周期分布和Annexin V-FITC/PI双染检测细胞凋亡，其结果显示MCRS1沉默可明显的抑制肺癌细胞的增殖、促进细胞凋亡并且延缓细胞周期进程。（2）系列体内外实验显示，MCRS1沉默可以逆转上皮间质转化（EMT）过程、降低肿瘤的侵袭和转移能力。进一步发现，MCRS1干扰后，可以削弱肿瘤细胞对顺铂和西妥昔单抗的耐药性，并减少肿瘤干细胞样CD44+ 细胞的产生。这些结果表明，MCRS1在肺癌发生和发展过程中发挥重要作用，可能是一个新的肺癌相关基因。其次，我们从mRNA和microRNA（miRNA）两个方面探索MCRS1促进肺癌侵袭和转移的分子机制。应用mRNA表达谱芯片检测和miRNA测序技术，分别筛选出MCRS1干扰后差异表达的mRNA和miRNA，并结合生物信息学分析和系列实验验证，其结果显示：（1）MCRS1可以调节细胞连接分子（ZO-1, Occludin和E-cadherin 等）和NOTCH3的表达，表明MCRS1通过调节细胞连接和Notch等多种信号通路促进肿瘤侵袭和转移。（2）MCRS1可以上调肿瘤相关基因miR-155表达；进一步正反干预实验均显示，miR-155可以转化MCRS1对细胞增殖和侵袭影响。这些研究结果提示，MCRS1可能通过调节一系列重要细胞连接分子表达及活化miR-155等多种途径促进肺癌发生和发展。最后，我们亦研究了MCRS1在非小细胞肺癌中的高表达的调控机制。（1）通过实时定量PCR检测MCRS1 DNA拷贝数和mRNA表达水平，结果发现：与非永生化支气管上皮细胞16HBE相比，MCRS1 DNA拷贝数和mRNA水平在非小细胞肺癌细胞系中发生扩增；与正常非癌肺组织相比，MCRS1 DNA拷贝数和mRNA水平在非小细胞肺癌组织也均增加。（2）miRNA表达谱测序显示，与肺永生化支气管上皮细胞16HBE相比，在EPLC-32M1、801D和A549肺癌细胞系中共有87个差异表达miRNAs。联合miRNA表达谱结果和miRNA 靶基因预测分析，发现miR-129*可以直接作用于MCRS1。实验证实，miR-129*与MCRS1表达呈相反趋势；用miR-129* mimics处理肺癌细胞，MCRS1 mRNA和蛋白水平均表达下调， 同时细胞增殖和侵袭能力明显降低。这些结果提示，基因拷贝数改变和miR-129*下调导致MCRS1在非小细胞肺癌中异常高表达。通过研究MCRS1在非小细胞肺癌中的功能及其表达调控机制，有助于深入了解其病理及生理功能，并可能为抑制肿瘤转移和克服耐药性开辟新途径。 |
| 英文摘要 | Lung cancer is the most common type of cancer in the world. With the increase of smoking and environment pollution, the morbidity and mortality rate of lung cancer are growing rapidly. Lung cancer has become the leading cause of cancer-related mortality in China. Both tumor suppressor genes and oncogenes are involved in the pathogenesis and progression of non-small cell lung cancer (NSCLC). Analyse of genetic abnormalities in tumors have been proved helpful in searching for new tumor suppressor genes and oncogenes. Chromosomal aberrations associated with mutations of tumor suppressor genes or gene amplifications of dominant oncogenes are common genetic abnormalities in tumors, including lung cancer.Previously, we showed, using karyotypic and loss of heterozygosity (LOH) analyses, that chromosome arm 12q abnormalities occurred in NSCLCs. Here, we examined mRNA expression of 20 genes within chromosome band 12q13 by quantitative real-time polymerase chain reaction in NSCLCs. Of these 20 genes, nine were upregulated, while two were downregulated. Among the 9 upregulated genes, mRNA values of RACGAP1, MCRS1, EIF4B, WNT1, and PTGES3 were significantly higher in NSCLCs compared with that from normal lung tissue. Subsequently, overexpression of microspherule protein 1(MCRS1) was confirmed at the protein level in tissues and cultured cells of lung cancer by immunostaining and Western blot. Interestingly, MCRS1 exhibits different localization in the mitotic cells of cultured immortalized human bronchial epithelial cells and lung cancer cells. These findings indicate that MCRS1 may be a novel cancer-related gene in NSCLC.MCRS1 is localized to the nucleus with known roles in transcription regulation, cellular transformation, and tumorigenesis. As MCRS1 overexpression in NSCLCs, we investigated its roles in NSCLC by RNAi and examined tumor growth, migration and invasion. (1) We found that MCRS1 silencing inhibited cell proliferation, increased apoptosis, and induced cell cycle arrest at the G1 phase in lung cancer cells. (2) MCRS1 knockdown also induced the morphological alteration, increased the monolayer integrity, decreased cellular invasion and metastasis, and attenuated stemness and drug resistance (cisplatin and cetuximab) in NSCLC cells. The levels of MCRS1 mRNA expression were associated with the tumor metastasis in NSCLC patients. These findings suggested that MCRS1 could play vital roles in the development and progression of lung cancer.To further explore molecular targets associated with MCRS1 activity in NSCLCs，we determined mRNA or microRNA (miRNA) expression profiles of cultured cells with or without RNAi mediated MCRS1 knockdown. (1) Downstream effectors of MCRS1 that were clarified by analysis of mRNA profiles and confirmed by the q-RT-PCR after MCRS1 knockdown, included cell junction molecules such as ZO-1, occluding, E-cadherin, and DSG2, as well as Notch family. (2) MCRS1 regulated miR-155 expression through assessing microRNA (miRNA) profiles after the MCRS1 silencing. RNAi experiments also confirmed that miR-155 expression could be induced by MCRS1 over-expression, and the enforced expression of miR-155 recapitulated effects of MCRS1 on the cell growth and invasion. Altogether, these results indicated that MCRS1 over-expression promoted the epithelial-mesenchymal transition (EMT) program and tumor invasion/metastasis in NSCLCs via influence on up-regulation of miR-155 and down-regulation of cell junction molecules.We also investigated the molecular mechanisms underlying MCRS1 alteration. (1) We analyzed MCRS1 DNA copy numbers. Alterations of MCRS1 DNA copy numbers were consistent with levels of MCRS1 mRNA, and MCRS1 DNA copy numbers were dramatically elevated in NSCLC tissues and culture cells compared with non-cancerous lung tissues and culture immortalized human bronchial epithelial cells, respectively. (2) Based on systematic studies including analysis of miRNA profiles, bioinformational prediction, comparison of mi |
| 语种 | 中文 |
| 公开日期 | 2013-12-31 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7762]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 刘敏霞. 新的肺癌相关基因MCRS1病理生理功能及其机制研究[D]. 北京. 中国科学院研究生院. 2013. |
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