【作者】 罗朝辉；

【导师】 李桂源； 向娟娟；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2012， 博士

【摘要】 MicroRNAs (miRNAs,小非编码RNA)能够与下游靶基因mRNA的3’UTR碱基配对并引导沉默复合物(RISC)降解mRNA或抑制：mRNA的翻译,参与生物合成和肿瘤的发展,其生物学功能机制是目前研究的热点领域之一。miRNA组学(microRNomics)属于基因组学的分支,包括miRNAs差异筛选、动态表达、分子结构、表达调控、靶基因预测及生物学功能分析等研究。鼻咽癌(Nasopharyngeal carcinoma, NPC)发生、发展及转移是一个多步骤多因素参与的复杂过程。迄今为止,对鼻咽癌组织进行niRNA分子差异表达谱分析的研究也是十分有限。在本论文中,我们旨在阐明鼻咽癌不同临床阶段的miRNA组学时空变化趋势以及miR-18a调控miRNAs组学作用的研究机制。[鼻咽癌不同临床阶段miRNA组学动态表达规律]利用显微切割分离纯化不同临床阶段的鼻咽癌组织标本和对照鼻咽上皮标本,采用illumina的miRNA芯片分析,MultiClassDif统计软件筛选出48个miRNAs分子在鼻咽癌不同临床阶段组学中表达存在差异。进一步运用生物学软件cluster3.0软件,将差异miRNA组的表达分为6种动态模式：其中3类差异miRNA组的模式归纳为不同的下降模式；另外3类差异miRNA组是不同的上升模式。进而运用targetScan软件,分析并预测差异miRNAs组靶基因,用数据库ftp://ftp.ncbi.nih.gov/repository/UniGene/组织特异性筛选差异miRNA组靶基因。然后与NCBI中鼻咽癌cDNA数据库(GEO: GSE12452)数据整合,得到以Smad2等为代表的差异miRNAs组的靶基因,并通过real-time PCR验证部分(?)niRNAs和靶基因在不同临床分期的鼻咽癌组织标本和对照样本中的表达。通过GO和pathway分析,上调差异miRNAs组的靶基因GO功能主要集中在粘附、凋亡和细胞死亡；下调差异miRNAs组的靶基因GO功能主要集中在细胞增殖、死亡和凋亡；上调差异miRNAs组的靶基因pathway主要集中在Adherens junction、Focal adhesion等与肿瘤转移相关的pathway,而下调差异miRNAs组的靶基因pathway主要集中在Pathways in cancer等。利用miRNAs与靶基因的负相关性,绘制了差异miRNAs组与靶基因的niRNAs-Genes网络图。miRNAs调控除了直接受Dicerl和Drosha的作用外,还受到转录因子的调控,因此,我们利用UCSC寻找差异miRNAs编码基因在基因组上的位置,确定ETS2等转录因子在调控miRNAs编码基因表达发挥循环反馈调控作用；并绘制差异miRNAs和转录因子的调控网络关系。为鼻咽癌发生、发展的分子机制研究提供新的思路。[MiR-18a具有促进鼻咽癌增殖、迁移和侵袭转移功能]MiRNA芯片筛选发现(?)niR-18a在鼻咽癌不同临床分期及淋巴结转移癌中存在显著差异。利用real-time PCR证实miR-18a随着临床分期进展而表达逐渐增加,并且在鼻咽癌细胞中高表达,原位杂交结果显示miR-18a与鼻咽癌的临床分期、淋巴结转移、EBV感染以及预后相关。MTT、伤口愈合实验以及transwell基质胶侵袭实验证明miR-18a过表达分别能够促进鼻咽癌细胞HK1、5-8F和6-10B的增殖、迁移能力和侵袭转移能力；反之抑制miR-18a则减少或降低鼻咽癌细胞的增殖、迁移能力和侵袭转移能力。采用慢病毒系统构建miR-18a的过表达和knockdown的稳定鼻咽癌HKl细胞系并通过real-time PCR验证。采用活体荧光示踪技术模拟miR-18a在裸鼠体内的促进鼻咽癌增殖和转移模型,揭示miR-18a能够促进裸鼠体内皮下移植瘤的增殖,miR-18a能够促进鼻咽癌细胞在裸鼠体内转移能力。[MiR-18a通过Dicerl促进鼻咽癌发展的致癌机制]通过targetScan等软件预测、以及荧光素酶报告基因实验证明Dicerl是miR-18a的靶基因,并采用real-time和western验证miR-18a能降低内源性Dicerl的表达。通过miRNA芯片miRNA Dicerl检测发现miR-18a能够下调78%的其他表达,恢复则逆转80%的miRNA的表达。其中miR-18a下调miR-143和miR-200家族表达最为显著。通过niR-18a处理Dicer1的过表达和knockdown的细胞,从正反两面证实miR-18a作为一个癌基因通过Dicer1发挥生物学功能。real-time PCR和western验证miR-18a可以调控miR-200家族及EMT标志物分子的变化；另外验证miR-18a通过Dicer1调控miR-143及靶基因K-Ras的表达,促进鼻咽癌的发展。综上所述,我们通过miRNA芯片筛选到一组与不同临床分期发展及淋巴转移可能起关键作用的miRNAs,揭示了鼻咽癌不同临床阶段的差异miRNAs组学变化趋势,通过与网络GEO数据整合,阐述了鼻咽癌差异miRNAs的靶基因的GO和pathway分析,进一步分析差异miRNAs表达调控的因素,利用USCS数据寻找调控差异miRNAs组学的转录因子以及他们之间的反馈调控关系。阐述了miR-18a能够促进鼻咽癌细胞的增殖,侵袭转移功能及促进裸鼠体内的成瘤速度和转移转移能力。揭示niR-18a通过Dicerl调节miRNAs组学的表达,通过生物学功能实验证明miR-18a通过Dicer1促进鼻咽癌的发生发展。阐述了miR-18a与niR-200家族和miR-143以及其他miRNAs之间的关系,揭示了miR-18a与Dicerl在鼻咽癌的临床分期、淋巴结转移、EBV感染以及预后的相关性。更多还原

【Abstract】 MicroRNAs,(or ’miRNAs’, which are small noncoding RNA molecules), can bind to the complementary sequences in the3’UTR of multiple target mRNAs by regulating mRNA stability and translation through the action of the RNA-induced silencing complex (RISC), miRNAs have been a hotspot in research for their involvement in biological processes and tumour development."MicroRNomics" is to describe a novel subdiscipline of genomics that studies including the identification, expression, structure, expression regulation, targets, and biological functions of miRNAs. The tumorigenesis and development of Nasopharyngeal Carcinoma (NPC) is a multistep process involving multiple genetic and environment factors. However, there has been limited research on microRNomics and the interaction of miRNAs in NPC. In this study, we are aiming to elucidate the spatiotemporal roles of miRNAs in different clinical stages of NPC and related regulatory mechanism on the microRNomics scale. We are also going to study the regulatory roles of miR-18a on the microRNomics of NPC.[The dynamic expression of miRNomics in Multi-stages of Nasopharyngeal Carcinoma]Laser capture microdissection (LCM) was used to separate the cancer tissues from the normal tissues. Illumina’s miRNA expression arrays were used for searching the differentially expressed miRNAs between the NPC and control samples.48miRNAs with significant change were obtained by the MultiClassDif statistical software. The differentially expressed miRNAs were clustered into the six expression models by the biology software cluster3.0software based on their similar expression patterns,3of which were divided into down-regulated patterns in Multi-stages and Lymphoid node metastasis of Nasopharyngeal Carcinoma; and3of which were divided into up-regulated models. The targetScan software was used for searching and predicting the target genes of these differentially expressed miRNAs. As more than thousands target genes were predicted and predicted target gene lists were then narrowed down by searching the data from ftp://ftp.ncbi.nih.gov/repository/UniGene/to find the nasopharyngeal-specific gene. We further performed data reduction strategy to overlay these nasopharyngeal-specific target genes with cDNA expression data (GEO:GSE12452). As the results, several representative target genes were selected, some of which were verified by real-time PCR in different clinical stages samples. The selected target genes were analyzed in the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway. The most enriched GO terms in the predicted target genes of up-regulated miRNAs were cell adhesion, apoptosis and cell death; in the predicted target genes of down-regulated miRNAs the enriched GO terms were focused on cell proliferation, death and apoptosis. The target genes of up-regulated miRNAs were mainly involoved in the adherens junction, Focal adhesion, which were related with cancer metastasis pathway. The target genes of down-regulated miRNAs were mostly implicated in pathways in cancer. Based on the inverse correlation of miRNAs and their target genes, we built miRNAs-Genes interactions into a bipartite network. The expression of miRNAs is directly regulated not only by Drosha and Dicer1, but also by transcription factors (TF). We then used UCSC Genome database to analyze the promoter regions of miRNA genes for the transcription factor (TF) binding sites. We then constructed TF-miRNA networks. We further investigated the TF-miRNAs-target genes feedback loop by integrating the TF-miRNA networks and miRNA-target genes networks. We found that some TFs such as ETS2could regulate the expression of miRNA expression and the miRNAs in turn suppress the expression of TF, forming feedback loops. These works will provide new ideas for the research in nasopharyngeal carcinoma.[MiR-18a promotes nasopharyngeal carcinoma cell growth, migration and invasion]MiR-18a was found significantly highly expressed in multistages and lymphoid node metastasis of NPC by using miRNAs array. The differential expression of miR-18a was verified by real-time PCR in the NPC samples and cancer cell lines. The in situ hybridization revealed that the expression of miR-18a was correlated with the clinical stages, the lymph node metastasis, EBV infection and prognosis of NPC patients. MTT, wound healing and transwell with matrix assays proved that miR-18a promoted HK1,5-8F and6-10B cell proliferation, mobility, invasion and metastasis ability, while inhibiting the expression of miR-18a decreased the ability of NPC cell to proliferate, migrate, invade and metastase. We then constructed the miR-18a overexpressed and knockdown cell lines by lentivirus based vectors. The efficency of transfection was verified by real-time PCR. In vivo roles of miR-18a in the cell growth and migration were assessed by the tumor formation following subcutaneous or intravenous injection into nude mice. The nude mice formed bigger subcutaneous tumors in mice which the cells overexpressed with miR-18a compared to the control group. The mobility and metastasis of cells in vivo were examined at different time points by using the IVIS imaging system (xenogen).[The carcinogenic mechanism of miR-18a mediated by Dicerl in Nasopharyngeal Carcinoma]We used three TargetScan to search for miRNA binding sites in the3’UTR of Dicerl. The luciferase assay, real-time PCR and western blot were performed to confirm that miR-18a binds to the3’UTR of Dicerl, suppressing the endogenous expression of Dicerl in NPC cell lines HK1and5-8F. MiRNA array assay were performed to investigate the miRNA profiles change caused by miR-18a. The overexpression of miR-18a caused78%globally downregulated expression of miRNAs and the global downregulation of miRNAs were restored by overexpression of Dicer1. MiR-200families and miR-143were shown significantly suppressed by miR-18a in the miRNA array. The oncogenic roles of miR-18a through targeting Dicer1were verified in the Dicer1overexpressed and knockdown cell. Real-time PCR and western blot assay showed that miR-18a can regulate the expression miR-200families and the EMT biomarkers. It also showed that miR-18a can regulate the expression of miR-143and its target gene K-Ras expression, accelerating the development of NPC.As described aboved, we obtained a group of miRNAs that may play important roles on development of NPC by using the data reduction strategies and we also revealed the dynamic expression profiles of these miRNAs. The selected target genes were analyzed in the GO biological process and KEGG biological pathway. We further investigated the mechanisms of miRNAs expression regulation by analyzing the TF binding sites of miRNA genes using UCSC Genome database. We also investigated the miRNAs expression regulation by miR-18a. MiR-18a showed obvious promoting effect on tumorigenesis of NPC by targeting Dicerl, in turn impairing the biogenesis of microRNome. In this paper, we elucidate the relationship of miR-18a and miR-200families and miR-143. The clinical features of NPC such as clinical stages of NPC, EB virus infection were also shown correlated to the expression of miR-18a.更多还原