鼻咽癌的miRNA组学动态表达特征及miR-18a通过Dicer1介导的致癌机制研究

发布时间：2018-05-29 06:00

  本文选题：MicroRNAs(miRNAs) + miRNAs组学　； 参考：《中南大学》2012年博士论文

【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。
[Abstract]:MicroRNAs (miRNAs, small non coding RNA) can match the 3 'UTR base of the downstream target gene mRNA and guide the silencing complex (RISC) to degrade mRNA or inhibit: mRNA translation, participation in biosynthesis and tumor development. Its biological function mechanism is one of the hot areas of current research,.MiRNA omics (microRNomics) belongs to genomics. Branch, including miRNAs differential screening, dynamic expression, molecular structure, expression and regulation, target gene prediction and biological function analysis. The occurrence, development and metastasis of nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is a complex process of multi step and multi factor participation. To date, the differential expression profiles of niRNA molecules in nasopharyngeal carcinoma tissues are analyzed. The study is also very limited. In this paper, we aim to elucidate the spatial and temporal trends of miRNA groups in different clinical stages of nasopharyngeal carcinoma and the mechanism of miR-18a regulating the role of miRNAs groups.
[miRNA dynamic expression pattern of nasopharyngeal carcinoma at different clinical stages]
The specimens of nasopharyngeal carcinoma in different clinical stages and the control of the nasopharyngeal epithelial specimens were separated and purified by microdissection. The Illumina miRNA chip was used to analyze the differences in the expression of 48 miRNAs molecules in different clinical stages of nasopharyngeal carcinoma. The cluster3.0 software of biological software was used to improve the expression of the 48 miRNAs molecules. The expression of the miRNA group is divided into 6 dynamic models: 3 types of differences in the miRNA group are divided into different modes of descent, and the other 3 types of miRNA groups are different ascending modes. Then targetScan software is used to analyze and predict the differential miRNAs target gene and to use the database ftp:// ftp.ncbi.nih.gov/repository/UniGene/ to organize the specificity. The target genes of different miRNA groups were screened and integrated with the data of the cDNA database (GEO: GSE12452) of nasopharyngeal carcinoma (GEO: GSE12452) in NCBI. The target genes of the differential miRNAs group represented by Smad2 were obtained, and the expression of some (?) niRNAs and target genes in the tissues of nasopharyngeal carcinoma and the control samples in different clinical stages were verified by real-time PCR. Through GO and pathway. The target gene GO function mainly focused on adhesion, apoptosis and cell death in the miRNAs group, and the target gene GO function mainly focused on cell proliferation, death and apoptosis in group miRNAs, and the target gene pathway mainly concentrated in Adherens junction, Focal adhesion, and path on tumor metastasis. The target gene pathway of the miRNAs group was mainly concentrated in the Pathways in cancer, and the negative correlation between the miRNAs and the target gene was used. The niRNAs-Genes network diagram of the difference miRNAs group and target gene was plotted, and the regulation of the.MiRNAs regulation of the difference miRNAs group and target gene was also regulated by the transcription factors. To find the location of the differential miRNAs coding gene in the genome, determine the role of ETS2 and other transcription factors to regulate the regulation of the miRNAs encoding gene expression, and to draw the regulatory network relationship between the differential miRNAs and the transcription factors. The molecular mechanism of the development of nasopharyngeal carcinoma is provided for the new idea.
[MiR-18a has the function of promoting proliferation, migration and invasion and metastasis of nasopharyngeal carcinoma.
MiRNA chip screening found significant differences in niR-18a in different clinical stages and lymph node metastasis of nasopharyngeal carcinoma. Real-time PCR showed that miR-18a increased with the progression of clinical staging, and expressed high expression in nasopharyngeal carcinoma cells. The results of in situ hybridization showed the clinical stage, lymph node metastasis, EBV of miR-18a and nasopharyngeal carcinoma, EBV Infection and prognosis related.MTT, wound healing experiment and Transwell matrix gel invasion test showed that miR-18a overexpression could promote the proliferation, migration and invasion and metastasis of nasopharyngeal carcinoma cells, respectively, and inhibit miR-18a to reduce or reduce the proliferation, migration and invasion and metastasis of nasopharyngeal carcinoma. Using the lentivirus system to construct the over expression of miR-18a and the stable nasopharyngeal carcinoma HKl cell line of knockdown and verified by real-time PCR. Using the living fluorescence tracing technique to simulate the proliferation and metastasis model of nasopharyngeal carcinoma in nude mice by miR-18a, which reveals that miR-18a can promote the proliferation of the transplanted tumor of nude mice, and miR-18a can promote nasopharynx. The metastatic ability of cancer cells in nude mice.
[MiR-18a promotes carcinogenesis of nasopharyngeal carcinoma through Dicerl]
Through targetScan and other software prediction, and the luciferase reporter gene experiment proved that Dicerl was the target gene of miR-18a, and real-time and Western proved that miR-18a could reduce the expression of endogenous Dicerl. MiRNA Dicerl detection by miRNA chip found that miR-18a could down 78% of other expressions, and the recovery reversed the expression of 80% of miRNA. MiR-18a down-regulation of miR-143 and miR-200 family expression is the most significant. Through niR-18a treatment of Dicer1 overexpression and knockdown cells, both positive and negative sides confirm that miR-18a acts as a oncogene through Dicer1 to play a biological function.Real-time PCR and Western verification miR-18a can regulate the changes in the family and biomarker molecules. It is proved that miR-18a can regulate the expression of miR-143 and target gene K-Ras through Dicer1, and promote the development of nasopharyngeal carcinoma.
To sum up, we screened a group of miRNAs which may play a key role in the development of different clinical stages and lymphatic metastasis by miRNA chip. We revealed the variation trend of miRNAs histopathological changes in different clinical stages of nasopharyngeal carcinoma. Through the integration of network GEO data, the GO and pathway analysis of the target genes of differential miRNAs in nasopharyngeal carcinoma were further elaborated. The factors that regulate the regulation of differential miRNAs expression are analyzed, and USCS data are used to find the transcription factors that regulate the differential miRNAs groups and the feedback regulation relationship between them. It is explained that miR-18a can promote the proliferation, invasion and metastasis of nasopharyngeal carcinoma cells, and promote the tumor formation speed and transfer and transfer ability in nude mice. It reveals that niR-18a passes through Dicerl modulation. The expression of miRNAs group studies shows that miR-18a promotes the occurrence and development of nasopharyngeal carcinoma through Dicer1. The relationship between miR-18a and niR-200 family and miR-143 and other miRNAs is expounded, and the correlation between miR-18a and Dicerl in the clinical stages, lymph node metastasis, EBV infection and prognosis of nasopharyngeal carcinoma is revealed.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63

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相关期刊论文 前1条

1 罗朝辉;张李洋;李征;江琛;代亚飞;刘晓萍;郑瑛;俞海波;向娟娟;李桂源;;miR-149促进鼻咽癌细胞侵袭和上皮-间质转变(英文)[J];中南大学学报(医学版);2011年07期

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