本文選題：MicroRNAs(miRNAs) + miRNAs組學��； 參考：《中南大學》2012年博士論文

【摘要】：MicroRNAs (miRNAs,小非編碼RNA)能夠與下游靶基因mRNA的3’UTR堿基配對并引導沉默復合物(RISC)降解mRNA或抑制：mRNA的翻譯,參與生物合成和腫瘤的發(fā)展,其生物學功能機制是目前研究的熱點領域之一。miRNA組學(microRNomics)屬于基因組學的分支,包括miRNAs差異篩選、動態(tài)表達、分子結構、表達調控、靶基因預測及生物學功能分析等研究。鼻咽癌(Nasopharyngeal carcinoma, NPC)發(fā)生、發(fā)展及轉移是一個多步驟多因素參與的復雜過程。迄今為止,對鼻咽癌組織進行niRNA分子差異表達譜分析的研究也是十分有限。在本論文中,我們旨在闡明鼻咽癌不同臨床階段的miRNA組學時空變化趨勢以及miR-18a調控miRNAs組學作用的研究機制。 [鼻咽癌不同臨床階段miRNA組學動態(tài)表達規(guī)律] 利用顯微切割分離純化不同臨床階段的鼻咽癌組織標本和對照鼻咽上皮標本,采用illumina的miRNA芯片分析,MultiClassDif統計軟件篩選出48個miRNAs分子在鼻咽癌不同臨床階段組學中表達存在差異。進一步運用生物學軟件cluster3.0軟件,將差異miRNA組的表達分為6種動態(tài)模式：其中3類差異miRNA組的模式歸納為不同的下降模式；另外3類差異miRNA組是不同的上升模式。進而運用targetScan軟件,分析并預測差異miRNAs組靶基因,用數據庫ftp://ftp.ncbi.nih.gov/repository/UniGene/組織特異性篩選差異miRNA組靶基因。然后與NCBI中鼻咽癌cDNA數據庫(GEO: GSE12452)數據整合,得到以Smad2等為代表的差異miRNAs組的靶基因,并通過real-time PCR驗證部分(?)niRNAs和靶基因在不同臨床分期的鼻咽癌組織標本和對照樣本中的表達。通過GO和pathway分析,上調差異miRNAs組的靶基因GO功能主要集中在粘附、凋亡和細胞死亡；下調差異miRNAs組的靶基因GO功能主要集中在細胞增殖、死亡和凋亡；上調差異miRNAs組的靶基因pathway主要集中在Adherens junction、Focal adhesion等與腫瘤轉移相關的pathway,而下調差異miRNAs組的靶基因pathway主要集中在Pathways in cancer等。利用miRNAs與靶基因的負相關性,繪制了差異miRNAs組與靶基因的niRNAs-Genes網絡圖。miRNAs調控除了直接受Dicerl和Drosha的作用外,還受到轉錄因子的調控,因此,我們利用UCSC尋找差異miRNAs編碼基因在基因組上的位置,確定ETS2等轉錄因子在調控miRNAs編碼基因表達發(fā)揮循環(huán)反饋調控作用；并繪制差異miRNAs和轉錄因子的調控網絡關系。為鼻咽癌發(fā)生、發(fā)展的分子機制研究提供新的思路。 [MiR-18a具有促進鼻咽癌增殖、遷移和侵襲轉移功能] MiRNA芯片篩選發(fā)現(?)niR-18a在鼻咽癌不同臨床分期及淋巴結轉移癌中存在顯著差異。利用real-time PCR證實miR-18a隨著臨床分期進展而表達逐漸增加,并且在鼻咽癌細胞中高表達,原位雜交結果顯示miR-18a與鼻咽癌的臨床分期、淋巴結轉移、EBV感染以及預后相關。MTT、傷口愈合實驗以及transwell基質膠侵襲實驗證明miR-18a過表達分別能夠促進鼻咽癌細胞HK1、5-8F和6-10B的增殖、遷移能力和侵襲轉移能力；反之抑制miR-18a則減少或降低鼻咽癌細胞的增殖、遷移能力和侵襲轉移能力。采用慢病毒系統構建miR-18a的過表達和knockdown的穩(wěn)定鼻咽癌HKl細胞系并通過real-time PCR驗證。采用活體熒光示蹤技術模擬miR-18a在裸鼠體內的促進鼻咽癌增殖和轉移模型,揭示miR-18a能夠促進裸鼠體內皮下移植瘤的增殖,miR-18a能夠促進鼻咽癌細胞在裸鼠體內轉移能力。 [MiR-18a通過Dicerl促進鼻咽癌發(fā)展的致癌機制] 通過targetScan等軟件預測、以及熒光素酶報告基因實驗證明Dicerl是miR-18a的靶基因,并采用real-time和western驗證miR-18a能降低內源性Dicerl的表達。通過miRNA芯片miRNA Dicerl檢測發(fā)現miR-18a能夠下調78%的其他表達,恢復則逆轉80%的miRNA的表達。其中miR-18a下調miR-143和miR-200家族表達最為顯著。通過niR-18a處理Dicer1的過表達和knockdown的細胞,從正反兩面證實miR-18a作為一個癌基因通過Dicer1發(fā)揮生物學功能。real-time PCR和western驗證miR-18a可以調控miR-200家族及EMT標志物分子的變化；另外驗證miR-18a通過Dicer1調控miR-143及靶基因K-Ras的表達,促進鼻咽癌的發(fā)展。 綜上所述,我們通過miRNA芯片篩選到一組與不同臨床分期發(fā)展及淋巴轉移可能起關鍵作用的miRNAs,揭示了鼻咽癌不同臨床階段的差異miRNAs組學變化趨勢,通過與網絡GEO數據整合,闡述了鼻咽癌差異miRNAs的靶基因的GO和pathway分析,進一步分析差異miRNAs表達調控的因素,利用USCS數據尋找調控差異miRNAs組學的轉錄因子以及他們之間的反饋調控關系。闡述了miR-18a能夠促進鼻咽癌細胞的增殖,侵襲轉移功能及促進裸鼠體內的成瘤速度和轉移轉移能力。揭示niR-18a通過Dicerl調節(jié)miRNAs組學的表達,通過生物學功能實驗證明miR-18a通過Dicer1促進鼻咽癌的發(fā)生發(fā)展。闡述了miR-18a與niR-200家族和miR-143以及其他miRNAs之間的關系,揭示了miR-18a與Dicerl在鼻咽癌的臨床分期、淋巴結轉移、EBV感染以及預后的相關性。
[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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R739.63

【參考文獻】

相關期刊論文 前1條

1 羅朝輝;張李洋;李征;江琛;代亞飛;劉曉萍;鄭瑛;俞海波;向娟娟;李桂源;;miR-149促進鼻咽癌細胞侵襲和上皮-間質轉變(英文)[J];中南大學學報(醫(yī)學版);2011年07期

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本文編號：1949769