【摘要】：目的微小RNA (microRNA, miRNA)是一類廣泛存在的在轉錄后水平調(diào)節(jié)基因表達的小分子非編碼RNA,參與多種生理和病理過程。近來研究表明, miRNAs主要是通過特異性識別信使RNAs (messenger RNAs, mRNAs)的3,-非翻譯區(qū)(3'-untranslated regions,3'-UTRs)發(fā)揮調(diào)節(jié)作用的。其中,某些miRNA還具有癌基因或抑癌基因的活性,在腫瘤發(fā)生和發(fā)展過程中發(fā)揮重要作用。前期本實驗室利用寡核苷酸微陣列技術,發(fā)現(xiàn)在人類喉鱗狀細胞癌組織中microRNA-1(miR-1)表達水平顯著下調(diào)。本課題進一步研究了miR-1對喉鱗狀細胞癌HEp2細胞生長表型的影響,并預測及驗證了miR-1直接作用的靶基因,以期探尋miR-1對喉鱗狀細胞癌發(fā)生和發(fā)展作用的分子機制。 方法在人類喉鱗狀細胞癌細胞系HEp2細胞中,過表達和封閉內(nèi)源性miR-1的功能后,利用MTT實驗和克隆形成實驗檢測細胞生長活性的變化。而后綜合利用生物信息學方法和cDNA微陣列技術篩選miR-1的候選靶基因,并利用熒光報告載體實驗驗證miR-1對靶基因的直接調(diào)控作用。通過real-time PCR、Western blot和免疫熒光實驗檢測miR-1過表達和封閉后的喉鱗癌HEp2細胞中靶基因mRNA和蛋白水平的表達變化,進一步驗證miR-1對靶基因的調(diào)控作用。同時,采用RNA干擾(RNA interference, RNAi)技術在HEp2細胞中沉默靶基因的表達,檢測其對細胞生長表型的影響。 結果在人類喉鱗癌HEp2細胞中,過表達miR-1的初始轉錄本pri-1后,細胞生長明顯受到抑制,而且克隆形成能力明顯降低；而以反義互補的寡核苷酸(ASO-1)封閉miR-1后,細胞生長活性增加,克隆形成能力也有所上升。靶基因篩選結果表明,纖維連接蛋白1(fibronectin1, FN1)是miR-1的候選靶基因,其3’-非翻譯區(qū)(3'-untranslated region,3'-UTR)包含miR-1的潛在結合位點。熒光報告載體實驗表明,miR-1能夠通過作用于FN1基因3'-UTR的特定結合位點,對其表達進行負性調(diào)節(jié)。此外,real-time PCR、Western blot和免疫熒光實驗證明,過表達miR-1可以下調(diào)靶基因的mRNA和蛋白水平,而封閉miR-1后,靶基因的mRNA和蛋白水平均有升高。采用RNAi技術沉默內(nèi)源性FN1后,細胞的生長活性和克隆形成能力下降,這與過表達miR-1的結果相一致。 結論在人類喉鱗狀細胞癌HEp2細胞中,miR-1能夠通過抑制細胞的生長活性,起到抑癌基因的作用；miR-1通過靶定FN1基因的3'-UTR區(qū)調(diào)節(jié)喉鱗癌HEp2細胞的生長活性；FN1可促進細胞的生長活性,可能發(fā)揮了癌基因的功能。通過研究miR-1在喉鱗癌細胞系中的具體作用機制,有利于我們對惡性腫瘤的發(fā)生和發(fā)展進行深入地了解,同時也為miRNAs作為腫瘤早期診斷和治療的分子標記物提供新的線索。
[Abstract]:Objective microRNAs are a class of small non-coding RNAs that regulate gene expression at posttranscriptional level and participate in many physiological and pathological processes. Recent studies have shown that miRNAs plays a regulatory role mainly by specifically identifying the 3'-untranslated regions of RNAs (messenger RNAs, mRNAs). Among them, some miRNA also have the activity of oncogene or tumor suppressor gene, which plays an important role in tumorigenesis and development. Using oligonucleotide microarray technique, we found that the expression of microRNA-1 (miR-1) was significantly down-regulated in human laryngeal squamous cell carcinoma (LSCC). The aim of this study was to investigate the effect of miR-1 on the growth phenotype of HEp2 cells in laryngeal squamous cell carcinoma (LSCC), and to predict and verify the target genes directly acting on miR-1, in order to explore the molecular mechanism of miR-1 in the pathogenesis and development of laryngeal squamous cell carcinoma (LSCC). Methods after overexpression and blocking the function of endogenous miR-1 in human laryngeal squamous cell carcinoma cell line HEp2, the changes of cell growth activity were detected by MTT assay and clone formation assay. Then the candidate target genes of miR-1 were screened by bioinformatics and cDNA microarray, and the direct regulation of target genes by miR-1 was verified by fluorescence report vector experiment. The expression of target gene mRNA and protein in laryngeal squamous cell carcinoma (LSCC) cells was detected by real-time PCR Western blot and immunofluorescence assay. It was further proved that miR-1 can regulate the target gene in laryngeal squamous cell carcinoma (LSCC) cells. At the same time, RNA interference (RNA interference, RNAi) technique was used to detect the expression of silencing target gene in HEp2 cells and its effect on cell growth phenotype. Results in human laryngeal squamous cell carcinoma (HEp2) cells, the cell growth was significantly inhibited and the clone formation ability was significantly decreased after overexpression of the initial transcribed pri-1 of miR-1, while the cell growth activity was increased after blocking miR-1 with antisense oligodeoxynucleotides (ASO-1). Cloning ability also increased. Target gene screening showed that fibronectin 1 (FN1) is a candidate target gene for miR-1, and its 3'-untranslated region 3G UTR contains the potential binding sites of miR-1. Fluorescence report vector experiments showed that the expression of FN1 gene 3'-UTR could be negatively regulated by the action of its specific binding site. In addition, Western blot and immunofluorescence assay showed that overexpression of miR-1 could down-regulate the mRNA and protein levels of the target gene, but the mRNA and protein levels of the target gene increased after blocking miR-1. After silencing endogenous FN1 by RNAi technique, the cell growth activity and clone formation ability decreased, which was consistent with the result of over-expression of miR-1. Conclusion in human laryngeal squamous cell carcinoma (HEp2) cells, miR-1 can inhibit the growth activity of human laryngeal squamous cell carcinoma (HEp2) cells by inhibiting the cell growth activity and acting as a tumor suppressor gene. MiR-1 can promote the growth activity of laryngeal squamous cell carcinoma HEp2 cells by targeting the 3'-UTR region of FN1 gene. It may function as a oncogene. By studying the specific mechanism of miR-1 in laryngeal squamous cell carcinoma cell line, it is helpful for us to understand the occurrence and development of malignant tumor and to provide new clues for miRNAs as a molecular marker for early diagnosis and treatment of cancer.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R739.65

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