【摘要】：目的微小RNA (microRNA, miRNA)是一類廣泛存在的在轉(zhuǎn)錄后水平調(diào)節(jié)基因表達(dá)的小分子非編碼RNA,參與多種生理和病理過(guò)程。近來(lái)研究表明, miRNAs主要是通過(guò)特異性識(shí)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(guò)程中發(fā)揮重要作用。前期本實(shí)驗(yàn)室利用寡核苷酸微陣列技術(shù),發(fā)現(xiàn)在人類喉鱗狀細(xì)胞癌組織中microRNA-1(miR-1)表達(dá)水平顯著下調(diào)。本課題進(jìn)一步研究了miR-1對(duì)喉鱗狀細(xì)胞癌HEp2細(xì)胞生長(zhǎng)表型的影響,并預(yù)測(cè)及驗(yàn)證了miR-1直接作用的靶基因,以期探尋miR-1對(duì)喉鱗狀細(xì)胞癌發(fā)生和發(fā)展作用的分子機(jī)制。 方法在人類喉鱗狀細(xì)胞癌細(xì)胞系HEp2細(xì)胞中,過(guò)表達(dá)和封閉內(nèi)源性miR-1的功能后,利用MTT實(shí)驗(yàn)和克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞生長(zhǎng)活性的變化。而后綜合利用生物信息學(xué)方法和cDNA微陣列技術(shù)篩選miR-1的候選靶基因,并利用熒光報(bào)告載體實(shí)驗(yàn)驗(yàn)證miR-1對(duì)靶基因的直接調(diào)控作用。通過(guò)real-time PCR、Western blot和免疫熒光實(shí)驗(yàn)檢測(cè)miR-1過(guò)表達(dá)和封閉后的喉鱗癌HEp2細(xì)胞中靶基因mRNA和蛋白水平的表達(dá)變化,進(jìn)一步驗(yàn)證miR-1對(duì)靶基因的調(diào)控作用。同時(shí),采用RNA干擾(RNA interference, RNAi)技術(shù)在HEp2細(xì)胞中沉默靶基因的表達(dá),檢測(cè)其對(duì)細(xì)胞生長(zhǎng)表型的影響。 結(jié)果在人類喉鱗癌HEp2細(xì)胞中,過(guò)表達(dá)miR-1的初始轉(zhuǎn)錄本pri-1后,細(xì)胞生長(zhǎng)明顯受到抑制,而且克隆形成能力明顯降低；而以反義互補(bǔ)的寡核苷酸(ASO-1)封閉miR-1后,細(xì)胞生長(zhǎng)活性增加,克隆形成能力也有所上升。靶基因篩選結(jié)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(jié)合位點(diǎn)。熒光報(bào)告載體實(shí)驗(yàn)表明,miR-1能夠通過(guò)作用于FN1基因3'-UTR的特定結(jié)合位點(diǎn),對(duì)其表達(dá)進(jìn)行負(fù)性調(diào)節(jié)。此外,real-time PCR、Western blot和免疫熒光實(shí)驗(yàn)證明,過(guò)表達(dá)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(shù)沉默內(nèi)源性FN1后,細(xì)胞的生長(zhǎng)活性和克隆形成能力下降,這與過(guò)表達(dá)miR-1的結(jié)果相一致。 結(jié)論在人類喉鱗狀細(xì)胞癌HEp2細(xì)胞中,miR-1能夠通過(guò)抑制細(xì)胞的生長(zhǎng)活性,起到抑癌基因的作用；miR-1通過(guò)靶定FN1基因的3'-UTR區(qū)調(diào)節(jié)喉鱗癌HEp2細(xì)胞的生長(zhǎng)活性；FN1可促進(jìn)細(xì)胞的生長(zhǎng)活性,可能發(fā)揮了癌基因的功能。通過(guò)研究miR-1在喉鱗癌細(xì)胞系中的具體作用機(jī)制,有利于我們對(duì)惡性腫瘤的發(fā)生和發(fā)展進(jìn)行深入地了解,同時(shí)也為miRNAs作為腫瘤早期診斷和治療的分子標(biāo)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.65

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