microRNA-1在喉鳞状细胞癌HEp2细胞中靶向调节fibronectin1基因
发布时间：2018-08-21 19:33
【摘要】：目的微小RNA (microRNA, miRNA)是一类广泛存在的在转录后水平调节基因表达的小分子非编码RNA,参与多种生理和病理过程。近来研究表明, miRNAs主要是通过特异性识别信使RNAs (messenger RNAs, mRNAs)的3,-非翻译区(3'-untranslated regions,3'-UTRs)发挥调节作用的。其中,某些miRNA还具有癌基因或抑癌基因的活性,在肿瘤发生和发展过程中发挥重要作用。前期本实验室利用寡核苷酸微阵列技术,发现在人类喉鳞状细胞癌组织中microRNA-1(miR-1)表达水平显著下调。本课题进一步研究了miR-1对喉鳞状细胞癌HEp2细胞生长表型的影响,并预测及验证了miR-1直接作用的靶基因,以期探寻miR-1对喉鳞状细胞癌发生和发展作用的分子机制。 方法在人类喉鳞状细胞癌细胞系HEp2细胞中,过表达和封闭内源性miR-1的功能后,利用MTT实验和克隆形成实验检测细胞生长活性的变化。而后综合利用生物信息学方法和cDNA微阵列技术筛选miR-1的候选靶基因,并利用荧光报告载体实验验证miR-1对靶基因的直接调控作用。通过real-time PCR、Western blot和免疫荧光实验检测miR-1过表达和封闭后的喉鳞癌HEp2细胞中靶基因mRNA和蛋白水平的表达变化,进一步验证miR-1对靶基因的调控作用。同时,采用RNA干扰(RNA interference, RNAi)技术在HEp2细胞中沉默靶基因的表达,检测其对细胞生长表型的影响。 结果在人类喉鳞癌HEp2细胞中,过表达miR-1的初始转录本pri-1后,细胞生长明显受到抑制,而且克隆形成能力明显降低；而以反义互补的寡核苷酸(ASO-1)封闭miR-1后,细胞生长活性增加,克隆形成能力也有所上升。靶基因筛选结果表明,纤维连接蛋白1(fibronectin1, FN1)是miR-1的候选靶基因,其3’-非翻译区(3'-untranslated region,3'-UTR)包含miR-1的潜在结合位点。荧光报告载体实验表明,miR-1能够通过作用于FN1基因3'-UTR的特定结合位点,对其表达进行负性调节。此外,real-time PCR、Western blot和免疫荧光实验证明,过表达miR-1可以下调靶基因的mRNA和蛋白水平,而封闭miR-1后,靶基因的mRNA和蛋白水平均有升高。采用RNAi技术沉默内源性FN1后,细胞的生长活性和克隆形成能力下降,这与过表达miR-1的结果相一致。 结论在人类喉鳞状细胞癌HEp2细胞中,miR-1能够通过抑制细胞的生长活性,起到抑癌基因的作用；miR-1通过靶定FN1基因的3'-UTR区调节喉鳞癌HEp2细胞的生长活性；FN1可促进细胞的生长活性,可能发挥了癌基因的功能。通过研究miR-1在喉鳞癌细胞系中的具体作用机制,有利于我们对恶性肿瘤的发生和发展进行深入地了解,同时也为miRNAs作为肿瘤早期诊断和治疗的分子标记物提供新的线索。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.65
本文编号：2196137
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.65
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