【摘要】：目的:檢測喉癌組織及細胞中miRNA-21的表達。構建反義微小RNAhsa-miR-21慢病毒表達載體,觀察反義寡核苷酸重組慢病毒（ASO-miR-21）靶向調節(jié)Ras基因對喉癌細胞增殖、細胞周期、凋亡和抑制其侵襲的影響。 方法:應用實時熒光定量PCR檢測喉癌組織及細胞中miRNA-21的表達。構建反義hsa-miR-21慢病毒表達載體，體外轉染喉鱗癌Hep-2細胞，采用MTT法及克隆形成實驗檢測反義miR-21（ASO-miR-21）重組慢病毒對喉癌細胞的增殖能力；應用流式細胞儀檢測喉鱗癌細胞細胞周期及凋亡情況。采用Boydenchambers法檢測喉鱗癌細胞的侵襲能力。采用免疫印跡法檢測RAS蛋白在喉鱗癌細胞中的表達。 結果:PCR檢測在喉癌組織與細胞中，miRNA-21表達顯著增高，并與臨床病理分期及分級相關。成功構建反義hsa-miR-21的慢病毒表達載體，采用ASO-miR-21慢病毒抑制喉癌細胞內高水平表達miR-21的活性后,可顯著抑制喉癌細胞增殖、誘導凋亡，及阻滯Hep-2細胞周期在G1期，并抑制其侵襲。Westernblot檢測轉染ASO-miR-21后的喉鱗癌細胞發(fā)現(xiàn)，Ras蛋白表達較對照組明顯減少。 結論:本研究發(fā)現(xiàn)喉癌組織與細胞中miRNA-21表達顯著增高，，轉染miR-21ASO可以明顯抑制喉癌細胞增殖、誘導凋亡，及阻滯Hep-2細胞周期在G1期，并抑制其侵襲。同時發(fā)現(xiàn)，miRNA-21能調節(jié)的Ras基因表達，參與其調控網(wǎng)絡。從而說明miR-21的發(fā)揮致癌作用，為喉癌及其他惡性腫瘤的基因治療提供的一個有效的靶標和理論依據(jù)。
[Abstract]:Objective: to detect the expression of miRNA-21 in laryngeal carcinoma tissues and cells. The expression vector of antisense RNAhsa-miR-21 lentivirus was constructed, and the effects of Ras gene targeted regulation by antisense oligonucleotide recombinant lentivirus (ASO-miR-21) on the proliferation, cell cycle, apoptosis and inhibition of invasion of laryngeal carcinoma cells were observed. Methods: real-time fluorescence quantitative PCR was used to detect the expression of miRNA-21 in laryngeal carcinoma tissues and cells. The antisense hsa-miR-21 lentivirus expression vector was constructed and transfected into laryngeal squamous cell carcinoma (Hep-2) cells in vitro. The proliferation ability of antisense miR-21 (ASO-miR-21) recombinant lentivirus on laryngeal carcinoma cells was detected by MTT assay and clone formation assay. The cell cycle and apoptosis of laryngeal squamous cell carcinoma were detected by flow cytometry. The invasive ability of laryngeal squamous carcinoma cells was detected by Boydenchambers method. The expression of RAS protein in laryngeal squamous cell carcinoma cells was detected by Western blot. Results: the expression of miRNA-21 was significantly increased in laryngeal carcinoma tissues and cells by PCR, and correlated with clinicopathological stage and grade. The lentivirus expression vector of antisense hsa-miR-21 was successfully constructed. After ASO-miR-21 lentivirus inhibited the activity of high level expression of miR-21 in laryngeal carcinoma cells, it could significantly inhibit the proliferation and induce apoptosis of laryngeal carcinoma cells. Westernblot assay showed that the expression of Ras protein in laryngeal squamous carcinoma cells after ASO-miR-21 transfection was significantly lower than that in the control group. Conclusion: the expression of miRNA-21 was significantly increased in laryngeal carcinoma tissues and cells. Transfection of miR-21ASO could significantly inhibit the proliferation of laryngeal cancer cells, induce apoptosis, and block the cell cycle of Hep-2 in G1 phase and inhibit its invasion. At the same time, it was found that miRNA-21 can regulate the expression of Ras gene and participate in its regulatory network. Therefore, miR-21 can play a carcinogenic role and provide an effective target and theoretical basis for gene therapy of laryngeal cancer and other malignant tumors.
【學位授予單位】：哈爾濱醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.65

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