MicroRNA-21在喉癌中的表达及其对喉癌侵袭与凋亡的调节

发布时间：2019-01-01 20:53

【摘要】：目的:检测喉癌组织及细胞中miRNA-21的表达。构建反义微小RNAhsa-miR-21慢病毒表达载体,观察反义寡核苷酸重组慢病毒（ASO-miR-21）靶向调节Ras基因对喉癌细胞增殖、细胞周期、凋亡和抑制其侵袭的影响。方法:应用实时荧光定量PCR检测喉癌组织及细胞中miRNA-21的表达。构建反义hsa-miR-21慢病毒表达载体，体外转染喉鳞癌Hep-2细胞，采用MTT法及克隆形成实验检测反义miR-21（ASO-miR-21）重组慢病毒对喉癌细胞的增殖能力；应用流式细胞仪检测喉鳞癌细胞细胞周期及凋亡情况。采用Boydenchambers法检测喉鳞癌细胞的侵袭能力。采用免疫印迹法检测RAS蛋白在喉鳞癌细胞中的表达。结果:PCR检测在喉癌组织与细胞中，miRNA-21表达显著增高，并与临床病理分期及分级相关。成功构建反义hsa-miR-21的慢病毒表达载体，采用ASO-miR-21慢病毒抑制喉癌细胞内高水平表达miR-21的活性后,可显著抑制喉癌细胞增殖、诱导凋亡，及阻滞Hep-2细胞周期在G1期，并抑制其侵袭。Westernblot检测转染ASO-miR-21后的喉鳞癌细胞发现，Ras蛋白表达较对照组明显减少。结论:本研究发现喉癌组织与细胞中miRNA-21表达显著增高，，转染miR-21ASO可以明显抑制喉癌细胞增殖、诱导凋亡，及阻滞Hep-2细胞周期在G1期，并抑制其侵袭。同时发现，miRNA-21能调节的Ras基因表达，参与其调控网络。从而说明miR-21的发挥致癌作用，为喉癌及其他恶性肿瘤的基因治疗提供的一个有效的靶标和理论依据。
[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.
【学位授予单位】：哈尔滨医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.65

【共引文献】