靶向AURKA的siRNA抑制喉癌HEp-2细胞生长的体内外研究

发布时间：2018-03-07 00:06

  本文选题：Aurora激酶　切入点：喉肿瘤　出处：《复旦大学》2011年博士论文　论文类型：学位论文

【摘要】：目的：研究Aurora激酶A (Aurora Kinase A, AURKA)在喉鳞状细胞癌中的表达及其临床意义,研究AURKA基因沉默在体外体内对喉癌HEp-2细胞生长的影响及其机制。 方法：收集37例喉鳞状细胞癌组织和邻近正常组织,应用实时荧光RT-PCR和Western blot技术检测AURKA mRNA和蛋白在喉癌及邻近正常组织中的表达,分析AURKA mRNA和蛋白与喉癌临床病理特征的关系。 构建针对AURKA的shRNA表达质粒并转染喉鳞癌细胞株HEp-2细胞,检测HEp-2细胞转染前后AURKA mRNA和蛋白表达,CCK-8实验检测细胞增殖,Transwell实验检测细胞侵袭,软琼脂实验观察克隆形成能力,流式细胞术检测细胞周期分布、凋亡和有丝分裂监控点,免疫荧光实验观察细胞间桥、多极纺锤体和多中心体等染色体不稳定性事件,Western blot检测细胞粘附和迁移的重要因子粘着斑激酶(FAK)和基质金属蛋白酶-2(MMP-2)蛋白表达。另外,将AURKA基因敲除的HEp-2细胞(HEp-2-S)接种于裸鼠,观察裸鼠移植瘤生长情况以及瘤体内FAK和MMP-2的表达,最后,将AURKA抑制剂VX-680注射入荷瘤裸鼠,观察VX-680对肿瘤生长的影响。 结果：32例(86.5%)病人肿瘤中的AURKA mRNA高于相应的喉正常黏膜,AURKA mRNA在喉癌组织中表达上调,显著高于喉正常黏膜组织中的表达。25对喉癌组织及其喉正常黏膜中,其中有16个(64.0%)病例AURKA蛋白T/N比值1.2,提示在喉癌组织中AURKA蛋白呈高表达。AURKA mRNA上调和蛋白的高表达与患者颈部淋巴结转移显著相关,AURKA mRNA还与临床Ⅲ/Ⅳ分期显著相关,而与患者性别、年龄、肿瘤部位、T分期和病理分级无相关性。喉癌HEp-2细胞中AURKA蛋白也呈高表达。 HEp-2细胞转染AURKA shRNA后,AURKA mRNA和蛋白表达明显下降。AURKA沉默后,抑制HEp-2细胞增殖活性、侵袭和克隆形成能力,以及裸鼠体内成瘤能力,使HEp-2细胞停滞于G2/M期,最终细胞凋亡；同时,AURKA沉默后,HEp-2细胞有丝分裂监测点被激活,减少HEp-2细胞染色体的不稳定性；此外,AURKA沉默后,在体外体内实验中,粘着斑激酶(FAK)、磷酸化FAK和基质金属蛋白酶-2(MMP-2)表达降低；最后,单独使用VX-680注射入荷瘤裸鼠,肿瘤生长虽然有所减慢,但是与对照组相比差异无显著性。 结论：在喉鳞状细胞癌中AURKA呈高表达,与颈部淋巴结转移和Ⅲ/Ⅳ分期相关,AURKA基因沉默在体外体内均能抑制喉癌HEp-2细胞的生长和侵袭,并与FAK、磷酸化FAK和MMP-2表达降低有关,本研究为靶向AURKA的喉癌基因治疗提供重要的理论依据。
[Abstract]:Aim: to study the expression and clinical significance of Aurora kinase A Aurora Kinase A (AURKAA) in laryngeal squamous cell carcinoma (LSCC), and to investigate the effect of AURKA gene silencing on the growth of HEp-2 cells in vitro and its mechanism. Methods: 37 cases of laryngeal squamous cell carcinoma and adjacent normal tissues were collected and the expression of AURKA mRNA and protein in laryngeal carcinoma and adjacent normal tissues was detected by real-time fluorescence RT-PCR and Western blot. To analyze the relationship between AURKA mRNA and protein and clinicopathological features of laryngeal carcinoma. ShRNA expression plasmid for AURKA was constructed and transfected into laryngeal squamous cell carcinoma (HEp-2) cell line. The expression of AURKA mRNA and protein in HEp-2 cells before and after transfection were detected by CCK-8 assay, cell proliferation was detected by Transwell assay, and clone formation ability was observed by soft Agar assay. Cell cycle distribution, apoptotic and mitotic monitoring sites were detected by flow cytometry, and intercellular bridges were observed by immunofluorescence assay. Chromosomal instability events, such as multipolar spindle and polycentrosome, were used to detect the expression of focal adhesion kinase (FAK) and matrix metalloproteinase-2 (MMP-2) protein, which are important factors of cell adhesion and migration. In addition, AURKA knockout HEp-2 cells were inoculated into nude mice. The growth of transplanted tumor and the expression of FAK and MMP-2 in nude mice were observed. Finally, AURKA inhibitor VX-680 was injected into nude mice to observe the effect of VX-680 on tumor growth. Results the expression of AURKA mRNA in tumor of 32 patients with normal laryngeal mucosa was higher than that of normal laryngeal mucosa, and the expression of Aurka mRNA in laryngeal carcinoma was significantly higher than that in normal laryngeal mucosa and normal laryngeal mucosa. The T / N ratio of AURKA protein was 1.2 in 16 patients with laryngeal carcinoma, indicating that the up-regulation of AURKA protein and the high expression of AURKA mRNA were significantly correlated with cervical lymph node metastasis, and also correlated with clinical stage 鈪，

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