人AURKA基因RNA干扰慢病毒载体的构建及其对A549细胞增殖的影响

Construction of the lentiviral vector containing the interfering RNA targeting against human AURKA gene andits effect on the proliferation of A549 cells

目的:针对AURKA基因构建RNA干扰重组慢病毒质粒并进行慢病毒包装,初步探讨AURKA基因的功能。方法:应用p GCLV-GFP慢病毒载体构建针对AURKA的shRNA载体,转染包装293T细胞,收集病毒上清液,转染肺腺癌细胞株A549。荧光显微镜下观察绿色荧光蛋白表达情况确定转染效率,应用Western blot技术检测AURKA基因在蛋白水平表达的变化,以明确RNAi的抑制率。应用Brd U法分析干扰AURKA前后A549细胞增殖情况的变化,运用克隆形成实验检测干扰AURKA后A549细胞株对长春新碱敏感性的变化。结果:针对AURKA基因的RNAi慢病毒表达载体构建成功,PCR和DNA测序鉴定实验表明插入位点与干扰片断的碱基序列完全正确。在293T细胞中进行慢病毒包装,获得高滴度病毒上清液。通过重组慢病毒载体将AURKA shRNA转导入A549细胞中,转染效率为100%。Western blot检测证实LV-AURKA慢病毒显著抑制AURKA的表达,Brd U检测显示AURKA基因表达下调抑制了A549细胞的增殖。克隆形成实验表明AURKA基因表达增强A549细胞对长春新碱的敏感性。结论:慢病毒载体介导的靶向AURKA的RNA干扰可有效抑制AURKA表达,降低肺腺癌A549细胞的增殖能力,并且增强A549细胞对长春新碱的敏感性。

Objective：To construct and package the recombination lentiviral vector containing the interfering RNA gene targeting against human AURKA gene, and preliminary investigate the AURKA gene function. Methods：The recombination pGCLV-GFP lentiviral vector plasmid containing the interfering RNA gene targeting against human AURKA gene was transfected into the 293T cells, then which was packaged. The viral supernatant was collected to transfect A549 cells. The transfection efficiency was determined by detecting the GFP expression under the fluorescent microscopy. The expression level of AURKA protein was examined by Western blot for identifying the inhibitory efficiency of RNAi. The effect of AURKA silencd by RNAi on the proliferation of A549 cell was analyzed by BrdU assay. Colony formation assay was used to explore the sensitivity of the transfected cells to Vincristine. Results：The recombination lentiviral vector containing the interfering RNA gene targeting against human AURKA gene was successfully constructed, and the sequence and insert site of the interference segment were confirmed by PCR and DNA sequencing. The lentivirus was packaged in 293T cells,and the high titer virus supernatant was obtained. The transfection efficiency of small hairpin RNA（shRNA） targeting AURKA into A459 cells was 100%. The result of Western blot showed that the LV-AURKA could significantly inhibit the expression of AURKA protein,the result of BrdU showed down-regulating of AURKA gene could inhibit the proliferation of A549 cells, and the colony formation assay showed that the expression of AURKA gene could enhance the sensitivity of A549 cells to vincristine. Conclusions：The interfering RNA gene targeting against human AURKA mediated by lentiviral vector can effectively inhibit the AURKA gene expression, decreases the proliferation ability of A549 cells, and enhance the sensitivity of A549 cells to vincristine.