EGFL7基因RNAi重组慢病毒的构建及其对喉癌细胞侵袭的抑制  

Construction of lentivirus vector of interference of EGFL7 gene and its inhibitive role on the invasion of laryngeal cancer cell

作　　者：王晓侠[1] 姚小宝[1] 嵇宪生[2] 陈菁华[2] 李蕾[2] 朱宏亮[1] 

机构地区：[1]西安交通大学医学院第一附属医院耳鼻咽喉科,西安710061 [2]解放军451医院耳鼻咽喉科

出　　处：《临床耳鼻咽喉头颈外科杂志》2011年第24期1135-1138,1141,共5页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：陕西省卫生厅基金资助项目(No:2010D40)

摘　　要：目的:构建类表皮生长因子域7(EGFL7)基因RNA干扰(RNAi)重组慢病毒,观察其对喉癌体外侵袭的抑制作用。方法:筛选确定的EGFL7基因RNAi有效靶序列,合成靶序列的Oligo DNA,与含H1启动子和绿色荧光蛋白(GFP)的pLV载体连接产生LV-sh EGFL7慢病毒载体,PCR筛选阳性克隆,测序鉴定。用重组慢病毒体外转导喉癌细胞,Western blot法检测喉癌细胞EGFL7蛋白表达。Boyden侵袭小室法实验观察转导shRNA后的喉癌细胞侵袭能力的变化。利用克隆形成实验检测EGFL7基因沉默对Hep-2细胞集落形成能力的影响。结果:成功构建EGFL7的慢病毒载体LV-sh EGFL7,包装慢病毒,浓缩病毒悬液的滴度为5×108 TU/L。转导siRNA的喉癌细胞EGFL7蛋白表达阴性。EGFL7siRNA转导后喉癌胞体外侵袭能力下降。EGFL7基因沉默组细胞克隆集落形成率与Hep-2组细胞及空载体组细胞比较,细胞克隆集落形成率显著减低。结论:成功构建人EGFL7基因RNAi慢病毒载体,EGFL7基因沉默对喉癌细胞体外侵袭有明显的抑制作用。沉默EGFL7后,Hep-2细胞集落形成能力显著减低,即下调EGFL7基因表达可在一定程度上抑制喉癌细胞的锚着不依赖性增殖能力。Objective：To construct a lentivirus vector of RNA interference（RNAi） of EGFL7 gene and observe its inhibitive role on the invision of laryngeal cancer cell.Method：The effective sequence of siRNA targiting EGFL7 gene was confirmed.Both sence and antisence Oligo DNA of the targiting sequence was designed,synthsized and cloned into the pLV vector,which contained H1 promotor and green fluorescent protein（GFP）.The resulting lentivirus vector containing EGFL7 shRNA was called LV-sh EGFL7,and it is confirmed by PCR and sequencing.After that,EGFL7 shRNA was transfected into Hep-2 cells and Western blot was used to test the expression of EGFL7.At last,boyden chanmber was used to observe the invision of the Hep-2 cells.Colony formation assay using a EGFL7 gene silencing on the Hep-2 cell colony forming ability.Result：PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of EGFL7（LV-sh EGFL7） producing EGFL7 shRNA was constructed successfully.The titer of concentrated virus was 5×108TU/L.Western blot showed that the expression of EGFL7 was negtive in the EGFL7 siRNA Hep-2 cells.And boyden chanmber showed the invisive capibility of Hep-2 cells thansfected EGFL7 siRNA were obviously decreased.EGFL7 gene silencing of cell colony formation rate of cloned Hep-2 cells and compared with empty vector cells,cell cloning and colony formation was significantly reduced.Conclusion：The lentivirus RNAi vector of EGFL7 was constructed successfully.And EGFL7 silence can inhibit invasion of laryngeal cancer in vitro.After silence EGFL7,Hep-2 cell colony formation was significantly lower,that is,gene expression can be down EGFL7 some extent laryngeal cancer cells inhibited anchorage independent growth capacity.

关 键 词：RNA干扰 EGFL7 喉肿瘤 慢病毒属 

分 类 号：R739.65[医药卫生—肿瘤]