慢病毒介导的类表皮生长因子域7基因沉默抑制喉癌Hep-2细胞增殖和侵袭的研究  

Effects of lentivirus-mediated epidermal growth factor-like domain 7 silencing on proliferation and invasion of human laryngeal carcinoma Hep-2 cells

作　　者：黎景佳[1] 叶进[1] 庄士民[1] 王涛[1] 王志远[1] 常利红[1] 张革化[1] 

机构地区：[1]中山大学附属第三医院耳鼻咽喉头颈外科,广州510630

出　　处：《中华耳鼻咽喉头颈外科杂志》2014年第8期643-648,共6页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：高校基本科研业务费中山大学青年教师培育计划（11ykpy43）;广东省自然科学基金博士启动项目（S2012040006622）

摘　　要：目的 探讨小干扰RNA（siRNA）重组慢病毒介导的类表皮生长因子域7（epidermal growth factor-like domain 7,EGFL7）基因沉默对体外培养喉癌Hep-2细胞增殖和侵袭潜能的影响.方法 采用RNA干扰技术,构建靶向EGFL7的shRNA重组慢病毒并转染Hep-2细胞,通过Real-timePCR和Western blot在mRNA及蛋白水平鉴定转染结果;实验分为空白对照组（NC）、空白慢病毒感染组（Lenti-NC）和EGFL7基因沉默慢病毒感染组（Lenti-EGFL7）,采用CCK-8法检测细胞增殖,流式细胞术检测细胞周期和凋亡,Transwell侵袭实验观察细胞侵袭能力.结果 Real-time PCR检测说明Lenti-EGFL7组mRNA相对表达量较NC组下降达94％（P＜0.001）,Western blot检测证实Lenti-EGFL7组EGFL7蛋白相对表达量为（0.07 ±0.04）较NC组的（0.39 ±0.12）明显减低（P ＜0.001）,表明成功构建稳定沉默EGFL7基因的喉癌Hep-2细胞株.CCK-8细胞增殖实验说明靶向沉默EGFL7表达导致喉癌细胞增殖明显抑制（P＜0.01）,流式细胞术检测证实,与NC组和Lenti-NC组相比,Lenti-EGFL7组S期细胞百分比明显升高（P＜0.01）,G1期细胞百分比下降（P＜0.05）,细胞凋亡检测证实Lenti-EGFL7组细胞凋亡率为（66.2±1.28）％较NC组的（6.09±3.28）％和Lenti-NC组的（9.86±2.13）％明显增加（P ＜0.001）.Transwell侵袭实验发现Lenti-EGFL7组穿过Matrigel到达下室的平均细胞数较NC组和Lenti-NC组均明显减少（P＜0.01）.结论 靶向沉默EGFL7基因能抑制体外培养喉癌Hep-2细胞的增殖并降低细胞侵袭能力.Objective To explore the effects of epidermal growth factor-like domain 7 （EGFL7） gene silencing on the proliferation and invasion ablity of laryngeal carcinoma cells.Methods A lentiviral vector expressing EGFL7 shRNA was constructed and transfected into human laryngeal carcinoma Hep-2 cells.The expressions of EGFL7 mRNA and protein were detected by Real-time PCR and Western blot,respectively.Cell proliferation was evaluated by CCK-8 assay,cell cycle and apoptosis were tested by flow cytometry,and cell invasion was detected by transwell invasion assay.Results The relative expression level s of EGFL7 mRNA and protein in EGFL7-SuRNA group were svgnificantly lower than control group （P ＜0.001）.Western blot analysis proved that the relative expression of EGFL7 protein in NC group,Lenti-NCgroup and Lenti-EGFL7 group was （0.39 ±0.12）,（0.36 ±0.14） and （0.07 ±0.04）,respectively.EGFL7 expression in Lenri-EGFL7 group was significantly inhibited than NC group （P ＜ 0.001）,which confirmed that the recombinant lentivirus was successfully transfected into Hep-2 cells.The proliferation of Hep-2 cells was significantly inhibited after transfection （P ＜ 0.01）.Compared with the NC group and Lenti-NC group,the proportion of cells in S phase was significantly increased in Lenti-EGFL7 group （P ＜ 0.01）,and the proportion in G1 phase was significantly decreased （P ＜ 0.05）.Cell apoptosis assay showed that the apoptotic rate in Lenti-EGFL7 group （66.2 ± 1.28） ％ was significantly increased in NC group （6.09-±3.28）％ and Lenti-NC group （9.86 ±2.13）％.In Transwell invision assay,the mean number of cells coming through the Metrigel in Lenti-EGFL7 group was significantly decreased than in the NC group and Lenti-NC group （P ＜ 0.01）.Conclusion The proliferation and invasion ablity of laryngeal carcinoma Hep-2 cells can be inhibited by siRNA mediated EGFL7 gene silencing.

关 键 词：RNA 小分子干扰 喉肿瘤 表皮生长因子 细胞增殖 肿瘤侵润 慢病毒属 肿瘤细胞 培养的 

分 类 号：R739.65[医药卫生—肿瘤]