摘要
目的构建携带干扰癌基因EphA3的小干扰RNA(siRNA)慢病毒载体,并研究其对肝癌细胞7402的生长和迁徙的影响。方法构建携带EphA3 siRNA的慢病毒载体质粒并将其与外源EphA3表达质粒共转染至人胚肾细胞293T中,Western印迹检测其干扰EphA3蛋白表达的效果。将pSIH-H1-小发夹RNA(shRNA)-1504及空载体分别与3种包装质粒共转染293T细胞,包装成慢病毒lentivirus-1504及对照慢病毒。将所获慢病毒分别感染肝癌细胞7402,并加入嘌呤霉素(puromycin)筛选,1周后将细胞收集。Western印迹检测EphA3蛋白的表达水平,然后分别应用MTT实验、平板克隆形成实验、软琼脂克隆形成实验以及划痕实验检测敲低EphA3对肝癌细胞增殖能力、存活能力、恶性程度和迁移能力的影响。结果 lentivirus-1504抑制了7402细胞EphA3的表达,敲低EphA3后抑制了7402细胞的增殖能力、存活能力、恶性程度和迁移能力。结论慢病毒lentivirus-1504能够明显抑制肝癌细胞7402的生长能力、存活能力、恶性程度和迁移能力,提示EphA3可能是一个新的肝癌治疗靶点。
Objective To construct the lentiviral vector carrying small interfering RNA（siRNA） which can inhibit proto-oncogene EphA3,and to study its effect on the growth and migration of liver cancer cell 7402.Methods After plasmids carrying EphA3 small-hairpin RNA（shRNA） were constructed and transfected into cells 293T together with plasmids carrying gene EphA3,the protein was collected to examine the expression level of EphA3.PSIH-H1-shRNA-1504 and shRNA vectors respectively with three viral packaging vectors were co-transfected into cells 293T to produce lentivirus-1504.Lentivirus-NC.Cells 7402 were infected with produced lentivirus and filtered with puromycin.The protein of these cells was collected after a week.Western blotting was used to examine the expression level of EphA3,MTT to examine the growth of cells,plate clonogenicity to examine the survival ability,softagar clonogenicity experiment to examine the malignant degree of tumer cells,and scarification experiment to examine the cell migration ability.Results Lentivirus-1504 could inhibit the expression of EphA3.Furthermore,the growth and survival ability,the malignant degree of 7402 were all obviously inhibited.Conclusion Lentivirus-1504 can inhibit the expression of EphA3 which significantly promotes the growth,survival ability and malignant degree of liver cancer cell 7402.EphA3 may become a potential target for cancer therapy.
出处
《军事医学》
CAS
CSCD
北大核心
2013年第4期254-258,共5页
Military Medical Sciences
基金
国家自然科学基金资助项目(30770834
30870961)
国家863计划资助项目(2008AA02Z123)
