论文部分内容阅读
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 a lentiviral vector carrying small interfering RNA (siRNA) interfering with the oncogene EphA3 and investigate its effect on the growth and migration of hepatocellular carcinoma 7402 cells. Methods Lentivirus vector carrying EphA3 siRNA was constructed and transfected into human embryonic kidney 293T cells with exogenous EphA3 expression plasmid. The expression of EphA3 protein was detected by Western blotting. 293T cells were co-transfected with pSIH-H1-shRNA shRNA-1504 and empty vector respectively with 3 kinds of packaging plasmids, and packaged into lentivirus-1504 and control lentivirus. The obtained lentivirus were respectively infected with 7402 hepatoma cells and puromycin screening was performed. After 1 week, the cells were collected. The expression level of EphA3 protein was detected by Western blotting. The effects of knocking down EphA3 on the proliferation, survival, malignancy and migration of hepatocellular carcinoma cells were detected by MTT assay, plate clone formation assay, soft agar colony formation assay and scratch assay. Results lentivirus-1504 inhibited the expression of EphA3 in 7402 cells. The knockdown of EphA3 inhibited the proliferation, survival, malignancy and migration of 7402 cells. Conclusion Lentivirus lentivirus-1504 can significantly inhibit the growth, survival, malignancy and migration of hepatocellular carcinoma 7402 cells, suggesting that EphA3 may be a new therapeutic target for hepatocellular carcinoma.