摘要
目的:观察RNA干扰沉默肾癌细胞株OS-RC-2的环氧合酶-2(COX-2)基因表达对肾癌细胞生长及体外侵袭能力的影响。方法:构建靶向COX-2的小干扰RNA(siRNA)真核表达质粒pSilencer2.0-U6-COX-2-siRNA。将对数生长期的OS-RC-2细胞分3组,干扰组转染pSilencer2.0-U6-COX-2-siRNA,空转组转染pSliencer2.0-U6,对照组不转染。培养72h后采用半定量RT-PCR、Westernblot检测COX-2mRNA和蛋白表达,CCK8增殖抑制实验观察细胞生长情况,改良Boyden小室法评估细胞体外侵袭能力。结果:酶切和测序证实pSilencer2.0-U6-COX-2-siRNA构建成功。3组细胞COX-2mRNA和蛋白的表达、细胞存活率及穿孔细胞数差异均有统计学意义(F分别为189.800,89.326,188.403和75.349,P均<0.001)。与其他2组比较,干扰组COX-2mRNA及蛋白表达下调(P<0.05),细胞存活率、穿孔细胞数降低(P<0.05)。结论:COX-2有望成为肾癌基因治疗的靶点。
Aim：To observe the effect of COX-2 gene silence on proliferation and invasion ability of human renal cell carcinoma cell OS-RC-2.Methods：The recombinant of pSliencer2.0-U6-COX-2-siRNA was established and transfected into OS-RC-2（interference group）.OS-RC-2 cells transfected with pSliencer2.0-U6 were the negative control,and OS-RC-2 cells without transfection were the blank control. COX-2 mRNA and protein were detected by RT-PCR and Western blot 72 h after the transfection.The cell proliferation and invasion ability was assayed by CCK-8 method and modified Boyden chamber experiment in vitro 24 h after the transfection.Results：The results of enzyme digestion analysis and DNA sequencing showed that pSilencer2.0-U6-COX-2-siRNA plasmid was successfully constructed. There were significant differences in COX-2 mRNA and protein levels,the rate of survival cells and the number of migrating cells among the 3 groups（F=189.800,89.326,188.403 and 75.349,respectively,P0.001）.Compared with negative and blank control, the indexes mentioned above decreased（P0.05）.Conclusion： COX-2 could be the target gene for gene therapy in renal cell carcinoma.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2010年第6期986-989,共4页
Journal of Zhengzhou University(Medical Sciences)
