摘要
目的探讨靶向抑制Fbxw8基因对结肠癌SW480细胞增殖的影响。方法 RNA干扰(RNAi)方法下调SW480细胞中Fbxw8的表达。通过Real-time PCR、western blotting方法分别检测Fbxw8 mRNA和蛋白表达变化;CCK-8、细胞克隆形成实验及BrdU掺入法检测下调Fbxw8对SW480细胞的增殖、DNA合成能力的影响。Western blotting检测下调Fbxw8对SW480细胞增殖相关基因表达的影响。结果 Fbxw8 siRNAs显著降低SW480细胞Fbxw8mRNA和蛋白的表达,CCK-8结果显示:与对照组(0.78±0.11)相比,两对Fbxw8 siRNAs均可显著抑制细胞的生长(0.46±0.07、0.49±0.03,P<0.05);细胞克隆形成实验显示与对照组相比,Fbxw8 siRNAs组细胞的克隆形成数目及大小显著降低。BrdU掺入实验显示与对照组(0.83±0.12)相比,Fbxw8 siRNAs均能显著下调DNA的合成能力(0.47±0.06、0.56±0.09,P<0.05);同时伴随着细胞周期蛋白cyclin E的下调而p27的表达升高。结论 Fbxw8 siR NA可以通过下调cyclin E、上调p27的表达而有效抑制SW480细胞增殖,为结肠癌的基因治疗提供新的候选靶点。
Objective To study the effect of siRNA targeted against Fbxw8 on cell proliferation of colon cancer SW480 cells.Methods siRNAs targeting Fbxw8 were transfected into SW480 cells by lipofectamine method.The expression of Fbxw8 mRNA and protein were detected by real-time PCR and western blotting respectively.Changes in cell proliferation and DNA synthesis were measured by CCK-8 and clone formation assay,bromodeoxyuridine（BrdU） incorporation.In addition,changes in expression of proliferation associated genes were studied by western blotting.Results Two Fbxw8 siRNAs decreased the expression of Fbxw8 mRNA and protein level,compared with the control group（0.78 ±0.1 l）,knockdown of Fbxw8 caused intubation of cell proliferation（0.46 ±0.07,0.49 ±0.03,P〈0.05）.Fbxw8 knockdown cells generated less numbers of colonies and formed significantly smaller colonies than the control group.BrdU test showed that compared the control group（0.83±0.12）,Fbxw8 siRNAs downregulated the DNA synthesis（0.47±0.06,0.56±0.09,P〈0.05）.Westren blotting showed that cyclin E was down-regulating and p27 was up-regulating.Conclusion Knockdown of Fbxw8 can reduce proliferation of SW480 cells through down-regulation of cyclin E and up-regulation of p27 protein,Fbxw8 has a potential value in gene therapy of colon cancer.
出处
《中国现代医生》
2015年第36期25-28,共4页
China Modern Doctor
基金
黑龙江省留学归国科学基金(LC2012C30)
黑龙江省卫生厅科研课题(2012-374)
