靶向Lin28B基因沉默对胰腺癌细胞增殖的影响

Effect of silencing Lin28B on proliferation of human pancreatic cancer cells

目的探讨靶向Lin28B基因沉默对细胞增殖的影响。方法设计Lin28B及GFP短发夹核糖核酸(shRNA)序列,装入Tet-on-pLKO-puro质粒,进行慢病毒包装,感染胰腺癌细胞Panc1,嘌呤霉素筛选并培养稳定干扰细胞株。实验组为Panc1-Tet-on-pLKO-sh-Lin28B;对照组为Panc1-Tet-on-pLKO-sh-GFP。筛选强力霉素最佳诱导浓度并诱导干扰序列表达,Western blot检测干扰效率,细胞增殖与活性实验CCK8检测沉默Lin28B对Panc1细胞增殖的影响。结果构建的Tet-on-pLKO-sh-Lin28B慢病毒可显著抑制Panc1细胞内Lin28B的表达。与对照组Panc1-Tet-onpLKO-sh-GFP相比,实验组Panc1-Tet-on-pLKO-sh-Lin28B的增殖速度减缓(P<0.05)。结论成功筛选出沉默Lin28B的稳定干扰细胞株,沉默Lin28B对胰腺癌细胞的增殖有抑制作用,为进一步研究Lin28B基因的功能或基因治疗提供有用技术。

Objective To investigate the effect of silencing Lin28B on proliferation of human pancreatic cancer cells.Methods Lin28B and green fluorecent protein short hairpin RNA（GFP shRNA）sequences were designed and inserted into Tet-on-pLKO-puro plasmid to package lentivirus to infect Panc1.Stable infected cells were obtained by puromycin screening.The doxycyline concentration was optimized to obtain best induction effect.Western blot was used to test the silencing effect and CCK8 experiment for proliferation.Results Tet-on-pLKO-sh-lin28B lentivirus could significantly reduce Lin28 Bexpression in Panc1 cells.At the same time,the proliferation rate of Panc1 cells was also much lower compared to Tet-on-pLKO-sh-GFP group（P〈0.05）.Conclusion Inducible Lin28B shRNA lentivirus has been successfully constructed and could silence target gene in Panc1 cells,which may provide a useful research tool for studying Lin28B.