KIF5B过表达和干扰慢病毒载体的构建及对结直肠癌细胞增殖的影响

Construction of KIF5B Overexpression and Interference lentivirus Vector and its Effect on Colorectal Cancer Cell Proliferation

目的构建马达蛋白KIF5B基因重组慢病毒过表达和RNA干扰载体并进一步构建稳定转染结直肠癌细胞模型,检测敲低KIF5B蛋白对结直肠癌细胞增殖的影响。方法将PCR扩增的人KIF5B基因序列和设计合成的shRNA序列分别插入GV-358和GV-248表达载体。采用慢病毒三质粒包装系统共转染HEK293T细胞,进行病毒包装和扩增。重组慢病毒感染结直肠癌细胞,荧光显微镜观察绿色荧光强度,Western blot法检测KIF5B过表达和沉默效率。CCK-8检测KIF5B干扰后HCT116和SW480细胞的增殖,并检测细胞内周期相关蛋白的表达。结果KIF5B过表达及shRNA载体测序与原设计序列完全符合。重组病毒感染结直肠癌细胞中,可见绿色荧光蛋白表达。KIF5B过表达组细胞中KIF5B-FLAG蛋白大量表达,KIF5B沉默细胞中,KIF5B的蛋白表达量显著下降。KIF5B被敲低后,HCT116和SW480细胞增殖加快,细胞内p21、p16两种蛋白表达下调。结论成功建立了KIF5B过表达和沉默的结直肠癌细胞模型,并且发现KIF5B表达被干扰后,可能会抑制p21与p16蛋白的表达,加速细胞周期,从而促进结直肠癌细胞增殖。

Objective To establish colorectal cancer cell models of KIF5B overexpression and silence by lentivirus-mediated transfection and investigate the effect of knockdown of KIF5B on the proliferation of colorectal cancer cells.Methods The PCR amplified human KIF5B gene and the designed and synthesized shRNA were inserted into the GV-358 and GV-248 expression vectors respectively.A lentivirus three-plasmid packaging system was used to co-transfect HEK293T cells for virus packaging and amplification.Recombinant lentivirus was used to infect colorectal cancer cells,the green fluorescence intensity was observed under a fluorescence microscope,and the overexpression and silencing efficiency of KIF5B were detected by Western blot.The proliferation rates of HCT116 and SW480 cells were detected by CCK-8 test.Expressions of cell cycle related proteins were testified in KIF5B+/+and KIF5B-/-colorectal cells by Western blot.Results The KIF5B overexpression and shRNA vector were successfully constructed and confirmed by sequencing.The expression of green fluorescent protein can be seen in colorectal cancer cells infected by the recombinant virus.The KIF5B-FLAG protein was expressed in large amounts in the KIF5B overexpression group,and the KIF5B protein expression in the KIF5B silenced cells was significantly decreased.In SW480 and HCT116 cells transfected with KIF5B shRNA,the proliferation rates were increased and expressions of p21,p16 were downregulated.Conclusion The colorectal cancer cell model with overexpression and silence of KIF5B was successfully established.When KIF5B was knockdown,p21 and p16 might be inhibited to accelerate cell cycle,leading to the enhanced growth of colorectal cancer cells.