关键词: EZH2基因；RNA干扰；SW480细胞；蛋白质印迹法；MTT法

Abstract:

Objective     To construct a recombinant short hairpin RNA (shRNA) expression vector targeting enhancer of zeste homolog 2(EZH2) gene and explore its effect on colorectal cancer cells. Methods    Two DNA sequences with short hairpin structure were designed according to the EZH2 cDNA sequence and cloned into pGFP-V-RS vector to construct a recombinant expression vector silencing EZH2 gene.After identification, the shRNA-expressing vector was then transfected into SW480 cells.Then transfected SW480 cells were divided into negative control group and gene silencing group.RT-PCR and Western blotting were used to detect inhibitory effect.MTT was used to detect cell proliferation. Results   A recombinant vector harboring shRNA of EZH2 was successfully constructed in this work.With respect to assessment of interference efficiency,the expression of EZH2 mRNA in negative control group was 5.8 times (P<0.01) of that in gene silencing group. And the expression of EZH2 protein in gene silencing group was significantly lower than that in negative control group (P<0.05 ).Moreover, the cell viability in gene silencing group was significantly reduced than that in negative control group (P<0.05). Conclusion    The present study demonstrates that a recombinant shRNA expression vector targeting EZH2 gene was successfully constructed, with significant inhibitory effect on proliferation of SW480 cells. This lays an experimental foundation for further exploring the mechanism underlying the action of EZH2 gene on tumor biology.

Key words: EZH2 gene; RNA interference; SW480 cells; Western blotting; MTT zeste