Construction of a recombinant shRNA expression vector targeting EZH2 gene and its inhibitory effect on colon adenocarcinoma SW480 cells

Abstract

To construct a recombinant short hairpin RNA (shRNA) expression vector targeting EZH2 gene, and to determine its effect on the proliferation of colon adenocarcinoma SW480 cells. The DNA sequence with short hairpin structure was 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. RT-PCR and Western blot were used to detect the inhibitory effect at both mRNA and protein levels. MTT was used to detect cell viability due to the alteration of EZH2 gene activity. At 48 h after transfection, the expression of EZH2 mRNA in the gene silencing group and negative control group were 0.339 ± 0.013 and 1.968 ± 0.072, respectively. The expression of EZH2 protein in the gene silencing group and negative control group were 0.229 ± 0.008 and 1.168 ± 0.053, respectively. The expression of EZH2 in the gene silencing group was significantly lower than that in the negative control group (P < 0.01, P < 0.05). At 48 and 72 h after transfection, the inhibition rate of cell growth in the gene silencing group was 30.7% and 25.9%, respectively, indicating that the cell growth was significantly inhibited in comparison with that in the blank control group (P < 0.05). A recombinant shRNA expression vector targeting EZH2 gene has been successfully constructed in this study, with a significant inhibitory effect on the proliferation of SW480 cells. This lays an experimental foundation for further exploring the mechanism underlying the action of EZH2 gene on tumor biology.