Construction of a RNAi lentiviral vector targeting ARK5 gene and its effect on the biological behavior of gastric cancer SGC7901 cells

Abstract

To construct a RNA interference lentiviral vector aimed at human ARK5 (AMPK-related protein kinase 5) gene and explore its effect on the biologic behavior of human gastric cancer SGC7901 cells. Targeting human ARK5 mRNA coding sequence, we designed three specific short hairpin RNAs (shRNAs) and constructed the lentiviral vector, then infected human gastric cancer SGC7901 cells with this vector. Afterwards, we used qPCR and Western blot for detecting the silencing effect on ARK5 gene, MTT colorimetric assay to measure the cell proliferation, cell scratch test for cell migration and Transwell for cell invasion, and flow cytometry analysis for apoptosis in cells treated with glucose starvation and TNF-α. Sequencing proved that the recombinant lentiviral vector containing ARK5-shRNA-3 was constructed successfully. Real time fluorescent quantitative PCR assay showed that the expression abundance of ARK5 gene in the normal control group, negative control group and ARK5-shRNA-3 infected group were 1.002+ 0.082, 1.001+ 0.050 and 0.140+ 0.003, respectively, showing a statistically significant difference (P<0.01). Cell scratch test showed that the cell migration rate of ARK5-shRNA-3 infected group was (38.5+ 4.3)%, significantly lower than that of the normal control group [(72.4+ 6.4)%] and negative control group [(75.1+ 7.1)%, P<0.01]. The results of Transwell test showed that the number of penetrating cells in the normal control group, negative control group and ARK5-shRNA-3 transfection group were 257.4±12.3, 245.7±11.6, 112.5±7.8, with a significant difference (P<0.01). After glucose starvation and TNF-α-treatment for 24 h, the cell death rate of the normal control group, negative control group and ARK5-shRNA-3 group were (11.7±3.2)%, (12.3±2.6)% and (30.8±4.3)%, respectively, showing that the cell apoptosis rate of ARK5-shRNA-3 transfected group was significantly higher than that of the normal control and negative control groups (P<0.01). We have successfully constructed a recombinant lentiviral vector which can efficiently silence ARK5 gene. Using it we can inhibit the proliferation, migration, invasion of tumor cells, and promote cell apoptosis under the condition of TNF-α treatment and glucose starvation.