Effect of c-myc gene silence on the expression of oncogenes and apoptotic genes in hepatocytes treated with PM(2).5

Abstract

Objective: To construct the c-myc gene silenced hepatocytes, study the effect of c-myc gene silence on expression of oncogenes and apoptosis genes in hepatocytes treated with PM2.5. Methods: According to the c-myc gene mRNA sequence provided by GenBank, three interfering sequences were designed and synthesized, the recombinant lentiviral vector was transfected into L02 hepatocytes. The real-time quantitative PCR and western blotting were used to identify the effect of c-myc gene silencing. L02 cells and c-myc gene silenced cells were used as experimental subjects. The normal L02 cells and c-myc silenced cells were treated with 50 μg/ml PM(2.5) water soluble solution, 10 μM positive control Cr(6+) and a blank control, the treatment period was 24 h. The mRNA levels of oncogenes (c-myc, c-fos, k-ras, p53) and apoptotic genes (Caspase-3, Caspase-8, Caspase-9) were detected by real-time PCR. The protein levels of oncogenes and apoptotic genes were detected by western blotting. Results: The mRNA level and protein level of c-myc decreased by 81% and 70% in c-myc silenced cells when compared with the normal L02 hepatocytes, the above results indicate that c-myc gene silenced cells were successfully constructed. After c-myc silenced cells were treated with PM2.5 water soluble solution, The mRNA levels of c-myc, c-fos, and k-ras decreased by 84.1%, 45.4%, and 54.6% (P<0.05) , p53 increased by 192.9% (P<0.05) , and the expression of Caspase-3, Caspase-8, and Caspase-9 decreased by 24.4%, 36.1%, 60.9% (P<0.05) . In the Cr(6+) positive control group, the expression of c-myc, c-fos, and k-ras decreased by 72.1%, 82.2%, and 54.0% (P<0.05) , p53 increased by 250.0% (P<0.05) , the expression of Caspase-3, Caspase-8, and Caspase-9 decreased by 34.6%, 36.0%, 68.9% (P<0.05) , respectively, when compared with the normal L02 hepatocytes (P<0.05) . Western blotting results showed that the protein levels of c-myc and c-fos increased, p53 decreased after PM(2.5) exposure; the protein levels of Caspase-3, Caspase-8, Caspase-9 increased after PM(2.5) exposure (P<0.05) . When in comparison with the c-myc silenced group, the protein levels of c-myc and c-fos decreased, p53 protein increased in PM(2).5 exposed group (P<0.05) . Conclusion: c-myc gene silenced cells were successfully constructed in this paper. PM(2.5) could promote the expression of oncogenes and apoptotic genes in L02 cells, and c-myc gene silencing can inhibit the expression of oncogenes and apoptotic genes after PM(2.5) treatment in L02 cells.