Application of RNA interference vector targeting mouse p16 gene in γ-irradiation-induced mouse embryonic fibroblast

Objective To explore the effects of silencing p16 gene by RNA interference （RNAi） mediated onγ-irradiation-induced mouse embryonic fibroblast（MEF）. Methods p16 RNAi vector was constructed by using pcDNATM6.2-GW/EmGFPmiR plasmid. p16 mRNA and protein levels of the irradia-tion-induced MEF before and after RNAi were detected by real-time PCR, Western blot and the percent-age of aging cells was detected by senescence associated-β-galactosidase（SA-β-Gal） staining. Results 24 h after RNAi, p16 mRNA levels of the irradiation-induced MEF decreased significantly in the irradiation＋p16 siRNA-1 and irradiation＋p16 siRNA-2 group as compared with the irradiation group （t=16.52 and 16.13, both P〈0.05）, protein levels of the irradiation＋p16 siRNA-1 and irradiation＋p16 siRNA-2 group also decreased as compared with the irradiation group. 5 days after irradiation the percentages of SA-β-Gal positive cells in the irradiation＋p16 siRNA-1 and irradiation＋p16 siRNA-2 group were（34.17±2.08）%and（33.83±1.75）%, significantly lower than that of the irradiation group[（68.83±1.26）%]（t=37.72 and 38.09, both P〈0.01）. Conclusion MEF aging can be effectively controlled by RNAi which can shut down the expression of silencing p16 gene and lay the foundations for its application in the related studies on cells senescence. Key words: Genes, p 16;  RNA interference;  Gamma rays;  Mouse embryonic fibroblast cell;  Aging