Effects of lentivirus-delivered short hairpin RNA targeting human papillomavirus 16 E7 gene on the expression of DNA methyltransferases in SiHa cells

Abstract

Objective To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) targeting human papillomavirus 16 (HPV16) E7 gene on the expression of 4 kinds of DNA methyl-transferases (DNMTs) , including DNMT1, DNMT3A, DNMT3B and DNMT3L, in HPV16-positive cervical cancer cell line SiHa. Methods The recombinant plasmid containing HPV16 E7 gene-targeting shRNA was constructed firstly. Then, the BLOCK-iTTM lentiviral RNAi expression system kit was used to package the lentiviral vector, which was transfected into 293T cells. The lentivirus-containing supernatants were collected at 48 and 72 hours after transfection. The SiHa cells were divided into 3 groups to be cultured with lentiviral supernatant containing HPV16 E7 gene-targeting shRNA recombinant plasmids mixed with complete medium at a ratio of 1∶1 (shRNA group) , lentiviral supernatant containing empty plasmids mixed with complete medium at a ratio of 1∶1 (negative control group) , and complete medium alone (blank control group) , respectively. Real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure mRNA expression of HPV16 E7 and 4 kinds of DNMTs in the above 3 groups at 0, 48, 96 hours after infection, and Western blot analysis to determine protein expression of the 4 DNMTs at 48, 96 hours after infection. Results There were no significant differences in the mRNA expression of HPV16 E7 and the 4 DNMTs among the shRNA group, negative control group and blank control group at 0 hour after infection (all P > 0.05) . At 48, 96 hours after infection, the mRNA expression of HPV16 E7 and the 4 DNMTs decreased significantly in the shRNA group compared with the negative control group and blank control group (all P 0.05) . Additionally, E7, DNMT1, DNMT3A, DNMT3B and DNMT3L gene-silencing efficiencies in the shRNA group were 71.13%, 50.53%, 13.72%, 46.27% and 17.92% at 48 hours, and 83.50%, 74.2%, 47.8%, 64.7% and 48.9% at 96 hours after infection, respectively. Western blot analysis showed that the protein expression of the 4 DNMTs significantly decreased in the shRNA group compared with the negative control group and blank control group at 48, 96 hours after infection (all P < 0.01) . Moreover, the protein expression of DNMT1, DNMT3A, DNMT3B and DNMT3L in the shRNA group gradually decreased over time, and was inhibited by 84%, 37.2%, 59.8% and 49.3% at 48 hours respectively, and by 73.1%, 68.7%, 55.5% and 65.5% at 96 hours after infection respectively. Conclusion Targeted silencing of E7 gene in HPV16-positive SiHa cells can interfere with the mRNA and protein expression of DNMT1, DNMT3A, DNMT3B and DNMT3L. Key words: Human papillomavirus 16; Papillomavirus E7 proteins; RNA, small interfering; Methyl-transferases; Cell line, tumor