Abstract: Yang Jia, Li Liming, Xu Cui, Long Jia, Wang Yao, Yang Xueyuan, Jiang Mingjun Central Laboratory, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China （Yang J, Li LM, Xu C, Long J, Yang XY, Jiang MJ）; Life Sciences and Technology Base Class, Nanjing Agricultural University, Nanjing 210095, China （Wang Y） Corresponding author: Jiang Mingjun, Email: drmingjunjiang@163.com 【Abstract】 Objective To evaluate the effects of lentivirus-delivered short hairpin RNA （shRNA） targeting human papillomavirus 16 （HPV16） E7 gene on the 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 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 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 of the 4 DNMTs at 48, 96 hours after infection. Results There were no significant differences in the mRNA 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 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）, but did not differ between 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 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 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 of DNMT1, DNMT3A, DNMT3B and DNMT3L.