Silencing of HPV11ET gene expression by different RNA interference technology

Objective To investigate the silencing effect of two kinds of RNA interference (RNAi)technology on the expression of HPV11 E7 gene in vitro.Methods A pair of small interference RNA(siRNA)targeting HPV11 E7 gene,named siRNA-11E7,was chemically synthesized,and three short hairpin RNA(shRNA)expression plasmids,i.e.pRNAT-11E7 1~#,2~#,3~#,were constructed.Then a murine melanoma cell line,B16,which stably expressed HPV11 E7 gene,was transfected respectively with the siRNA- 11E7 and pRNAT-11E7 1~#,2~#,3~#,etc.The level of E7 mRNA expression was detected in these cells by real-time fluorescent quantitative PCR before and after the transfection.Results siRNA-11E7 could inhibit the E7 gene mRNA expression in B16 cells by 12.60%,31.41%,41.93%,42.93%,74.35%,32.71% and 29.00% at 6 h,12 h,24 h,48 h,72 h,96 h and 120 h after the transfection,respectively.The optimal dose of siRNA was 25 nmol/L with an inhibition rate of 70.06%,and the lowest effective dose was 3.13 nmol/L with an inhibition rate of 47%.The 0.4μg of pRNAT-11E7 1~#,2~#,3~# and a mix of the 3 plasmids could decrease the target gene expression by 44.52%,59.26%,89.62%,54.09%,respectively,while the negative plasmid had no inhibitory effect on the gene expression,pRNAT-11E7 3~# could decrease the E7 gene expres- sion by 0,17.50%,40.90%,42.40%,61.90%,51.50% and 0,at 6 h,12 h,24 h,48 h,72 h,96 h and 120 h after the transfection,respectively.The optimal dose ofpRNAT-11E7 was 0.2μg.Conclusions Both siRNA and shRNA expression plasmids could efficiently and safely silence the HPV11E7 mRNA expression in a time-and dose-dependent manner,and the optimal action dose and time is likely to be identified.