Inhibitory Effect Of SiRNAs Targeting Of HPV E6/E7 Gene Of HPV16-positive Cell Lines

Objective:The E6/E7 oncoprotiens play important roles in initiation and malignant progression of human cervical carcinoma,when high-risk types of human papillomaviruses (HPVs) integrate into human chromosome.For our study, siRNAs targeting of the HPV16 E6/E7 oncogenes were used to treat on SiHa and CaSki, two human cervical carcinoma cell lines positive for high risk type of HPV16, to evaluate the special inhibitory effect and time-efficiency of siRNA .Methods:1. Design and synthesize 3 specific small interfering RNA s (siRNAs) targeting of each of E6/E7 genes separately,(named E6-1 siRNA, E6-2 siRNA, E6-3 siRNA, E7-1 siRNA, E7-2 siRNA and E7-3 siRNA ), andβ-actin siRNA for positive control, NControl siRNA for negative control. Some of NControl siRNA were modified by FAM and transfected into SiHa and CaSki cells by lipofectmineTM2000. The green fluorescence was observed in cells by fluorescence microscope.2. The expression levels of mRNA and protein ofβ-actin were detected by RT-PCR and Western blot after treatment withβ-actin siRNA in both cells.3. The expression levels of mRNA and protein of E6 or E7 gene were detected by RT-PCR and Western blot after treatment with separate E6-1, E6-2, E6-3 and E7-1, E7-2, E7-3 siRNAs in SiHa in order to screen the most effective siRNAs.4.To investigate the special inhibitory effect and time-efficiency of siRNA, the expression levels of mRNA and protein of the target gene were measured by RT-PCR and Western blot at 24 h, 48 h, 72 h and 96 h after treatment with the two most effective siRNAs.Results:1.The fluorescence photos indicated that the green fluorescence was observed in both two cell lines by fluorescence microscope.2.Compared with control group, the expression levels ofβ-actin gene downregulated remarkably after treatment withβ-actin siRNA in both cells(P<0.05). While no distinct change was detected in the group transfected by lipofectemineTM2000 without siRNAs. 3. The results of RT-PCR and Western Blot suggested that each siRNA would lead to downregulation of expression of target genes respectively(P<0.05), but the capability of downregulation was different. Acording to our experimental data, E6-1 siRNA and E7-2 siRNA were screened as the most effective siRNAs.4. Treatment with either E6-1 or E7-2 siRNAs that were screened at former step induced a marked decrease in the respective E6/E7 mRNA and protein levels in both cells(P<0.05),compared with non-treated control group ,by RT-PCR and Western blot at 24 h, 48 h, 72 h and 96 h after transfection. However the other corresponding gene remained constant at each time pointsConclusions:1.Both of the two human cervical carcinoma cell lines can be transfected with siRNA by lipofectmine TM2000 successfully.2. Inhibitory efficiency varies with sequence of siRNAs targeting of the same gene.3. Treatment with either E6 or E7 siRNAs induced a special and efficient inhibition at respective expression levels of target gene in both cells(P<0.05), and sustained at least 96 h.