Interference Of HPV6bE7 Gene Expression Induced By Small Interfering RNA

Human Papilloma Virus (HPV) infections cause cutaneous or mucosal verrucous hyperplasia, sexually transmitted diseases and malignant tumors which spread all over the world. Condyloma Acuminatum (CA), the most common sexually transmitted disease in our country is due primarily to HPVs infection .usually types 6 and 11.The treatment of CA is time-consuming and tiresome with frequent relapse. As we know that medication, the physical or immune therapies, such as operation, laser, podophyl, antiviral drugs, corrosive agents, anticancer medicines, often failed to eradicate HPVs completely.So people still seek for ideal preventional and therapeutic methods.Short or small interfering RNA(siRNA) which has been discovered recently evocates a noval stratige for infectious disease. The 19-23 basal duplex riboneucleotides named siRNA, can effectively degradate endogenic mRNA via RNA-induced silencing complex (RISC), and the phenomenon is called RNA interference(RNAi).In this study,we investgated the interference of HPV6bE7 gene expression induced by dicer siRNA and hairpin siRNA expression plasmid in vitro and in vivo. This study might be beneficial for the application of RNAi techniques in controlling HPVs infectious diseases.Materials and MethodsCell lines and plasmids: B16 cell, 293Tcell, pcDNA3.1 (+)-GFP/HPV6bE7 recombinant plasmid.Reagents:The duplex siRNA and hairpin siRNA targeting to HPV6bE7 gene were designed, sharing no homology with exons of known human genes, synthesized by Hangzhou New Ray Jay Bioraed Corporation and Shanghai KangChen Biotechnical Corporation.Methods:Cell line construction:B16 and 293T cells were transfected with recombinant plasmid pcDNA3. 1 (+)-GFP/HPV6bE7 via Lipofectamine²⁰⁰⁰, and the positive cell clones were selected by fluorescence protein observation and RT-PCR. In vitro: the Specific dicer siRNA (siRNA-HPV6bE7) and hairpin siRNA expression plasmid (shRNA- HPV6bE7) were transfected to the above cells using Oligofectamine and Lipofectamine²⁰⁰⁰. The quantitative real-time PCR was performed to measure the inhibitory rate of target gene expression by comparing HPV6.bE7 mRNA concentrations before and after transfection, including dose-efficiency and time-efficiency. In vivo: pcDNA3. l(+)-GFP/HPV6bE7/B16 positive cells(5 X 105cells) were injected to the subcutan of right back of C57BL/6J mice. Ten days later, the tumor developed like a bean, and the mice were divided to six groups randomly, including 100nmol/L siRNA(in tumor site), 200nmol/L siRNA (in tumor site),400nmol/L siRNA (tail vein), siRNA control, 100ug shRNA(in tumor site), shRNA control.The mice in the first to third groups were injected with siRNA alternatively for three times, and the fifth groups were injected for one time, we took the entire tumor tissue for mRNA extraction and RT-PCR after sacrifice.Results1. Transfection of recombinated plasmid to cells:24h after pcDNA3.1(+)-GFP/HPV6bE7 transfecting to B16 and 293T cells,the green fluorescence from the transfected cells in the fluorescent microscope can be seen.2. The morphological change of cells transfected with siRNA:24h after B16 and 293T cells being transfected with pcDNA3. l(+)-GFP/ HPV6bE7, no obvious change can be seen.3.The interference of duplex siRNA for HPV6bE7 gene:3.1 Dose-efficiency:In B16 cell, at 48h after 1, 5, 10, 25, 50. 100 nmol/L siRNA transfection, the inhibitory rates were 9. 14%,38. 94%, 76. 40%> 83. 35%, 87. 05%. 66.02%. In 293T cell, at 48h after 1, 5. 10, 25, 50 nmol/L siRNA transfection, the inhibitory rates were 46.92%, 77.08%, 78.87%, 51.31%, 49.36%. The scrambled siRNA had no inhibitory effect on HPV6bE7 gene.3.2 Time-efficiency. In B16 cell,the inhibitory rates of 50 nmol/L siRNA at 12, 24, 48, 72 and 96h post-transfection were 0, 32.47%, 74.72%, 70. 02%, 8. 91%. In 293T cell, the inhibitory rates of 25 nmol/L siRNA at 24, 48, 72 and 96h were 26. 66%, 45. 93%, 65. 93%, 33.10%.The inhibitory rates of 10 nmol/L siRNA at 24, 48, 72 and 96h were 20.31%, 41.16%, 35. 23%, 1. 57%.4. The interference of hairpin siRNA expression plasmid for HPV6bE7 gene:4.1 Dose-efficiency: At 24h post-transfecting with B16 cell,the transfectory efficiency were 30% 60%. At 72h post-transfection,the inhibitory rates of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3. 2^g were 51. 51%, 50.95%, 64.66%, 71.13%, 75.01%, 63.14%, 65.34%. The scrambled shRNA expression plasmid had no inhibitory effect on HPV6bE7 gene.4.2 time-efficiency:0.8M-g shRNA transfected with B16 cell, the inhibitory rates at 6, 12, 24, 48, 72> 96, 120h were 0, 15.43%, 33.71%, 67.31%, 68.08%, 48.84%, 14.56%.5.In vivo:the pcDNA3.1/ HPV6bE7-GFP/B16 cells were injected subcutaneously to C57BL/6J mice. Ten days later, all the mice developed tumors. Then siRNA and shRNA were injected in the tumor sites or via tail veins. The inhibitory rate of 100 and 200 duplex siRNA in the tumor sites were 36.58%, 34.04%,400 nmol/L via tail vein injection was 52. 18%. The inhibitory rate of 10 Hg shRNA in the tumor site was 55. 95%.Discussions and Conclusions1. After screening and passaging, the pcDNA3. 1(+)-GFP/HPV6bE7 transfected B16 and 293T cells still can express HPV6bE7 gene strongly, make sure for the next process.2.No obvious morphologic changes could be seen after transfection.3. The study of duplex siRNA on cells showed that the 50¹0 nmol/L siRNA had highest inhibition.Besides,the inhibitory rates of same concentration of siRNA in B16 and 293T were not equal. The effect of siRNA in cells was known to be transient, lasting at least 72h.4. The study of 0.05 to 3. 2^g hairpin siRNA expression plasmid on B16 cell showed that the inhibitory rates were between 50% to 75%, 0. 8M-g had the highest inhibition.And the effect last 12h to 120h.5. In vivo , whether in the tumor site or via tail vein, both duplex siRNA and hairpin siRNA expression plasmid showed inhibitory effect on HPV6bE7.6. Our data showed specific interference of HPV6bE7 gene by duplex and hairpin siRNA expression plasmid both in vitro and in vivo, and may provide an useful profile for further investigation of inhibition of HPV viral protein in clinic trials, and may also be a promising controllingapproach for HPV infection,such as Condyloma Acurainatum.