Inhibition Of HPV18 E6,E7 Gene Expression In Hela Cervical Cancer Cells By RNA Interfernce

ObjectiveCervical cancer is the most common anogenital cancer and represents the second fatality rate cancer in females worldwide. The strong association between human papillomavirus (HPV) infection and the development of cervical cancer has been well established. Almost all of cervical cancer cells contain certain HPVs, most commonly HPV16 and HPV18. HPV18 positive cervical cancer is characterized by high invasion, rapid development to infiltrating carcinoma from cervical dysplasia, high lymphatic metastasis and recurrence rate. The current treatment methods such as radical surgery, chemotherapy have sevious adverse reaction and have the possibility of deprivation of females' breeding function. With the requirement for high quality of life, it is urging to explore a new, minimal adverse reaction method to treat cervical cancer.Hela cervical cancer cells contain human papillomavirus 18 genome. It has been confirmed the high risk HPV18 encode E6 and E7 oncoproteins, which are essential for malignant transformation as well as maintenance of the malignant phenotype of cervical cancer. HPV E6 and E7 proteins modulate cellular cycle regulator factor P53 and Retinoblastoma protein (pRB). The E6 and E7 oncoproteins play critical role in the generation and development of cervical cancer and become ideal targets for treatment of cervical cancer.RNA interference(RNAi) may serve as a novel and powerful technology for the research of gene function and gene treatment. It can silence gene expressing by degrading mRNA highly specifically and efficiently, and it has been widely applied for the research in gene function of virus and gene treatment on virus associated tumors. And exciting progress has been achieved. But the researches on HPV18 by RNAi and the change of cellular cycle regulator factor after the inhibition of HPV oncogene are limited. In this study, investigations are carried out targeting HPV18 E6 by RNAi.â‘ small interference RNA (siRNA) targeting highly conservative HPV18 E6 was designed and synthesized and transfected into Hela. The aim is to change tumor property of Hela by inhibiting expression of oncogene E6 and E7.â‘¡Construction short hairpin RNA(shRNA) expressing vector targeting extron and intron of HPV E6. Investigate the level of expression of E6 and E7 to approach the feasibility of gene treatment on cervical cancer.â‘¢Investigation the change of cellular P53 in order to further know pathogenesis of cervical cancer.Method1. Designing siRNA targeting HPV18 E6According to Tuschl's principle, search targeted sequences from down stream of AUG start codon. And the sequences were analyzed for homology by BLAST.2. Transfection of HPV-siRNA to Hela cellsHPV-siRNA and siNeoFX were diluted with RPMI-1640 without serum and antibiotics and incubated for 10 min at room temperature seperately. The two mixtures were then combined and incubated for 10 min at room temperature. Then the entire mixture was added to 24 wells plate followed by adding cells. After incubation, further investigations were carried out.3. Detection of transfection efficiencyThe number of cells was counted under inverted fluorescence microscope. Transfection efficiency was confirmed by calculating the percentage of fluorescent cells.4. Detection of the effect of HPV-siRNA on HPV E6,E7 mRNA and HPVE6,E7,P53,HPA protein in Hela cells.Total RNAs were Extracted and the change of HPV18 E6 and E7 mRNA in cells were detected by RT-PCR. The change of HPV18 E6, E7, P53, HPA protein were detected by Western Blot.5. Detection of cells morphage was carried out by phase contrast microscope. 6. Cell viability was examined with MTTCells were transfected with siRNA for 48h, then dispensed into a 96-well plate at 750 cells/well, after which cell viability was determined at 5 days after plating. 20μl/well MTT was added and incubated in 37℃, 5% CO₂ incubator for 4h. After emptying the wells, 150μl DMSO was added to each well. A value was detected and the cell proliferation rate was calculated. Cell proliferation rate=(the average A value of siRNA group/the average A value of control group)×100%.7. The colony forming ability assayCells were transfected with siRNA, incubated for 48h, trypsinized, and then suspended in RPMI-1640 containing 0.3% agar and 10% FBS at adensity of 300 cells/ml. Next, 1ml of the cell suspension was placed over 1ml of RPMI-1640 containing 0.5% agar and 10% FBS in 12-well plates. After plating, 1ml of RPMI-1640 containing 10% FBS was added to the soft agar cultures. Cells were allowed to grow for 14 days and colonies consisting of more than 50 cells were counted. Clony forming rate=(the average clones number/the added cells number)×100%, The clony formation inhibitive rate (1-the average clones number of siRNA group/the average clones number of control group)×100%.8. Detection of cell cycle distribution by FCMHeLa cells were transfected with the indicated siRNA. After 48h of incubation, all the cells were collected, stained with propidium iodide containing RNase for 30 min. The cells were washed with PBS and analyzed by flow cytometry.9. Construction and verification of shRNA expressing vectorOligonucleotides were synthesized and annealed for 3 min at 94℃. The plasmid pGenesil was cut with BamHI and Hindâ…¢and run agarose gel electrophoresis. The larger fragment was retrieved and ligated with annealed oligonucleotides. The recombinant plasmid pHPV was transformed into E.coli DH-5α. The positive clones were selected, and the recombinant plasmids were extracted and verified the insert with restriction enzymes and DNA sequencing.10. Investigation of the inhibition of HPV E6, E7, P53 by pHPVpHPV was transfected and the stablely transfected Hela were selected by G418. HPV E6, E7 mRNA were detected by RT-PCR, and HPV E6, E7, P53 protein were detected by Western Blot.Result1. Designing siRNA targeting HPV18 E6The sequence targeting extron of HPV18 E6 is 434-452, and the intron is 287-305. No obvious homology with human genome was found.2. Detection of transfection efficiencyCells infected FAM-siRNA were green fluorescent cells under inverted fluorescent microscope and the transfection effiency was about 85%.3. The effect of HPV-siRNA on HPV18 E6, E7 mRNA and on HPV18 E6, E7, P53 and HPA protein in Hela cellsThe size of HPV E6 amplified fragment is 196 bp, and E7 is 332 bp. HPV-siRNA decreased HPV E6, E7 mRNA to 33.33%, 36.78% as compared with control group respectively. HPV E6, E7 and HPA protein were decreased to 37.98%, 33.84% and 41.78%n P53 was increase by 2.104 fold as compared with control group respectively.4. The effect of HPV-siRNA on cells morphageCells transfected siRNA became round and smaller. More granules in the cells and vacuolus could be seen in cytoplasm. Some cells suspended in the culture medium.5. The effect of siRNA on cells proliferation and clony formation ability of HeLaCells proliferation rate and clony formation ability were decreased. siRNA was found to inhibit cell proliferation of HeLa cells to 31.40% and clony formation to 65.25% as compared with control group.6. The effect of siRNA on Hela cells cycleG₀/G₁ stage cells of siRNA group were increased to 77.47% compared with 60.39% of control group. S stage cells of siRNA group were decreased to 19.92% compared with 35.83% of control group.7. The recombinant shRNA expressing vectors pHPV1, pHPV2 were constructed successfully and verified by restriction enzymes and DNA sequencing.8. The effect of pHPV on HPV E6, E7 and P53 expression in Hela.pHPV1 decreased the level of HPV E6, E7 mRNA to 30.75%, 37.58% respectively. and decreased HPV E6, E7 protein to 36.75%, 31.34%, increased P53 protein to 2.991 fold as compared with control respectively. pHPV2 decreased the level of HPV E6, E7 mRNA to 54%, 77.3% and decreased HPV E6, E7 protein to 51.81%, 83.22%, increased P53 protein to 1.878 fold as compared with control respectively.Conclusion1. Synthesized siRNA could inhibit expression of HPV E6, E7 and induce P53 to accumulation.2. Synthesized siRNA could inhibit Hela cell growth and promote cell cycle arrest at G₀/G₁ stage.3. shRNA expressing vectors pHPV1, pHPV2 could inhibit expression of HPV E6, E7 and induce P53 to accumulation. pHPV targeting E6 extron has more efficient inhibition. 4. HPV18 genome 434-452 and 287-305 are RNAi efficient targets.