Distribution Of HPV Genotypes In The Patients With Cervical Carcinoma And Its Precursors And Transcriptional Gene Silencing Of HPV 16 Oncogenes

Cervical carcinoma is the second most common malignancy in women worldwide,and it severely threaten women' health.Of all the malignant tumors,the incidence of cervical cancer has the most specific factor.Various evidences reveal that the human papillomavirus(HPV)is the single most important etiologic agent in cervical carcinoma.The definite pathogeny and the slowness progression character of cervical cancer are important for the prevention and cure of cervical cancer.HPV genotypes have been classified into high-risk(HR)and low-risk(LR)types according to the relativity of HPV and cervical cancer.A total of 15 HPV genotypes were classified as high-risk types.However,the data on many of the genotypes are limited and some of them are still uncertain.There were variant distributions of HPV genotypes among the different areas,and only few epidemiologic data on HPV genotypes in cervical carcinoma and its precursors were from Mainland China and none from Zhejiang Province.On the HPV detection method,there are some problems in the methods.For example,HCâ…¡cannot distinguish HPV genotypes; Southern blot has low sensitivity;PCR-hybridization includes limited HPV genotypes; and microchip need high cost.So it is important to establish a suitable method to get information of human papillomavirus genotypes distribution in the patients with cervical carcinoma and its precursors.Significant progress has been made in the vaccine of cervical cancer.As it is known that vaccine prevent cervical cancer by the prevention of HPV infection. However,HPV is a high prevalent virus and most of the HPV infection can be cleaned y host defense.So the high cost of the vaccine made it difficult to popularize the vaccine in the Chinese women.It is more realistic to prevent cervical cancer by means of persistent high-risk HPV infection treatment.To date,the developing RNA interfering(RNAi)technology is the most effective gene silencing,and it is suitable for the treatment of persistent high-risk HPV oncogenes.There were some reports about HPV oncogenes silencing with RNAi in posttranscriptional level.However,like other virus,HPV also has ability of variation,which indicates new pathway research is necessary.Recently,it is reported that small interfering RNA(siRNA)homologous to the promoter can induce silence of downstream genes,often accompanied by DNA methylation and other epigenetic mechanisms.So firstly it is important to get information about significance of DNA methylation in different cervical lesions HPV 16 infection status.And after that,it is deserved to use a siRNA homologous to the promoter of HPV16 E6/E7,to explore a new pathway in transcriptional level on HPV oncogene silencing.In this study,we establish a cost-effective HPV genotype detection method (PCR-RFLP),which can distinguish most of genital HPV genotypes.With the method, we detected HPV infection and genotypes in the cervical cancer and it precursors from Zhejiang Province,to get most important genotypes in cervical cancer and its precursors.Then,we detected HPV 16 DNA methylation in asymptomatic infection, cervical cancer and its precusors,to know the significance of HPV 16 DNA methylation in the progress of cervical neoplasia.After that,we designed a siRNA homologous to the promoter of HPV16 E6/E7,to explore a new pathway in transcriptional level on HPV oncogenes silencing. PARTâ… Establishment of a HPV genotype detection methodObjective:To establish a HPV genotype detection methodMethod:Based on the genital HPV sequences,we chose the suitable enzymes to establish PCR-restriction fragment length polymorphism(RFLP)assay.HCâ…¡was used to test sensitivity of PCR-RFLP,and DNA sequencing was used to test the accuracy.Result:Establish a HPV genotype detection method(PCR-RFLP).Establish common HPV genotypes electrophoresis graphs.The accordance of the PCR-RFLP and HCâ…¡was 92.4%in HPV detection.The genotypes PCR-RFLP identified was verified by DNA sequencing.Conclusion:Our PCR-RFLP assay is an accurate and cost-effective method,can distinguish most of the genital HPV genotypes.The method is suitable for HPV genotype detection. PARTâ…¡Distribution of HPV genotypes in the patients with cervical cancer and its precursorsObjective:To investigate the distribution of HPV genotypes in the patients with cervical cancer and its precursors from Zhejiang Province.Method:Cervical Cancer(N=181),cervical intraepithelial neoplasia(CIN)â…¡-â…¢(N=345),and CINâ… (N=105)from Zhejiang Province were detected for HPV genotypes by the improved PCR-RFLP.Age-matched samples of 217 women without cervical neoplasia were detected as control.Result:The prevalence of HPV was 95.0%in CC,88.4%in CINâ…¡-â…¢,and 73.3%in CINâ… ,while only 41.9%in control.High-risk/low-risk HPV ratio showed a significant trend of increase with increased grade of CIN and transformation to carcinoma.A total of 24 HPV genotypes were detected in CC and its precursors.Of those,HPV 16,18,and 58 were the major HPV genotypes in CC,while HPV 16,58,33,and 18 in CIN.High-risk/low-risk HPV ratio showed a significant trend of increase with increased grade of CIN and transformation to carcinoma.The prevalence of HPV16 also increased with increased grade of CIN and transformation to carcinoma.Conclusion:1.The prevalence of HPV was 95.0%in CC,88.4%in CINâ…¡-â…¢,and 73.3%in CINâ… ,which indicated that prevalence of HPV infection in the patients with CC and its precursors in Zhejiang Province is higher than that in other areas in China.2.In conclusion,the predominant HPV genotypes in the patients with cervical cancer were HPV 16,58,and 18,and the major HPV in CIN were HPV 16,58, 33 and 18.It indicated that other than HPV16 and 18,the major HPV genotypes in the patients with cervical cancer and CIN in Zhejiang Province was HPV58,probably as well as 33. PARTâ…¢The methylation of HPV 16 LCR in asymptomatic infection,cervical cancer and its precursorsObjective:To obtain detailed methylation information on crucial regions of HPV 16 LCR and clarify the significance in clinical cervical lesionsMethod:70 clinical samples,including 26 cases of cervical carcinoma,13 of CINâ…¢, 17 of CINâ… -â…¡,and 14 of asymptomatic HPV 16 infections,were detected on the methylation status of HPV 16 promoter and enhancer core by bisulfite modification and pyrosequencing.Result:For the general methylation status of HPV 16 LCR,the proportion of methylated samples was highest in cervical carcinoma,followed by asymptomatic infection.The methylation of CpGs in the promoter region was more common than in the enhancer core region.Conclusion:1.HPV 16 LCR showed hypomethylation in CIN,compared with that in asymptomatic HPV infections,which indicate hypomethylation is probably associated with the initiation of neoplasia.2.HPV 16 LCR showed hypermethylation in cervical cancer,which suggested the hypermethylation HPV16 of cervical cancer maybe the reflection of host defense.3.The methylation of CpGs in the promoter region was more common than in the enhancer core region of HPV16,which indicated that the methylation of promoter is more important than that of the enhancer core in the regulation of transcription. PARTâ…£Promoter-targeting siRNA induces gene silencing of HPV16 E6/E7Objective:To explore whether HPV16 E6/E7 can be induced by promoter-targeting siRNA in SiHa cellsMethod:The siRNA targeting the promoter of human papillomavirus(HPV)16 E6/E7 was designed and transfected it into cervical cancer cell line SiHa. Then the E6 and E7 expression,cellular growth,proliferation,apoptosis, senescence and DNA methylation in HPV16 promoter were observed.Result:E6/E7 mRNA and protein expression were simultaneously reduced,cell growth and proliferation were inhibited,and cell death including apoptosis and senescence was induced,especially senescence was remarkably increased in SiHa cell treated by promoter-siRNA.Meanwhile,a low frequency of promoter methylation was also found when E6/E7 gene expression was inhibited by promoter-targeting siRNA.Conclusion:1.The promoter-targeing siRNA could effectively and simultaneously knocks down both extraneous viral genes E6 and E7 at the transcriptional level,and consequently inhibited the proliferation and induced the death in HPV 16 infected cells.2.The frequency of promoter methylation was probably not always consistent with the expression of downstream genes,which indicated other regulation pathway anticipating in the progression.