The Role Of APOBEC3 Protein In The Pathogenesis Of Cervical Cancer Induced By HPV

Purpose HPV infection can lead to cervical intraepithelial neoplasia(CIN)and even cervical cancer.Persistentin fection of high risk HPV subtypes is recognized as a main cause of invasive cervical cancer.Among the high risk HPV genotypes,HPV 16 accounts for most cases of invasive cervical cancer and its precursor lesions.HPV integration is an essential genetic event in cervical carcinogenesis.Under certain circumstances,HPV DNA can integrate into the host genome,while 90% of detected infections are cleared in 2-3 years.The loss of the viral E2 gene is a common consequence of HPV integration,which may lead to an elevated transcription and expression of E6 and E7 gene due to the fact that the two oncoproteins are negatively controlled by the viral E2 protein.Recent studies have identified the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3(APOBEC3s)as a new member of human innate immunity,given its potent antiviral function.The APOBEC3 s can convert deoxycytidines to deoxyuridines by deamination activity within single stranded DNA and/ or RNA.C-to-U deamination eventsmanifest as C-to-T transitions and C-to-G transversions in host genome.Although increasing evidence revealed that APOBEC3 A and APOBEC3 G are potential HPV DNA mutators,APOBEC3 s,the “iceberg” of HPV induced hypermutation is little known.Further elucidation of APOBEC3 s induced hypermutation is in urgent need,especially in clinical samples.The aim of this study is to examine the HPV DNA hypermutation in clinical samples.By further analyzing hypermutation,we can demonstrate the associations between grades of squamous intraepithelial lesions and mutation load.Moreover,demonstrate the possible role of APOBEC3 s in the progression of HPV induced cervical cancer.Methods 1.APOBEC3 s expression was analyzed using quantitative reverse transcription-polymerasechain reaction(q RT-PCR).Total RNA was extracted from Caski,C33 A and Ha Cat cervical cancer cell lines with the TRIZOL reagent in accordance with the manufacturer’s specifications.We intend to explore the associations between the APOBEC3 m RNA expression and the HPV related cervical cancer.2.The Ethics Committee of Dalian Women’s and Children’s Hospital approved this study.A total of 700 women from the outpatient clinic underwent cervical examines(Pap smear and HPV genotyping).Patients with HPV positive and/ or HPV 16 positive are recorded.A statistical analysis was performed about the positive rate of HPV and HPV 16.Written informed consent was obtained from all patients in this study.3.A Total of 46 biopsies of cervical exfoliated cells were collected from patient using a Cervex-brushcombi and stored in Thinprep collection media.Total DNA was extracted from a 200 ml aliquot of the suspended cell samples using the QIAamp DNA Blood Mini kit(Qiagen,Valencia,CA),resulting in a final elution volume of 100 ml Tris-EDTA buffer,followed by PGMY-PCR,and reverse blot hybridization-based HPV genotyping.Histological diagnosis was made using hematoxylin-eosin-stained sections obtained by punch biopsy according to the World Health Organization classification.Forty-six HPV16-positive DNA samples were randomly selected and subjected to E2 nested PCR.Subsequently,Sanger sequencing of their DNA was carried outto identify mutation in each gene.Mutation load,mutation frequency and dinucleotide preference of hypermutation was further analyzed.Results1.q RT-PCR was performedto determine expression levels of APOBEC3 A and APOBEC3 B in cervical cancer cell lines.Results showed high expression levels of APOBEC3 A and B m RNA in HPV positive cervical cancer cell line,and HPV negative cell line showed low expression levels of them.2.In the 700 cases,the HPV testing positive were 110,the HPV positive rate was 15.71%,of which 46 cases were positive for HPV16,making up 41.82%,proving that HPV16 is the predominant oncogenic types so far.3.Among the 46 samples,26 were found to havepositive E2 gene under our experimental condition.4.To further investigate hypermutation in these samples,a nested PCR detecting E2 gene was performed.Results showed that E2 hypermutation was observed in 11 out of 26 samples.The rest 15 samples has less than 4 mutation in each molecular compared to the controlled plasmid.5.To confirm hypermutation signature,we performed TA clone and DNA sequencing.Results showed that 1155 G/C->A/T mutations were identified in 10890 dinucleotides.6.E2 gene sequencing of G/C mutation revealed a preference of dinucleotides,approximately 80% of mutations were Cp C and Tp C,suggesting that the APOBEC3 proteins may be the major sources of mutation.7.The hypermutation load was significantly higher in H-SIL,indicating a positive correlation between grades of squamous intraepithelial lesionand hypermutation load.Conclusion 1.APOBEC3 A and 3B m RNA expressed abundantly in HPV positive cervical cancer cell line,while expressed in a relatively low level in the other HPV negative cell lines.2.The viral E2 gene could be hypermutated by APOBEC3 Aand 3B,the mutation load of which was associated with the development and carcinogenesis of cervical cancer.