engleski

Detection of HPV16 integration in the human genome by amplification of papillomavirus oncogene transcripts (APOT)

Infection with high-risk human papillomavirus (HR HPV) has been confirmed as the necessary cause for the development of cervical cancer. However, the infection alone is not sufficient, and additional factors that contribute to the malignant transformation are under investigation. Cervical cancer develops through three distinctive precancerous stages, namely cervical inthraepithelial neoplasias grade 1 to 3 (CIN 1-3). The turning point in the cervical carcinogenesis is the integration of viral DNA into the human genome. The integration stabilizes and even enhances the expression of two major viral oncoproteins E6 and E7 that are interacting with a number of cellular factors involved in the regulation of cell cycle. Lower grade lesions (CIN 1) almost exclusively contain episomal forms of HPV 16, while in the higher grade lesions and cancer increasing number of HPV genomes is found in the integrated form. Therefore, integration of HR HPV should be considered as a possible prognostic factor for the severity of the precancerous lesions. Although integration is random, the site of integration in the genome might have an additional effect on the cellular genes that could be involved in the process of carcinogenesis. To determine the frequency of integration of HPV 16 DNA in the different grades of precancerous lesions (CIN 1-3), 20 samples of RNA isolated from cervical swabs was tested for the presence of HPV 16 oncogene transcripts by the APOT method. APOT consists of reverse transcription of total RNA isolated from cervical swabs with oligodT primer that contains a linker sequence, followed by 2 consecutive PCR amplifications of the reversely transcribed cDNA by a combination of HPV specific primer and linker specific primer. The episomal transcripts are 1050 bp in size, while all amplicons that differ from that size point to the presence of integrated transcript, which has to be further confirmed by sequencing. In 9 samples we were not able to amplify oncogene transcripts of HPV 16. In 6 of 11 (54.5 %) successfully analyzed samples, only episomal transcripts were amplified, in 4 of 11 (36.4 %) integrated transcripts, and in 1 sample both integrated and episomal transcript were amplified. In one sample with integrated transcript of HPV 16, the site of integration in the human genome was determined, on chromosome 17, at locus 17q23 in the region of VMP1 gene, which has a role in the apoptosis and autophagy.

human papillomavirus; integration

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