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Title/Abstract/Keywords

Mechanism of progression in cervical intraepithelial neoplasia

Rotondo, John Charles (2015) Mechanism of progression in cervical intraepithelial neoplasia. PhD Thesis , Università degli Studi di Ferrara.

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    Abstract

    The cervical intraepithelial neoplasia (CIN) grade 1, 2 and 3 are the precursor lesions of cervical cancer. Due to the lack of useful study models, the molecular mechanisms involved in CIN progression are still largely unknown. The overall aim of my study was to investigate the molecular mechanisms occurring in CIN lesion progression. This goal was achieved by investigating gene expression profiles and methylation status of gene promoters in a novel study model of tumor progression, i.e. primary colonies of CIN2 and CIN3 keratinocytes derived from CIN2 and CIN3 lesions. To this purpose, the first aim of my study was to develop a rapid and simple cell culture protocol enables primary colonies of HPV16-CIN2 and HPV16-CIN3 keratinocytes to be derived from small tissue fragments of CIN2 and CIN3 lesions. The primary colonies of CIN2 and CIN3 keratinocytes were then investigated for presence epithelial and cervical markers showing cytokeratin -14, -17, -19 expression. In the second part of my study primary colonies of HPV16-CIN2 and HPV16-CIN3 keratinocytes were chosen to be investigated by microarray analysis. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16-CIN2 keratinocytes and in HPV16-CIN2 keratinocytes compared with HPV16-CIN3 keratinocytes. Thirty-seven candidate genes with continuously decreasing or increasing expression during CIN progression were identified. Specifically, 23 down-expressed genes involved in antiviral immune response and differentiation and 14 over-expressed genes involved in proliferation and tumor invasiveness were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of HPV16-CIN toward cancer. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression. Finally, in the third part of my study, to verify whether down-expression of genes in CIN2 and CIN3 keratinocytes depended on DNA promoter methylation, I investigated the methylation status of RARB and IRF6 promoter, both tumor suppressor genes, in relationship with expression of their two pathway-correlated genes, p63 and c-JUN. The epigenetic analysis revealed a hypermethylation of RARB and IRF6 gene promoters in CIN2 and CIN3 keratinocytes compared to normal keratinocytes as well as a progressive hypermethylation of RARB promoter region from normal to CIN2 keratinocytes and from CIN2 to CIN3 keratinocytes. Consistently, a gradual up-regulation of p63 and c-JUN from CIN2 to CIN3 keratinocytes was detected. It is conceivable that the hypermethylation of RARB and IRF6 promoter region may cause a consequent RARB and IRF6 down-regulation and this reduction of expression could enhance c-JUN and p63 expression in CIN keratinocytes. In conclusion, in my study the molecular mechanisms occurring in CIN lesions progression were investigated. In particular, it was investigated gene expression profiles and methylation status of gene promoters in a novel study model, i.e. primary colonies of CIN2 and CIN3 keratinocytes derived from CIN2 and CIN3 lesions. Gene expression analysis revealed 37 down-expressed or over-expressed genes which may contribute to CIN progression. One of these genes, the phosphoglycerate dehydrogenase, which resulted over-expressed at both mRNA and protein level in CIN2 and CIN3 keratinocytes and in CIN2, CIN3 and cancer tissues, respectively, is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression. Aberrant promoter hypermethylation of RARB and IRF6 genes also may be a potential epigenetic prognostic marker for CIN progression.

    Item Type:Thesis (PhD Thesis)
    Date:27 February 2015
    Tutor:Tognon, Mauro
    Coordinator:Capitani, Silvano
    Institution:Università degli Studi di Ferrara
    PhD:XXVII Anno 2012 > SCIENZE BIOMEDICHE
    Divisions:Dipartimento > Morfologia ed embriologia
    Subjects:Area 05 - Scienze biologiche > BIO/13 Biologia applicata
    Uncontrolled Keywords:HPV, CIN, cervical intraepithelian neoplasia, gene expression, methylation
    Deposited on:28 Jul 2015 11:01

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