Downregulation of 5-hydroxymethylcytosine is associated with the progression of cervical intraepithelial neoplasia

Abstract

Around the world, cervical cancer is one of the most common neoplastic diseases among women, and the prognosis of patients in an advanced stage remains poor. To reduce the mortality rate of cervical cancer, early diagnosis and treatment are essential. DNA methylation is an important aspect of gene regulation, and aberrant DNA methylation contributes to carcinogenesis and cancer progression in various cancers. Although 5-methylcytosine (5mC) has been analyzed intensively, the function of 5-hydroxymethylcytosine (5hmC) has not been clarified. The purpose of our study was to identify the molecular biomarkers for early diagnosis of cervical tumors due to epigenetic alterations. To assess the clinical relevance of DNA methylation, we used immunohistochemistry (IHC) to characterize the level of 5hmC in 102 archived human cervical intraepithelial neoplasia (CIN) samples and cervical cancer specimens. The level of 5hmC was significantly decreased between CIN2 and CIN3. The progression of cervical tumors is caused by a reduction of TP53 and RB1 because of HPV infection. We observed that Tp53 and Rb1 were knocked down in mouse embryonic fibroblasts (MEF), a model of normal cells. The level of 5hmC was reduced in Tp53 -knockdown cells, and the expression levels of DNA methyltransferase 1 (DNMT1) and ten-eleven translocation methylcytosine dioxygenase 1 (TET1) were induced. In contrast, there was no significant change in Rb1 -knockdown cells. Mechanistically, we focused on apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) 3B (A3B) as a cause of 5hmC reduction after TP53 knockdown. In the human cell line HHUA with a wild-type TP53 gene, A3B was induced in TP53 -knockdown cells, and A3B knockdown recovered 5hmC levels in TP53 knockdown cells. These data indicate that TP53 suppression leads to 5hmC reduction in part through A3B induction. Moreover, IHC showed that expression levels of A3B in CIN3 were significantly higher than those in both normal epithelium and in CIN2. In conclusion, 5hmC levels are decreased between CIN2 and CIN3 through the TP53-A3B pathway. Since A3B could impair genome stability, 5hmC loss might increase the chances of accumulating mutations and of progressing from CIN3 to cervical cancer. Thus, these epigenetic changes could predict whether CINs are progressing to cancer or disappearing.

Phototheranostics of Cervical Neoplasms with Chlorin e6 Photosensitizer

“…Squamous intraepithelial lesion of the cervix, characterized by squamous epithelial lesions (SIL), cervical intraepithelial neoplasia (CIN) in the form of atypical transformation of the squamous epithelium of the cervix without invasion into the stroma, is a precancerous condition [ 8 , 9 , 10 ]. Squamous intraepithelial cervical lesion is divided into the low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) [ 11 ].…”

“…Active screening among the female population for precancerous lesions and their treatment, if detected, especially in developing countries, provides very high chances of reducing mortality from cervical cancer and, given the prevalence of cervical pathology in young patients of reproductive age, the possibility of maintaining fertility [ 11 ].…”

“…To date, several methods have been proposed for diagnosing squamous intraepithelial lesions of the cervix. The main ones are extended colposcopic examination, cytological examination of scrapings of the ecto- and endocervix, molecular biological methods that allow identifying the HPV genotype with determining the degree of viral load, polymerase chain reaction (PCR), real-time PCR, and nucleic acid sequence-based amplification (NASBA) [ 10 , 11 , 12 ].…”

Immunohistochemical detection of global epigenetic DNA modifications using antibodies to 5-methylcytosine and 5-hydroxymethylcytosine. Impact of antigen retrieval protocols.

“…Reactivation of epigenetically silenced genes is initiated by the TET (ten-eleven translocation) enzyme family, which can convert 5-mC to 5-hydroxymethylcytosine (5-hmC) Detection methods for gene-speci c 5-mC and 5-hmC DNA modi cations are mostly based on methylation speci c polymerase chain reaction (PCR) assays or sequencing analyses of isolated DNA (Rein et al 1998;Storebjerg et al, 2018). The overall hyper-and hypomethylation status of an individual cell can be determined by using antibodies directed against 5-mC and 5-hmC in immunohisto(cyto)chemical procedures applying uorescence or bright eld microscopy (Singh et al 2018; Kato et al, 2020). Immunohistochemical studies targeting 5-mC and 5-hmC have been performed on different types of biological samples, including tumour tissues (Szulwach et al 2011;Singh et al 2018; Kato et al 2020).…”

“…The overall hyper-and hypomethylation status of an individual cell can be determined by using antibodies directed against 5-mC and 5-hmC in immunohisto(cyto)chemical procedures applying uorescence or bright eld microscopy (Singh et al 2018; Kato et al, 2020). Immunohistochemical studies targeting 5-mC and 5-hmC have been performed on different types of biological samples, including tumour tissues (Szulwach et al 2011;Singh et al 2018; Kato et al 2020). Discrepancies in staining patterns, the presence of positive nuclei adjacent to negative ones and the impact of immunocytochemical pretreatment protocols on the detection sensitivity of 5-mC and 5-hmC have been reported (Pendina et al 2017;Wang et al 2019; Estévez-Torres and Baigl 2011).…”