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Background: Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers., Methods: The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients., Results: Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis., Conclusions: The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.

Recently long non-coding RNAs were identified as new factors involved in gene expression regulation. To gain insight into expression pattern of these factors related to E7 HPV18 oncogene, this study uses HeLa cell culture transfected with E7-siRNA. Gene expression profile was investigated using microarray analysis. After analysing the microarray results, we identified 15,387 RNA species differentially expressed in E7-siRNA-transfected cells compared with controls (fold change >2). The expression profiles of lncRNA species highlighted 731 lncRNAs and 203 lincRNAs. We selected two lincRNAs (LINC01101 and LINC00277) and we evaluated the expression profile in HPV-induced neoplasia. Both lincRNAs investigated display a significantly reduced pattern of expression in cervical lesions and cancer, associated with clinical parameters. A connection between HPV presence and lincRNAs was noted. hrHPV-positive samples exhibit significantly reduced LINC01101 and LINC00277 expression level (P < 0.05). These results provide new insights into involvement of lncRNA in HPV-induced cervical cancer, enriching our understanding of their potential role in this pathology., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The aim of this study was to identify and associate the sequence variations of human Papillomavirus 16 (HPV16) genes from women who live in two different areas of Romania and associate them with malignant progression. One hundred twenty-four HPV16-positive cervical isolates were collected, and the E2, E4, E5, E6 and E7 viral genes were sequenced. Two new missense mutations in the E6 gene (C279G and A305C) were found (together or alone, in association with other mutations) in 44 of 124 cases. The most frequently simultaneously mutated genes were E4/E2 hinge, E5 and E6 (p = 0.0004) in squamous cell carcinoma (SCC) samples. Also, for SCC patients, the best-correlated mutation patterns were obtained for E4/E2 hinge-E5 (r = 0.7984; p < 0.0001). No sample was found to have all of the investigated viral genes concurrently mutated. Phylogenetic analysis was performed to characterize the viral variants. Similar results were found for SCC and cervical intraepithelial neoplasia III (CINIII) cases. After all of the target gene sequences were assembled, all patients were found to be infected with viruses of the HPV16- European-German (EG) lineage, and two clusters were identified, the first (55/96 variants) from Moldavia and the second (41/96 variants) from Bucharest. The distinct cluster derived from EG in Moldavia could partially explain the increased frequency of SCC in this area. This study has generated a comprehensive set of sequence variation data on HPV16 circulating in Romania to join the existing data and highlight the important role of HPV16 variants during cervical carcinogenesis.

The aim of this study was to investigate the role of methylenetetrahydrofolate reductase (MTHFR) polymorphisms and MTHFR methylation pattern in cervical lesions development among women from Romania, a country with high prevalence of human papillomavirus (HPV) cervical infections. To achieve this goal, blood samples and cervical cytology specimens (n = 77)/tumour tissue specimens (n = 23) were investigated. As control, blood and negative cytological smears (n = 50) were used. A statistically significant association was found between T allele of C677T polymorphism and cervical lesions, heterozygote women presenting a threefold increased risk (normal/cervical lesions and tumours: wild homozygote 34/41 (0.68/0.41), heterozygote 14/51 (0.28/0.51), mutant homozygote 2/8 (0.04/0.08); OR = 3.081, P = 0.0035). Using χ square test for the control group, the HPV-negative and HPV-positive patients with cervix lesions, a significant correlation between viral infection and T allele of C677T polymorphism (P = 0.0287) was found. The MTHFR promoter was methylated in all HGSIL and tumour samples, significant differences being noted between HPV-positive samples, control group and cases of cervical dysplastic lesions without HPV DNA (P < 0. 0001) and between samples from patients with high-risk (hr)HPV versus low-risk (lr)HPV (P = 0.0026). No correlations between polymorphisms and methylation were observed. In Romania, individuals carrying T allele are susceptible for cervical lesions. MTHFR promoter methylation is associated with cervical severity lesions and with hrHPV., (© 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

High risk human papillomaviruses (hr-HPV) are known to be the etiological agents of cervical cancer disease. On the other hand, other cofactors are considered to be important in cervix carcinogenesis. Mutations in mitochondrial DNA (mtDNA) as well as alterations in mtDNA content have been reported in numerous cancers examined to date. The D-loop region has been shown to be a mutational "hot spot" in human cancer. In order to evaluate the role of mtDNA mutations in cervical lesions progression, cervical specimens (from 79 women, 29-65 years old) were investigated. DNA was isolated (High Pure PCR Template, Roche Diagnostics) from cervical cells from patients with different cytology (normal cervical epithelium, ASCUS-Atypical Squamous Cells of Undetermined Significance, LGSIL-Low-Grade Intraepithelial Lesion, HGSIL-High-Grade Intraepithelial Lesion and SCC-Squamous Cell Carcinoma) and tested for HPV DNA presence (Linear Array HPV Genotyping Test, Roche Diagnostics). To elucidate a causative role of mtDNA in cervical lesions, mtDNA mutations were investigated using Mutector mtDNA kit (TrimGen Corporation). In patients with normal and ASCUS cytology, mtDNA mutations were absent. 16.66% of LGSIL patients presented mutations in D-loop region whereas 28.57% HGSIL cases showed mutations in mtDNA. Mutations were detected in 66.66% cases of SCC cases. These studies provide strong evidence that instability in the D-loop region of mtDNA may be involved in cervical dysplasia. We suggested that mtDNA mutations may play a role in cervical precursor lesions and cancer but their role in the mechanism of carcinogenesis remains to be solved.

Cervical cancer is a leading cause of mortality in women. Molecular and epidemiological data have unequivocally confirmed that high-risk human papillomaviruses (HPVs) are a major etiological agent of this malignancy, as host epigenetic alterations are induced in response to viral infection. The present study evaluated the methylation status of CpG islands surrounding miR-124a, miR-34b and miR-203 in 29 cervical cancer precursor lesions, 31 cervical tumors and 30 normal control samples, with the aim of identifying potential markers of cervical cancer. Direct quantitative methylation-specific PCR (qMSP) was used to evaluate the degree of methylation in the samples. HPV DNA was detected and genotyped using the Linear Array HPV Genotyping Test. Data were statistically analyzed using the Kruskal-Wallis test. Differences in miRNA hypermethylation between the tumor and control samples were highly significant for all the genes tested (p<0.0001). Significant results were also obtained regarding the hypermethylation of miR-124a and miR-203 in the precursor lesions compared to the control samples. Among the 29 patients with precursor lesions, 68.97% (20/29) presented high risk (hr)-HPV genotypes and 31.03% (9/29) were diagnosed with low risk (lr)-HPV. Significant results (p=0.0266) were obtained for miR-124a (hr-HPV group, mean 41.32; lr-HPV group, mean 6.74), revealing a strong association between the methylation process and the hr-HPV genotype. Borderline results (p=0.058) were obtained for miR-203 (hr-HPV group, mean 44.05; lr-HPV group, mean 3.33). These results confirm the involvement of epigenetic alterations in cervical oncogenesis. The lr-HPV precursor lesions had a methylation percent pattern similar to that of the normal samples, while the results for the hr-HPV precursor lesions and tumors indicate a possible involvement of the hr-HPV genotype in the miRNA methylation process.

Human papillomaviruses (HPV) are considered the etiological agents of cervical cancer, especially high-risk genotypes. TGF-beta (transforming growth factor-beta) is well known for its anti-proliferative effects but the neoplastic cells often lose their sensitivity to TGF-beta. A characteristic alteration associated with malignant progression is the loss of responsiveness to TGF-beta1-induced cell growth inhibition. The aim of the present study was to establish the possible role of some members of TGF-beta signalling pathway during cervical cancer development and the possible relationship with HPV infection. In order to establish TGF-beta gene expression levels in cervical oncogenesis, TGF-beta1, TGF-beta1 receptors and Smad2 were investigated in precancerous and cervical cancer samples (Quantitative Real-Time PCR). The study revealed that 84.5% of patients were positive for HPV DNA. The most prevalent HPV genotypes were high-risk HPV 16 and 18 in single or co-infections. Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia to cervical carcinoma. Furthermore, we observed that cervical lesions without HPV infection expressed significantly less TGF-beta1. TGF-betaRI and Smad2 gene expression levels were found to be decreased in SCC and AC samples in contrast with CIN1 and CIN2/3 samples. Our results showed that in human cervical cancer the disruption of TGF-beta/Smad signalling pathway might contribute to the malignant progression of cervical dysplasia. These data emphasize the importance of canonical TGF-beta pathway integrity in carcinogenesis.