Irina Huica

Quantitative analysis of the relationship between microRNA‑124a, -34b and -203 gene methylation and cervical oncogenesis

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.

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and promoter methylation in cervical oncogenic lesions and cancer.

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.

Type-specific human papillomavirus detection in cervical smears in Romania.

Anton G, Peltecu G, Socolov D, Cornitescu F, Bleotu C, Sgarbura Z, Teleman S, Iliescu D, Botezatu A, Goia CD, Huica I, Anton A‐C. Type‐specific human papillomavirus detection in cervical smears in Romania. APMIS 2010.

LINC01101 and LINC00277 expression levels as novel factors in HPV‐induced cervical neoplasia

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.

Mechanisms of Oncogene Activation

The main modifications that characterize cancer are represented by alterations in onco‐ genes, tumor-suppressor genes, and non-coding RNA genes. Most of these alterations are somatic and the process is a multistep one. Tumors often arise from an initial trans‐ formed cell, and after subsequent genetic alterations different cytogenetically clones lead to tumor heterogeneity. Oncogenes encode proteins that control cell processes such as proliferation and apopto‐ sis. Among these proteins are transcription factors, chromatin remodelers, growth fac‐ tors, growth factor receptors, signal transducers, and apoptosis regulators. Oncogenes activation by structural alteration (chromosomal rearrangement, gene fusion, mutation, and gene amplification) or epigenetic modification (gene promoter hypomethylation, mi‐ croRNA expression pattern) confers an increased or a deregulated expression. Therefore, cells with such alterations possess a growth advantage or an increased survival rate. Giv‐ en the fact that expression profiling of these alterations determines specific signatures as‐ sociated with tumor classification, diagnosis, staging, prognosis, and response to treatment, it highlights the importance of studying oncogenes activation mechanisms and the great potential that they hold as therapeutic tools in the near future.

The Involvement of Epigenetic Mechanisms in HPV‐Induced Cervical Cancer

High‐risk human papillomavirus (HPV) genotypes infection associates with cervical dysplasia and carcinogenesis. hr‐HPV transforming potential is based on E6 and E7 viral oncoproteins actions on cellular proteins. A persistent infection with hr‐HPV leads to progression from precursor lesions to invasive cervical cancer inducing changes in host genome and epigenome. Pathogenesis and development of cancer associated with both genetic and epigenetic defects alter transcriptional program. An important role for malignant transformation in HPV‐induced cervical cancer is played by epigenetic changes that occur in both viral and host genome. Furthermore, there are observations demon‐ strating that oncogenic viruses, once they integrated into host genome, become susceptible to epigenetic alterations made by host machinery. Epigenetic regulation of viral gene expression is an important factor in HPV‐associated disease. Gene expression control is complex and involves epigenetic changes: DNA methylation, histone modification, and non‐coding RNAs activity. Persistent infection with hr‐HPV can cause viral DNA integration into host genome attracting defense mechanisms such as methylation machinery. In this chapter, we aim to review HPV infection role in chromatin modification/ remodeling and the impact of HPV infection on non‐coding RNAs in cervix oncogene‐ sis. The reversible nature of epigenetic alterations provides new opportunities in the development of therapeutic agents targeting epigenetic modification in oncogenesis.