EVALUATION OF EXPRESSION OF 4 MIRNAS IN CYTOLOGICAL SAMPLES AS AN ADDITIONAL METHOD OF CERVICAL CANCER DIAGNOSIS

Introduction. Cervical cancer is the 4th most common cancer among women. The main screening method for cervical cancer is cytological examination of the cervical epithelium. This method allows to evaluate the level of cervical dysplasia (malignant potential) but it has several limitations and flaws. Development and implementation of new methods of molecular and genetic analysis in clinical practice can increase informational value of the traditional cytological examination and therefore objectivity in choosing treatment options.Objective is to develop and verify a new method of differential diagnosis of severe intraepithelial dysplasia and invasive cervical cancer.Materials and methods. The method is based on analysis of small non-coding RNA molecules (miRNAs) extracted from the material of traditional Pap smears. Based on literature search, 18 “marker” microRNA molecules were chosen and their expression levels were estimated in 166 samples of Pap smears from cervical canals with different cytological diagnoses. The analysis was performed using reverse transcription polymerase chain reaction.Results. Estimation of ratios between expression levels of miRNA pairs: 126/375; 20а/375; 126/145 allows to differentiate with high confidence borderline states of severe intraepithelial dysplasia and invasive cervical carcinoma (coefficients of quantitative interpretation of the error curve were 0.8, 0.75, 0.72, respectively).Conclusions. Analysis of miRNAs in Pap smear samples is a promising additional method of cervical cancer diagnosis. The method is objective and can be proposed as a supporting technique in cases when cytological examination doesn’t allow to differentiate between borderline pathological states of the cervical epithelium. Implementation of the method in clinical practice requires methodological optimization and additional validation using more clinical material

Histone deacetylase inhibitors cause TP53-dependent induction of p21/Waf1 in tumor cells with TP53 mutations

The p21/Waf1 protein is one of the main regulators of cell cycle arrest and one of the best-known transcriptional targets of the TP53 protein. Here, we demonstrated that there is activation of expression of the p21/Waf1 gene when the cells were treated with sodium butyrate (NaBu), which is a natural histone deacetylase inhibitor, and investigated whether this phenomenon depends on the presence of a functionally active TP53 protein. For this purpose, we compared the effect of NaBu treatment of human cell lines with different TP53 mutation profiles, including wild-type TP53, single nucleotide substitutions, and the complete absence of the TP53 gene. NaBu activated the TP53 protein via hyperacetylation at the lysine residue K382, without significant changes in the level of protein expression. Western blotting showed that the addition of NaBu triggers a significant increase in the p21/Waf1 protein level in both TP53 wild-type cells and in cells with single nucleotide substitutions in the central DNA-binding core domain (DBD) of the TP53 protein. At the same time, no p21/Waf1 protein induction was observed in cells with complete deletion of the TP53 gene. However, NaBu was not able to induce p21/Waf1 production when the expression of TP53 was transiently knocked down by the p53 siRNA. Overall, our results suggest that NaBu-dependent induction of p21/Waf1 does require the presence of TP53 protein, but, unexpectedly, it can occur regardless of mutational changes in the domain responsible for the TP53 binding to DNA. One possible explanations is that NaBu increases the level of TP53 acetylation and the modified protein is able to establish a new network of protein–protein interactions or trigger conformational changes affecting the TP53-dependent transcriptional machinery even when its DNA binding ability is impaired

ANALYSIS OF A MIRNA SET (MIR-21, -181A, AND -146A) AS A METHOD OF DIFFERENTIAL DIAGNOSIS OF THYROID NODULES

Introduction. In clinical practice, differential diagnosis of nodular thyroid diseases poses a serious problem which can be solved by development of new, safe, and specific thyroid tumor markers. Small regulatory RNAs (microRNA, miRNA) are a class of molecules that control gene expression at the post-transcriptional level. miRNAs, both intracellular and secreted into the extracellular space, can be used as markers of various diseases, including cancer. Stability of extracellular miRNAs is determined by binding to proteins and lipoproteins, or by “packing” into membrane microvesicles – exosomes. It is considered that exosomes with specific miRNA content are a result of active and biologically significant secretion, while release of other forms of miRNA is associated with apoptotic or necrotic cell death. This determines diagnostic value of exosomal fraction of circulating miRNAs, which may reflect presence or clinically significant properties of a tumor.The study objective was to explore a method of exosomal miRNA isolation, identify marker miRNAs, and estimate diagnostic value of their analysis.Methods. We used serum samples from 57 patients with nodular thyroid diseases and 13 healthy donors. Exosomes were isolated from serum by ultracentrifugation and analyzed by atomic force microscopy, laser correlation spectroscopy, and western blotting. Analysis of exosomal miRNAs was carried out by RT-PCR.Results. We have identified a specific correlation between certain miRNAs and status of thyroid nodular disease. Expression profiles of three miRNAs (miRNA-21,  miRNA-146a,  and miRNA-181a) exhibited specific characteristics for different forms of nodular thyroid disease and their analysis may have diagnostic value.Conclusions. Exosomes isolated by ultracentrifugation from serum are a source of RNA suitable for subsequent analysis of miRNA. The levels of different miRNAs in serum exosomes may differ by 1–2 times. «Marker» exosomal miRNAs have specific profiles in circulating exosomes of patients with different thyroid nodules. Clinical significance of testing exosomal miRNAs in patients with benign and malignant nodules of the thyroid gland can be increased by a parallel assessment of several molecules and analysis of the profile of their representation in exosomes. MiRNA-181a, -146a, and -21 form a diagnostic combination of «marker» molecules present in the circulating exosomes, which can be extended and used for diagnosis (differential diagnosis) of thyroid nodules