[The Minor T Allele of the Single Nucleotide Polymorphism rs 13360222 Decreases the Activity of the HAVCR2 Gene Enhancer in a Cell Model of Human Macrophages].

The TIM-3 receptor, encoded by the Hepatitis A Virus Cellular Receptor 2 (HAVCR2) gene, is an immune checkpoint and plays an important role in preventing the development of autoimmune reactions. This receptor is expressed on the surface of various immunocytes and its functions in myeloid cells remain poorly understood, compared to the role of T cell specific TIM-3 that is actively studied in the context of the search for promising therapeutic targets in cancer immunotherapy. During this study, we performed deletion analysis of the promoter region of the HAVCR2 gene, as well as functional characterization of its enhancer, and studied the effect of a number of single nucleotide polymorphisms (SNPs) on the activity of these regulatory elements in the relevant model of human macrophage-like cells-U937 activated monocytes. We have shown that the SNPs rs10515746(A) and rs4704853(A) located in the HAVCR2 gene promoter and associated with the development of a number of pathologies, do not affect the activity of the promoter in activated monocytes. However, a minor T variant of SNP rs13360222 located in the enhancer in the third intron of the gene, significantly reduces the ability of the enhancer to activate the HAVCR2 promoter, presumably due to weakening of the binding of nuclear receptor ESR2 to the respective region.

[Serum of Mice Immunized with Mt1-MMP Metalloproteinase Reduces Migration Potential of Pancreatic Cancer Cells].

Expression levels of matrix metalloproteinases, in particular MT1-MMP, are elevated in pancreatic cancer (PC) cells, and this is associated with increased tumor proliferation, invasion, and migration. MT1-MMP is considered a promising target for drug therapy of PC, but the use of inhibitors and therapeutic antibodies to MT1-MMP is limited because maximal efficiency is only observed in a narrow time interval, at the early asymptomatic stages of the disease. This problem could be solved by immunization to MPs at the moment of detection of the primary tumor. This therapeutic effect could be provided by specific antibodies that can be re-produced in case of relapses. Here, we selected the optimal mode for immunization of mice with MT1-MMP fragments that allows us to obtain a high titer of specific antibodies in the blood serum. The obtained antiserums effectively inhibited MT1-MMP enzymatic activity, migration of PANC-02 PC cells through the collagen matrix, and activation of the main inducers of epithelial -mesenchymal transition, TGF-ß and MMP-2. These results maybe useful in the development of drugs for PC treatment, and the approach we propose might form the basis for design of antitumor drugs with prolonged action.

[Noncoding Polymorphism rs6832151 Is an Attractive Candidate for Genome Editing Aimed at Finding New Molecular Mechanisms of Autoimmune Diseases].

Currently only a small fraction of the proteins encoded in the human genome serve as pharmaceutical targets. Genome-wide association studies are a powerful tool to uncover new genetic loci responsible for predisposition to complex diseases, such as autoimmune disorders. However, further work is still required to identify causative single-nucleotide polymorphisms (SNPs) which directly mediate the disease risk at these loci, and to determine their target genes. These genes can be located millions base pairs away from the regulatory SNPs. Here, by using bioinformatic tools and databases, we identified five intergenic autoimmunity-associated polymorphisms with high probability of being causative, for which the target genes are still unknown. We tested their ability to influence gene expression using luciferase reporter system. The polymorphism rs6832151 affected the reporter expression in the CEM human T-cell line upon the highest enhancer activity. Target genes of this SNP could be further identified by introducing point mutations to the genome and comparison of transcriptomes of the derivative cell sublines carrying alternative alleles of rs6832151.

Constitutive Expression of NRAS with Q61R Driver Mutation Activates Processes of Epithelial-Mesenchymal Transition and Leads to Substantial Transcriptome Change of Nthy-ori 3-1 Thyroid Epithelial Cells.

The Q61R mutation of the NRAS gene is one of the most frequent driver mutations of thyroid cancer. Tumors with this mutation are characterized by invasion into blood vessels and formation of distant metastases. To study the role of this mutation in the growth of thyroid cancer, we developed a model system on the basis of thyroid epithelial cell line Nthy-ori 3-1 transduced by a lentiviral vector containing the NRAS gene with the Q61R mutation. It was found that the expression of NRAS(Q61R) in thyroid epithelial cells has a profound influence on groups of genes involved in the formation of intercellular contacts, as well as in processes of epithelial-mesenchymal transition and cell invasion. The alteration in the expression of these genes affects the phenotype of the model cells, which acquire traits of mesenchymal cells and demonstrate increased ability for survival and growth without attachment to the substrate. The key regulators of these processes are transcription factors belonging to families SNAIL, ZEB, and TWIST, and in different types of tumors the contribution of each individual factor can vary greatly. In our model system, phenotype change correlates with an increase in the expression of SNAIL2 and TWIST2 factors, which indicates their possible role in regulating invasive growth of thyroid cancer with the mutation of NRAS(Q61R).

Isolation of Large Amounts of Highly Pure Mitochondria for "Omics" Studies.

Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.