Immunocompetent Mice As a Model for Preclinical Studies of mRNA Vaccine Immunogenicity.

Conducting preclinical studies of mRNA vaccines is complicated by the lack of relevant animal models of the human immune system. Immunocompetent mice are widely used in biomedical research. However, critical differences in the genetics and immune system of mice and humans prevent the study of unique human immune responses in mice. Within the framework of this work, the possibility of modeling the cytotoxic T-cell response to mRNA vaccines encoding the S-protein of the SARS-CoV-2 virus was investigated. High-affinity peptides from S-protein were analyzed for the most frequent allelic variants of human MHC-I, two immunocompetent mouse lines (C57BL/6, BALB/c) and an outbred mouse model of IRC. The results of computer modeling have shown that mouse models can be used in preclinical studies of mRNA vaccines against SARS-CoV-2. Mouse MHC-I is able to present virus peptides that are highly affine for human MHC-I. Moreover, the immunogenicity of some of them has already been confirmed by examining blood samples from patients who have had COVID-19.

SARS-CoV-2 Mutations Lead to a Decrease in the Number of Tissue-Specific MicroRNA-Binding Regions in the Lung.

RNA interference in vertebrates acts as an antiviral mechanism only in undifferentiated embryonic stem cells and is mediated by microRNAs. In somatic cells, host microRNAs also bind to the genomes of RNA viruses, regulating their translation and replication. It has been shown that viral (+)RNA can evolve under the influence of host cell miRNAs. In more than two years of the pandemic, the SARS-CoV-2 virus has mutated significantly. It is quite possible that some mutations could be retained in the virus genome under the influence of miRNAs produced by alveolar cells. We demonstrated that microRNAs in human lung tissue exert evolutionary pressure on the SARS-CoV-2 genome. Moreover, a significant number of sites of host microRNA binding with the virus genome are located in the NSP3-NSP5 region responsible for autoproteolysis of viral polypeptides.

Differences in Presentation of SARS-CoV-2 Omicron Strain Variant BA.1-BA.5 Peptides by HLA Molecules.

In this work, we analyzed the binding affinities of mutated peptides of Omicron strain variants BA.1-BA.5 and the worldwide prevalent HLA alleles. Bioinformatics analysis was conducted with the use of T-CoV web portal. We showed that, for all five viral variants, mutations cause a significant reduction in the number of tightly binding peptides for HLA-B*07:02 and HLA-C*01:02 molecules. At the same time, there were novel potential mutant epitopes (binding affinity less than 50 nM) in case of HLA-A*32:01 allele. Interestingly, mutations caused multidirectional effect on the binding affinities of the viral peptides and HLA-DRB1*03:01. Specifically, Spike protein mutations in the BA.1 variant caused more than 100-fold decrease in PINLVRDLPQGFSAL binding affinity, 10-fold decrease in affinity in the case of BA.2, BA.4, and BA.5 variants, and 30% increase in affinity for the BA.3 variant.

Factors Involved in miRNA Processing Change Its Expression Level during Imitation of Hypoxia in BeWo b30 Cells.

One of the main complications of pregnancy and causes of maternal and perinatal mortality is preeclampsia. The pathophysiology of preeclampsia is associated with the development of placenta and fetal hypoxia and secretion of a number of effective molecules. The human choriocarcinoma cell line BeWo b30 is often used as a model of the placental barrier. It was shown that oxyquinoline derivatives can mimic hypoxia by suppressing HIF-prolyl hydroxylases and the accumulation of HIF-1α. This effect also leads to a change in the expression of microRNAs and their target genes. However, with hypoxia in cells, not only the level of individual miRNAs but also the ratio of miRNA isoforms (isomiRs) can change, presumably due to inaccuracies in the work of the Drosha and Dicer enzymes. In this work, we showed a change in the expression of the factors involved in the maturation of miRNAs when simulating hypoxia in BeWo b30 cells with an oxyquinoline derivative, which may be one of the causes for the change in the ratio of miRNA isoforms.

Differences in the Drosha and Dicer Cleavage Profiles in Colorectal Cancer and Normal Colon Tissue Samples.

Human colorectal adenocarcinoma cell line Caco-2 is often used as a model of healthy intestinal epithelium, in particular, in miRNA studies. The work of the enzymes Drosha and Dicer is an integral part of the process of miRNA formation. Inaccuracies in the work of these enzymes lead to a change in the nucleotide sequences of miRNAs with the formation of new isoforms, which, in turn, can change intracellular regulatory mechanisms. In the framework of this study, it was shown that the quantitative estimates of inaccuracies in Drosha and Dicer activity significantly differ between the specimens of normal colon tissue and malignant colorectal tumors.

Transcriptome Guided Drug Combination Suppresses Proliferation of Breast Cancer Cells.

One of actively developing trends in modern pharmacology is the use of the transcriptome analysis for drug repositioning. We have previously detected two molecular markers of relapses in patients with malignant breast tumors: ELOVL5 and IGFBP6. Poor prognosis is associated with low expression of these markers. Here we analyze the effects of simvastatin and a new potential proteasome inhibitor K7174 inducing expression of IGFBP6 and EVOVL5 on the proliferation of breast cancer cells MDA-MB-231 and DU4475. Compound K7174 potentiates the inhibitory effect of simvastatin on the proliferation of DU4475 cells characterized by low expression of ELOVL5-IGFBP6 pair, but not on the proliferation of MDA-MB-231 cells with high expression of these markers.

Non-Invasive Evaluation of Extracellular Matrix Formation in the Intestinal Epithelium.

Differentiation of colorectal cancer Caco-2 cells was assessed using Affymetrix Human Gene 1.0 ST arrays and by the main electrical parameters measured by bioimpedance spectroscopy. Transepithelial electrical resistance (TEER) was maximum on day 7, then decreased by day 11, and remained stable. The baseline resistance was maximum on day 4, minimum on day 7, but then gradually increased over 2 weeks, which can be explained by the formation of the basement membrane components or the apical mucous layer. Caco-2 cells express components of laminin-111 and laminin-511. A synchronous increase in the expression of mucin 3 mRNA (MUC3A/MUC3B) and mucin 17 mRNA (MUC17) and reduced expression of miR-21 and miR-622 microRNA genes were observed. Possible use of the described approach for studying the formation of extracellular matrix is discussed.

[Expression of Stroma Components in the Lymph Nodes Affected by Prostate Cancer Metastases].

The architecture of stroma is crucial for normal lymph node functioning, as well as for the systemic and local immune response. Data from previous studies in metastatic lymph nodes suggest that changes in the composition of extracellular matrix proteins may occur, not only around the lesion site, but throughout the lymph node stroma. In the present study, the extracellular matrix status was compared between the affected and metastasis-free lymph nodes in prostate cancer. It was found that the presence of tumor cells was associated with significant changes in the expression of genes encoding extracellular matrix components, including α4, ß1 and γl laminin chains, osteonectin, and collagen, as well as with decrease in the expression of lymphatic endothelial cell biomarkers LYVE1 and NRP2. This result suggests that the normal stromal architecture is significantly disrupted in metastatic lymph nodes and may indicate the development of immune tolerance to the tumor cells.

New Fluorescent Reporter Systems for Evaluation of the Expression of E- and N-Cadherins.

During metastatic growth, cells of solid tumors undergo phenotypical changes related to epithelial-mesenchymal transition. Epithelial-mesenchymal transition is regarded as a potential target for prospective antitumor drugs. Fluorescent reporter systems for evaluation of the expression of markers of epithelial and mesenchymal status (E- and N-cadherins) were created. The described approaches can be used for creation of analogous reporter systems.

Role of IGFBP6 Protein in the Regulation of Epithelial-Mesenchymal Transition Genes.

Protein IGFBP6 plays an important role in the pathogenesis of many malignant tumors, including breast cancer. The relationship between IGFBP6 protein and the expression of genes associated with the epithelial-mesenchymal transition is studied. Gene IGFBP6 knockdown does not trigger the epithelial-mesenchymal transition in MDA-MB-231 cells, but modifies significantly the expression of many genes involved in this process. A decrease of IGFBP6 expression can involve a decrease in the expression of N-cadherin and transcription factor Slug.

In Vitro Model for Studying of the Role of IGFBP6 Gene in Breast Cancer Metastasizing.

IGFBP6 gene plays an important role in the pathogenesis of breast cancer. In this work, we performed knockdown of IGFBP6 gene in MDA-MB-231 cells and obtained a stable cell line. Knockdown of IGFBP6 gene was confirmed by the real-time PCR. The influence of IGFBP6 gene on migration and proliferation of breast cancer cells was studied. Knockdown of IGFBP6 gene reduced migration activity of MDA-MB-231 cells and increased their proliferation rate. This in vitro cell model can be used for the further analysis of the role of IGFBP6 gene in the pathogenesis of breast cancer.

Plasma Level of hsa-miR-619-5p microRNA Is Associated with Prostatic Cancer Dissemination beyond the Capsule.

Profiles of circulating microRNA in the plasma of patients with prostate cancer with pathomorphological stages pT2, pT3, and pT4 are analyzed. The level of circulating microRNA hsa-miR-619-5p is elevated in patients with extracapsular spreading of the tumor, increasing significantly from stage pT2 to stage pT4.

Target Cell Glycosylation Determines the Biodistribution of Plant Lectin Viscumin.

We studied the possibility of using viscumin lectin (MLI) for targeted delivery of antitumor drugs. Affinity of MLI for more than 600 oligosaccharide structures was determined and the glycosylation profiles of cell surface in various mouse tissues were analyzed. It was found that biodistribution of MLI was determined by not only expression of oligosaccharides specifically recognized by the lectin in tissue cells, but also by the structure of glycan in general.

Detection of Potential Metastatic Prostate Cancer Circulating Biomarkers by Comparison of miRNA Profiles in DU145 Cells and Culture Medium.

We studied the profile of miRNA secreted into culture medium by DU145 prostate cancer cells and identified a subset of miRNAs characterized by the absence of correlation of their content in the cell and medium, which is likely a result of specific secretion. Three of these miRNA, hsa-miR-4417, hsa-miR-3175, and hsa-miR-6782-5p, exhibit the highest expression and are candidate circulating biomarkers for metastatic activity of prostate cancer. Two of these miRNA are coded by introns of genes linked with genome stability maintenance and chromatin remodeling regulation.

Changes in the Level of Circulating hsa-miR-297 and hsa-miR-19b-3p miRNA Are Associated with Generalization of Prostate Cancer.

We performed diagnostic classification of plasma specimens from patients with non-metastatic and metastatic prostate cancer based on pairs of miRNA that have no individual diagnostic significance. Of 230 miRNA detected in plasma specimens, 3 pairs were diagnostically significant. The miRNA pair hsa-miR-19b-3p and hsa-miR-297 demonstrated highest sensitivity and specificity. Among common target genes of these miRNA, CFL2 gene associated with cell mobility was detected.

MicroRNA hsa-miR-4674 in Hemolysis-Free Blood Plasma Is Associated with Distant Metastases of Prostatic Cancer.

We analyzed microRNA profile in hemolysis-free blood plasma of patients with prostatic cancer. The metastatic form of prostatic cancer was found to be associated with increased levels of hsa-miR-22-3p, hsa-miR-663a, and hsa-miR-4674 in comparison with non-metastatic form. Common candidate target genes of these microRNA include JUNB, KMT2A, and XPO6.

Plasma Levels of hsa-miR-619-5p and hsa-miR-1184 Differ in Prostatic Benign Hyperplasia and Cancer.

Peripheral blood plasma profiles of circulating microRNA expression were analyzed in patients with prostatic cancer and benign hyperplasia. In prostatic cancer, significant increase in hsa-miR-619-5p and hsa-miR-1184 microRNA expression and significant decrease in hsalet-7b-5p and hsa-let-7c-5p microRNA expression were observed. The role of the relationship between the microRNA expression and the activities and functions of host genes with introns encoding these microRNA is discussed.

Role of L1CAM in the Regulation of the Canonical Wnt Pathway and Class I MAGE Genes.

Molecule L1CAM is specific for nerve cells and tumors of various localizations. The expression of L1CAM is significantly higher in melanoma in comparison with benign nevi and correlates with the progress of melanoma and transition from radial to vertical growth. Monoclonal antibodies to L1CAM effectively and specifically attenuate melanoma growth, though stimulates the epithelial-mesenchymal transition. shRNA-mediated knock-down of L1CAM showed the involvement of L1CAM in regulation of activity of the canonical Wnt pathway and expression of genes of class I melanoma-associated antigens (MAGE).

Highly informative marker sets consisting of genes with low individual degree of differential expression.

Genes with significant differential expression are traditionally used to reveal the genetic background underlying phenotypic differences between cancer cells. We hypothesized that informative marker sets can be obtained by combining genes with a relatively low degree of individual differential expression. We developed a method for construction of highly informative gene combinations aimed at the maximization of the cumulative informative power and identified sets of 2-5 genes efficiently predicting recurrence for ER-positive breast cancer patients. The gene combinations constructed on the basis of microarray data were successfully applied to data acquired by RNA-seq. The developed method provides the basis for the generation of highly efficient prognostic and predictive gene signatures for cancer and other diseases. The identified gene sets can potentially reveal novel essential segments of gene interaction networks and pathways implied in cancer progression.

Suppression of ITGB4 Gene Expression in PC-3 Cells with Short Interfering RNA Induces Changes in the Expression of ß-Integrins Associated with RGD-Receptors.

We studied the effect of transfection of PC-3 prostate cancer cells with a plasmid encoding shRNA complimentary to a fragment of integrin ß4 (ITGB4). The results attest to considerable changes in the transcriptome of transfected cells. For instance, compensatory changes in the expression of integrin family genes were found.