The interaction of cold atmospheric plasma (CAP) with biotargets is accompanied by chemical reactions on their surfaces and insides, and it has great potential as an anticancer approach. This study discovers the molecular mechanisms that may explain the selective death of tumor cells under CAP exposure. To reach this goal, the transcriptional response to CAP treatment was analyzed in A549 lung adenocarcinoma cells and in lung-fibroblast Wi-38 cells. We found that the CAP treatment induced the common trend of response from A549 and Wi-38 cells—the p53 pathway, KRAS signaling, UV response, TNF-alpha signaling, and apoptosis-related processes were up-regulated in both cell lines. However, the amplitude of the response to CAP was more variable in the A549 cells. The CAP-dependent death of A549 cells was accompanied by DNA damage, cell-cycle arrest in G2/M, and the dysfunctional response of glutathione peroxidase 4 (GPx4). The activation of the genes of endoplasmic reticulum stress and ER lumens was detected only in the A549 cells. Transmission-electron microscopy confirmed the alteration of the morphology of the ER lumens in the A549 cells after the CAP exposure. It can be concluded that the responses to nuclear stress and ER stress constitute the main differences in the sensitivity of tumor and healthy cells to CAP exposure.

Oncolytic virotherapy is a rapidly evolving approach that aims to selectively kill cancer cells. We designed a promising recombinant vaccinia virus, VV-GMCSF-Lact, for the treatment of solid tumors, including glioma. We assessed how VV-GMCSF-Lact affects human cells using immortalized and patient-derived glioma cultures and a non-malignant brain cell culture. Studying transcriptome changes in cells 12 h or 24 h after VV-GMCSF-Lact infection, we detected the common activation of histone genes. Additionally, genes associated with the interferon-gamma response, NF-kappa B signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathways showed increased expression. By contrast, genes involved in cell cycle progression, including spindle organization, sister chromatid segregation, and the G2/M checkpoint, were downregulated following virus infection. The upregulation of genes responsible for Golgi vesicles, protein transport, and secretion correlated with reduced sensitivity to the cytotoxic effect of VV-GMCSF-Lact. Higher expression of genes encoding proteins, which participate in the maturation of pol II nuclear transcripts and mRNA splicing, was associated with an increased sensitivity to viral cytotoxicity. Genes whose expression correlates with the sensitivity of cells to the virus are important for increasing the effectiveness of cancer virotherapy. Overall, the results highlight molecular markers, biological pathways, and gene networks influencing the response of glioma cells to VV-GMCSF-Lact.

Cold atmospheric plasma (CAP) is an intensively-studied approach for the treatment of malignant neoplasms. Various active oxygen and nitrogen compounds are believed to be the main cytotoxic effectors on biotargets; however, the comprehensive mechanism of CAP interaction with living cells and tissues remains elusive. In this study, we experimentally determined the optimal discharge regime (or semi-selective regime) for the direct CAP jet treatment of cancer cells, under which lung adenocarcinoma A549, A427 and NCI-H23 cells demonstrated substantial suppression of viability, coupled with a weak viability decrease of healthy lung fibroblasts Wi-38 and MRC-5. The death of CAP-exposed cancer and healthy cells under semi-selective conditions was caspase-dependent. We showed that there was an accumulation of lysosomes in the treated cells. The increased activity of lysosomal protease Cathepsin D, the transcriptional upregulation of autophagy-related MAPLC3B gene in cancer cells and the changes in autophagy-related proteins may have indicated the activation of autophagy. The addition of the autophagy inhibitor chloroquine (CQ) after the CAP jet treatment increased the death of A549 cancer cells in a synergistic manner and showed a low effect on the viability of CAP-treated Wi-38 cells. Downregulation of Drp1 mitochondrial protein and upregulation of PINK1 protein in CAP + CQ treated cells indicated that CQ increased the CAP-dependent destabilization of mitochondria. We concluded that CAP weakly activated pro-survival autophagy in irradiated cells, and CQ promoted CAP-dependent cell death due to the destabilization of autophagosomes formation and mitochondria homeostasis. To summarize, the combination of CAP treatment with CQ could be useful for the development of cold plasma-based antitumor approaches for clinical application.

The GAS5 gene encodes a long non-coding RNA (lncRNA) and intron-located small nucleolar RNAs (snoRNAs). Its structure, splice variants, and diverse functions in mammalian cells have been thoroughly investigated. However, there are still no data on a successful knockout of GAS5 in human cells, with most of the loss-of-function experiments utilizing standard techniques to produce knockdowns. By using CRISPR/Cas9 to introduce double-strand breaks in the terminal intronic box C/D snoRNA genes (SNORDs), we created monoclonal cell lines carrying continuous deletions in one of the GAS5 alleles. The levels of GAS5-encoded box C/D snoRNAs and lncRNA GAS5 were assessed, and the formation of the novel splice variants was analyzed. To comprehensively evaluate the influence of specific SNORD mutations, human cell lines with individual mutations in SNORD74 and SNORD81 were obtained. Specific mutations in SNORD74 led to the downregulation of all GAS5-encoded SNORDs and GAS5 lncRNA. Further analysis revealed that SNORD74 contains a specific regulatory element modulating the maturation of the GAS5 precursor transcript. The results demonstrate that the maturation of GAS5 occurs through the m6A-associated pathway in a SNORD-dependent manner, which is a quite intriguing epitranscriptomic mechanism.

Small nucleolar RNAs (snoRNAs) are a highly expressed class of non-coding RNAs known for their role in guiding post-transcriptional modifications of ribosomal RNAs and small nuclear RNAs. Emerging studies suggest that snoRNAs are also implicated in regulating other vital cellular processes, such as pre-mRNA splicing and 3′-processing of mRNAs, and in the development of cancer and viral infections. There is an emerging body of evidence for specific snoRNA’s involvement in the optimal replication of RNA viruses. In order to investigate the expression pattern of snoRNAs during influenza A viral infection, we performed RNA sequencing analysis of the A549 human cell line infected by influenza virus A/Puerto Rico/8/1934 (H1N1). We identified 66 that were upregulated and 55 that were downregulated in response to influenza A virus infection. The increased expression of most C/D-box snoRNAs was associated with elevated levels of 5’- and 3’-short RNAs derived from this snoRNA. Analysis of the poly(A)+ RNA sequencing data indicated that most of the differentially expressed snoRNAs synthesis was not correlated with the corresponding host genes expression. Furthermore, influenza A viral infection led to an imbalance in the expression of genes responsible for C/D small nucleolar ribonucleoprotein particles’ biogenesis. In summary, our results indicate that the expression pattern of snoRNAs in A549 cells is significantly altered during influenza A viral infection.

Glioma is the most common and heterogeneous primary brain tumor. The development of a new relevant preclinical models is necessary. As research moves from cultures of adherent gliomas to a more relevant model, neurospheres, it is necessary to understand the changes that cells undergo at the transcriptome level. In the present work, we used three patient-derived gliomas and two immortalized glioblastomas, while their cultivation was carried out under adherent culture and neurosphere (NS) conditions. When comparing the transcriptomes of monolayer (ML) and NS cell cultures, we used Enrichr genes sets enrichment analysis to describe transcription factors (TFs) and the pathways involved in the formation of glioma NS. It was observed that NS formation is accompanied by the activation of five common gliomas of TFs, SOX2, UBTF, NFE2L2, TCF3 and STAT3. The sets of transcripts controlled by TFs MYC and MAX were suppressed in NS. Upregulated genes are involved in the processes of the epithelial–mesenchymal transition, cancer stemness, invasion and migration of glioma cells. However, MYC/MAX-dependent downregulated genes are involved in translation, focal adhesion and apical junction. Furthermore, we found three EGFR and FGFR signaling feedback regulators common to all analyzed gliomas—SPRY4, ERRFI1, and RAB31—which can be used for creating new therapeutic strategies of suppressing the invasion and progression of gliomas.

Extracellular vesicles (EVs) produced by various cell types are heterogeneous in size and composition. Changes in the RNA sets of EVs in biological fluids are considered the basis for the development of new approaches to minimally invasive diagnostics and the therapy of human diseases. In this study, EVs were obtained from the blood of healthy donors by centrifugation, followed by ultracentrifugation. It was shown that EVs consist of several populations including small exosome-like vesicles and larger microvesicle-like particles. The composition of EVs’ RNAs was determined. A549 lung adenocarcinoma cells were incubated with EV and the NGS analysis of differentially expressed genes was performed. During the incubation of A549 cells with EVs, the levels of mRNA encoding components for the NF-kB signaling pathway increased, as well as the expression of genes controlled by the NF-kB transcription factor. Overall, our results suggest that components of EVs trigger the NF-kB signaling cascade in A549 cells, leading to the transcription of genes including cytokines, adhesion molecules, cell cycle regulators, and cell survival factors. Our data provide insight into the interaction between blood EVs and human cells and can be used for designing new tools for the diagnosis and treatment of human diseases.

Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. The study of the pathogenesis of GBM, as well as the development of targeted oncolytic drugs, require the use of actual cell models, in particular, the use of 3D cultures or neurospheres (NS). During the formation of NS, the adaptive molecular landscape of the transcriptome, which includes various regulatory RNAs, changes. The aim of this study was to reveal changes in the expression of microRNAs (miRNAs) and their target mRNAs in GBM cells under conditions of NS formation. Neurospheres were obtained from both immortalized U87 MG and patient-derived BR3 GBM cell cultures. Next generation sequencing analysis of small and long RNAs of adherent and NS cultures of GBM cells was carried out. It was found that the formation of NS proceeds with an increase in the level of seven and a decrease in the level of 11 miRNAs common to U87 MG and BR3, as well as an increase in the level of 38 and a decrease in the level of 12 mRNA/lncRNA. Upregulation of miRNAs hsa-miR: -139-5p; -148a-3p; -192-5p; -218-5p; -34a-5p; and -381-3p are accompanied by decreased levels of their target mRNAs: RTN4, FLNA, SH3BP4, DNPEP, ETS2, MICALL1, and GREM1. Downregulation of hsa-miR: -130b-5p, -25-5p, -335-3p and -339-5p occurs with increased levels of mRNA-targets BDKRB2, SPRY4, ERRFI1 and TGM2. The involvement of SPRY4, ERRFI1, and MICALL1 mRNAs in the regulation of EGFR/FGFR signaling highlights the role of hsa-miR: -130b-5p, -25-5p, -335-3p, and -34a-5p not only in the formation of NS, but also in the regulation of malignant growth and invasion of GBM. Our data provide the basis for the development of new approaches to the diagnosis and treatment of GBM.

Human influenza remains a serious public health problem. This data article reports the transcriptome analysis data of human cell lines infected with influenza A/Puerto Rico/8/1934 (H1N1) virus. Mock-infected cells were included as controls. Human embryonic fibroblasts (MRC-5) and immortalized cell lines (A549, HEK293FT, WI-38 VA-13) were selected for RNA sequencing using Illumina NextSeq500 platform. Raw data were applied to the bioinformatic pipeline, which includes quality control with FastQC and MultiQC, adapter and quality trimming with Cutadapt, filtering to the genome of influenza A with STAR, transcript quantification with Salmon tool (GRCh38_RefSeq_Transcripts). Differential expressed genes were identified using R package DESeq2 with FDR-adjusted p-value < 0.001 and absolute value of log2(FC) > 1. Lists of differentially expressed genes is provided. The raw and processed RNA-seq data presented in this article were deposited to the European Nucleotide Archive via the ArrayExpress partner repository with the dataset accession number E-MTAB-9511 .

CRISPR technologies are nowadays widely used for targeted knockout of numerous protein-coding genes and for the study of various processes and metabolic pathways in human cells. Most attention in the genome editing field is now focused on the cleavage of protein-coding genes or genes encoding long non-coding RNAs (lncRNAs), while the studies on targeted knockout of intron-encoded regulatory RNAs are sparse. Small nucleolar RNAs (snoRNAs) present a class of non-coding RNAs encoded within the introns of various host genes and involved in post-transcriptional maturation of ribosomal RNAs (rRNAs) in eukaryotic cells. Box C/D snoRNAs direct 2'-O-methylation of rRNA nucleotides. These short RNAs have specific elements in their structure, namely, boxes C and D, and a target-recognizing region. Here, we present the study devoted to CRISPR/Cas9-mediated editing of box C/D snoRNA genes in Gas5. We obtained monoclonal cell lines carrying mutations in snoRNA genes and analyzed the levels of the mutant box C/D snoRNA as well as the 2'-O-methylation status of the target rRNA nucleotide in the obtained cells. Mutations in SNORD75 in the obtained monoclonal cell line were shown to result in aberrant splicing of Gas5 with exclusion of exons 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results suggest that SNORD75 contains an element for binding of some factors regulating maturation of Gas5 pre-lncRNA. We suggest that METTL3/METTL14 is among such factors, and m6A-methylation pathways are involved in regulation of Gas5 splicing. Our results shell light on the role of SNORDs in regulating splicing of the host gene.

The phenomenon of chemotherapy-resistant cancers has necessitated the development of new therapeutics as well as the identification of specific prognostic markers to predict the response to novel drugs. Primary cancer cells provide a model to study the multiplicity of tumourigenic transformation, to investigate alterations of the cellular response to various molecular stimuli, and to test therapeutics for cancer treatment.

Short nuclear regulatory RNAs play a key role in the main stages of maturation of the precursors of the major RNA species. Small nuclear RNAs (snRNAs) form the core of the spliceosome and are responsible for the splicing of pre-mRNA molecules. Small nucleolar RNAs (snoRNAs) direct post-transcriptional modification of pre-rRNAs. A promising strategy for the development of non-coding RNA (ncRNAs) mimicking molecules is the introduction of modified nucleotides, which are normally present in natural ncRNAs, into the structure of synthetic RNAs. We have created a set of snoRNAs and snRNA analogs and studied the effect of base modifications, specifically, pseudouridine (Ψ) and 5-methylcytidine (m5C), on the immune-stimulating and cytotoxic properties of these RNAs. Here, we performed a whole-transcriptome study of the influence of synthetic snoRNA analogs with various modifications on gene expression in human cells. Moreover, we confirmed the role of PKR in the recognition of snoRNA and snRNA analogs using the short hairpin RNA (shRNA) technique. We believe that the data obtained will contribute to the understanding of the role of nucleotide modification in ncRNA functions, and can be useful for creating the agents for gene regulation based on the structure of natural snoRNAs and snRNAs.

Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs’ fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches.

Fragments of small nucleolar RNAs (snoRNAs) were found among various non-coding RNAs (ncRNAs) circulating in human blood. Currently, the function of such cell-free sno-derived-RNAs is not clearly defined. This work is aimed at identifying regulatory pathways controlled by extracellular snoRNAs. In order to determine the molecular targets and pathways affected by artificial snoRNAs, we performed Illumina array analysis of MCF-7 human adenocarcinoma cells transfected with box C/D RNAs. The genes related to the innate immune response and apoptotic cascades were found to be activated in transfected cells compared with control cells. Intriguingly, the transfection of MCF-7 cells with artificial box C/D snoRNAs also increased the transcription of several microRNAs, such as mir-574, mir-599 and mir-21. Our data demonstrated that extracellular snoRNAs introduced into human cells may function as gene expression modulators, with activation of microRNA genes being one of the regulatory mechanisms.

Human blood contains a great variety of membrane-covered RNA carrying vesicles which are spherical or tubular particles enclosed by a phospholipid bilayer. Circulating vesicles are thought to mediate cell-to-cell communication and their RNA cargo can act as regulatory molecules. In this work, we separated blood plasma of healthy donors by centrifugation and determined that vesicles precipitated at 16,000 g were enriched with CD41a, marker of platelets. At 160,000 g, the pellets were enriched with CD3 marker of T cells. To characterize the RNA-content of the blood plasma sub fractions, we performed high throughput sequencing of the RNA pelleted within vesicles at 16,000 g and 160,000 g as well as RNA remaining in the vesicle-free supernatant. We found that blood plasma sub fractions contain not only extensive set of microRNAs but also fragments of other cellular RNAs: rRNAs, tRNAs, mRNAs, lncRNAs, small RNAs including RNAs encoded by mtDNAs. Our data indicate that a variety of blood plasma RNAs circulating within vesicles as well as of extra-vesicular RNAs are comparable to the variety of cellular RNA species.

PURPOSE:Endometrial carcinoma is the most common gynecologic malignancy which is associated with a poor prognosis when diagnosed at an advanced stage; therefore, the discovery of efficacious new drugs is required to reinforce conventional chemotherapy. Short-term cultures of primary cells from endometrial tumors could be used for testing new anticancer therapeutics as well as for the development of personalized cancer therapy strategy. Here, the antitumor effect of a recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, was examined against primary human endometrial cancer cells. MATERIALS AND METHODS:Primary cell cultures of malignant and normal human endometrium were performed by enzymatic digestion of endometrial tissue from biopsy material. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the messenger ribonucleic acid (mRNA) state of estrogen (ERs) and progesterone (PRs) hormone receptors and aromatase (Cyp 19) in cell cultures. Dynamic monitoring of cell adhesion and proliferation was made using the iCELLigence system (ASEA Biosciences). The sensitivity of cell cultures to conventional anticancer drugs and the lactaptin analog was estimated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and the iCELLligence system. RESULTS:Established short-term primary cultures of endometrial cancer cells were ERα/ERβ/PR-positive and sensitive for RL2. The IC 50 values of doxorubicin and cisplatin were determined for all of the primary cultures designed. KE normal cells displaying low Cyp19 mRNA levels and high ERβ and PR mRNA levels were more resistant to RL2 treatment as well as to cisplatin and doxorubicin. CONCLUSIONS:Our results indicate that the recombinant analog of lactaptin, RL2, exerts cytotoxic effects against primary hormone-dependent endometrial tumor cells in vitro with features of apoptosis.

Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2′-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs.

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.

We translocated 80 individuals of Phrynocephalus guttatus guttatus into non-occupied isolated sand dune in Southern Kalmykya in June 1998. Observations during the following two years signify to the viability of the population. The demographic structure remained stabile since 1999, signifying to the congruence of habitat quality and ecological demands of the lizard species. We evaluated differences in the rate of postnatal development of males and females. In result, the number of sexually active males prevailed over the number of receptive females during the reproductive period, increasing probability of females’ pregnancy. Maximum life span of Ph. g. guttatus was estimated as up to four years.