KDM5 Family Demethylase Inhibitor KDOAM-25 Reduces Entry of SARS-CoV-2 Pseudotyped Viral Particles into Cells.

We studied the effect of KDM5 family demethylase inhibitors (JIB-04, PBIT, and KDOAM-25) on the penetration of SARS-CoV-2 pseudotyped viruses into differentiated Caco-2 cells and HEK293T cells with ACE2 hyperexpression. The above drugs were not cytotoxic. Only KDOAM-25 significantly reduced virus entry into the cells. The expression of ACE2 mRNA in Caco-2 significantly increased, while TMPRSS2 expression did not significantly change under these conditions. In differentiated Caco-2 cells, KDOAM-25 did not affect the expression of BRCA1, CDH1, TP53, SNAI1, VIM, and UGCG genes, for which an association with knockdown or overexpression of KDM5 demethylases or with the action of demethylase inhibitors had previously been shown. In undifferentiated Caco-2 cells, the expression of BRCA1, SNAI1, VIM, and CDH1 was significantly increased under the action of KDOAM-25.

Chemical Induction of Trophoblast Hypoxia by Cobalt Chloride Leads to Increased Expression of DDIT3.

Choriocarcinoma cells BeWo b30 are used to model human placental trophoblast hypoxia using cobalt (II) chloride and hydroxyquinoline derivative (HD) as chemical inducers of hypoxia-inducible factor (HIF). In this study, it was shown that both substances activate the hypoxic pathway and the epithelial-mesenchymal transition and inhibit the pathways of cell proliferation. However, CoCl2 caused activation of the apoptosis pathway, increased the activity of effector caspases 3 and 7, and increased the expression of the unfolded protein response target DDIT3. The mTORC1 pathway was activated upon exposition to CoCl2, while HD suppressed this pathway, as it happens during real trophoblast hypoxia. Thus, effect of CoCl2 on BeWo cells can be a model of severe hypoxia with activation of apoptosis, while HD mimics moderate hypoxia.

Intracellular Transport of Ribosome-Inactivating Proteins Depends on Annexin 13.

In the present study, we assessed the role of annexin 13 membrane-binding protein (ANXA13) in the intracellular transport of vesicles containing type II ribosome-inactivating proteins (RIP-IIs). A modified human intestinal epithelial cell line HT29 was used, in which the expression of ANXA13 was significantly reduced. The cytotoxic effect of ricin and viscumin was evaluated by modification of 28S ribosome RNA. The observed differences in the activity of toxins on the parental and modified HT29 lines indicate that ANXA13 plays a different role in the intracellular transport of vesicles containing the RIP-IIs.

Relationship between the Expression Level of PSMD11 and Other Proteasome Proteins with the Activity of Ricin and Viscumin.

The role of proteasome proteins and proteins of the ERAD system in the cytotoxicity of type II ribosome-inactivating proteins ricin and viscumin was investigated. For this, the cell line of colorectal adenocarcinoma HT29, as well as the HT29-sh002 line obtained on its basis, were used. On the basis on the proteome analysis of these lines and the estimation of the proportion of inactivated ribosomes, it was shown that the contribution of the proteasome to the degradation of the catalytic subunits of toxins is different. The role of the Cdc37 co-chaperone in maintaining the stability of A subunit of viscumin in the cytoplasm is shown.

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.

[Impedance Spectroscopy and Transcriptome Analysis of Choriocarcinoma BeWo b30 as a Model of Human Placenta].

Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.

Detection of Low-Abundant MicroRNAs with Hybridization Microchips.

The effect of low concentrations of miRNA on the ability of GeneChip miRNA 4.0 hybridization chips to evaluate their representation in the sample was studied. It is shown that the evaluation of the expression of 61 miRNAs is statistically significantly associated with the multiplicity of plasma dilution. Only 12 miRNAs showed very high Pearson correlation coefficient (>0.95) and they all decreased in response to dilution. High abundance of has-miR-4532 miRNA in plasma was demonstrated. This miRNA was never detected during sequencing of similar samples. It was concluded that in case of miRNA expression <1.12±0.33 units in log2 scale, dilution was not followed by further decrease in the signal intensity in GeneChip miRNA 4.0 chips.

Oxyquinoline-Dependent Changes in Claudin-Encoding Genes Contribute to Impairment of the Barrier Function of the Trophoblast Monolayer.

Natural response to hypoxia critically depends on rapid stabilization of hypoxia-inducible factor (HIF). Under normoxic conditions, HIF-prolyl hydroxylases mark α-subunits of HIF for degradation, while hypoxia results in stabilization of HIF-α. Oxyquinoline derivatives suppress activity of HIF-prolyl hydroxylases leading to HIF activation in the cell. Here we show that 24-h incubation of BeWo b30 choriocarcinoma cells (a model of trophoblast in the placental barrier) with oxyquinoline derivative leads to a decrease in transepithelial electrical resistance (TEER) of the cell monolayer, while the permeability of the monolayer for FITC-dextran (70 kDa) remains unchanged. These findings suggest that the overall barrier function is preserved, while the structure of intercellular tight junctions can undergo minor changes. Using Affymetrix Human Transcriptome Array 2.0, we showed that the treatment with oxyquinoline derivative was followed by a decrease in the expression of claudins 6 and 7 (CLDN6, CLDN7), occludin (OCLN), contact adhesion molecule 3 (JAM3), and angiomotinlike protein 1 (AMOTL1).

Metabolic Reprogramming of Trophoblast Cells in Response to Hypoxia.

Hypoxia of trophoblast cells is an important regulator of normal development of the placenta. However, some pathological states associated with hypoxia, e.g. preeclampsia, impair the functions of placental cells. Oxyquinoline derivative inhibits HIF-prolyl hydroxylase by stabilizing HIF-1 transcription complex, thus modeling cell response to hypoxia. In human choriocarcinoma cells BeWo b30 (trophoblast model), oxyquinoline increased the expression of a core hypoxia response genes along with up-regulation of NOS3, PDK1, and BNIP3 genes and down-regulation of the PPARGC1B gene. These changes in the expression profile attest to activation of the metabolic cell reprogramming mechanisms aimed at reducing oxygen consumption by enabling the switch from aerobic to anaerobic glucose metabolism and the respective decrease in number of mitochondria. The possibility of practical use of the therapeutic properties of oxyquinoline derivatives is discussed.

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.

[Expression of SLC30A10 and SLC23A3 Transporter mRNAs in Caco-2 Cells Correlates with an Increase in the Area of the Apical Membrane].

Drug bioavailability studies commonly employ in vitro barrier tissue models consisting of epithelial and endothelial cells. These experiments require that the cell barrier quality be assessed regularly, which is usually performed using various labeled substrates and/or evaluation of transepithelial (transendothelial) electrical resistance (TEER). This technique provides information on the integrity of the monolayer, but not on differentiation-induced changes in the cell morphology. The present work shows that impedance spectroscopy can be applied to monitor both the integrity of the monolayer and the morphological changes of Caco-2 cells. The growth kinetics of the apical membrane was determined by calculating the electrical capacitance of the cell monolayer. In the course of differentiation, the most pronounced changes in the expression levels were observed for the mRNAs that encode SLC30A10 and SLC23A3 transporters. Their increase correlated with an increase in the apical membrane area, indicating that SLC30A10 and SLC23A3 mRNA levels assessed by qRT-PCR may be employed as cell differentiation biomarkers in Caco-2 models.

Expression of microRNA Genes MIR221, MIR222, and MIR181B1 Increases during Induction of Inflammation in the Endothelial Barrier Model.

We studied expression profile of microRNA and their target genes in human umbilical vein endothelial cells (HUVEC) during proinflammatory activation with TNFα. TNFα-induced activation of HUVEC was accompanied by a decrease in the expression of CDKN1B, HIST1H3D, and OIP5 genes that are the common target genes for mature microRNA encoded by MIR221, MIR222, and MIR181B1 genes, whose expression increases in activated cells. Proteins encoded by HIST1H3D and OIP5 genes are associated with chromatin compaction and cell cycle. Our results suggest that fetal endothelial microRNA can appear in the maternal blood during various pathological states, e.g., under conditions of preeclampsia.

TNFα-Induced Expression of Transport Protein Genes in HUVEC Cells Is Associated with Enhanced Expression of Transcription Factor Genes RELB and NFKB2 of the Non-Canonical NF-κB Pathway.

Endothelial HUVEC cells used as an in vitro model of the endothelial monolayer in placental barrier were activated by TNFα in a dose of 2 ng/ml for 24 h. Significant changes in the expression of genes of the SLC family transport protein were observed: an increase in the expression of SLC7A2, SLC12A2, SLC9B2, SLC25A37, SLC16A9, and SLC41A2 and a decrease in the expression of SLC40A1. These transporters participate in the transport of iron, magnesium, sodium, potassium, and chloride ions, protons, and amino acids. It was also found that SLC7A2, SLC12A2, SLC9B2, SLC25A37, and SLC41A2 genes have binding sites for transcriptional factor RelB that together with NFKB2 is the main effector of the non-canonical NF-κB pathway. The expression of RELB and NFKB2 genes was also significantly enhanced in TNFα-activated HUVEC cells, which can attest to the important role of the non-canonical NF-κB pathway in the regulation of gene expression of transport proteins in response to TNFα stimulation.

hsa-miR-1973 MicroRNA is Significantly and Differentially Expressed in MDA-MB-231 Cells of Breast Adenocarcinoma and Xenografts Derived from the Tumor.

The gene expression profiles of MDA-MB-231 cell line of breast cancer and xenografts derived from this tumor in murine model were analyzed using GeneChip Human Transcriptome array 2.0 platform (Affymetrix). A more than 1000-fold increase in the MIR1973 gene expression was observed in the xenografts compared to the original cell line. Real-time reverse transcription PCR showed that the content of mature hsa-miR-1973 microRNA encoded by this gene was elevated in the xenografts by more than 300 times. According to microarray analysis, none of hsa-miR-1973 target genes available in the databases changed the expression in the cell line and xenografts. A possible role of hsa-miR-1973 is reduction of apoptosis in the tumor.

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.

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.