Neuromedin U secreted by colorectal cancer cells promotes a tumour-supporting microenvironment.

BACKGROUND: Neuromedin U (NMU) was identified as one of the hub genes closely related to colorectal cancer (CRC) progression and was recently shown to be a motility inducer in CRC cells. Its autocrine signalling through specific receptors increases cancer cell migration and invasiveness. Because of insufficient knowledge concerning NMU accessibility and action in the tumour microenvironment, its role in CRC remains poorly understood and its potential as a therapeutic target is still difficult to define. METHODS: NMU expression in CRC tissue was detected by IHC. Data from The Cancer Genome Atlas were used to analyse gene expression in CRC. mRNA and protein expression was detected by real-time PCR, immunoblotting or immunofluorescence staining and analysed using confocal microscopy or flow cytometry. Proteome Profiler was used to detect changes in the profiles of cytokines released by cells constituting tumour microenvironment after NMU treatment. NMU receptor activity was monitored by detecting ERK1/2 activation. Transwell cell migration, wound healing assay and microtube formation assay were used to evaluate the effects of NMU on the migration of cancer cells, human macrophages and endothelial cells. RESULTS: Our current study showed increased NMU levels in human CRC when compared to normal adjacent tissue. We detected a correlation between high NMUR1 expression and shorter overall survival of patients with CRC. We identified NMUR1 expression on macrophages, endothelial cells, platelets, and NMUR1 presence in platelet microparticles. We confirmed ERK1/2 activation by treatment of macrophages and endothelial cells with NMU, which induced pro-metastatic phenotypes of analysed cells and changed their secretome. Finally, we showed that NMU-stimulated macrophages increased the migratory potential of CRC cells. CONCLUSIONS: We propose that NMU is involved in the modulation and promotion of the pro-metastatic tumour microenvironment in CRC through the activation of cancer cells and other tumour niche cells, macrophages and endothelial cells. Video abstract.

The New Model of Snail Expression Regulation: The Role of MRTFs in Fast and Slow Endothelial-Mesenchymal Transition.

Endothelial-mesenchymal transition (EndMT) is a crucial phenomenon in regulating the development of diseases, including cancer metastasis and fibrotic disorders. The primary regulators of disease development are zinc-finger transcription factors belonging to the Snail family. In this study, we characterized the myocardin-related transcription factor (MRTF)-dependent mechanisms of a human snail promoter regulation in TGF-ß-stimulated human endothelial cells. Although in silico analysis revealed that the snail promoter's regulatory fragment contains one GCCG and two SP1 motifs that could be occupied by MRTFs, the genetic study confirmed that MRTF binds only to SP1 sites to promote snail expression. The more accurate studies revealed that MRTF-A binds to both SP1 elements, whereas MRTF-B to only one (SP1near). Although we found that each MRTF alone is capable of inducing snail expression, the direct cooperation of these proteins is required to reinforce snail expression and promote the late stages of EndMT within 48 hours. Furthermore, genetic and biochemical analysis revealed that MRTF-B alone could induce the late stage of EndMT. However, it requires a prolonged time. Therefore, we concluded that MRTFs might cause EndMT in a fast- and slow-dependent manner. Based on MRTF-dependent Snail upregulation, we recognized that TGF-ß1, as an MRTF-B regulator, is involved in slow EndMT induction, whereas TGF-ß2, which altered both MRTF-A and MRTF-B expression, promotes a fast EndMT process.

Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages.

This study tested the hypothesis that Mycobacterium tuberculosis (Mtb) uses a cholesterol oxidase enzyme (ChoD) to suppress a toll-like receptor type 2- (TLR2-) dependent signalling pathway to modulate macrophages' immune response. We investigated the impact of Mtb possessing or lacking ChoD as well as TBChoD recombinant protein obtained from Mtb on the expression and activation of two key intracellular proteins involved in TLR2 signalling in human macrophages. Finally, the involvement of TLR2-related signalling proteins in an inflammatory/immunosuppressive response of macrophages to Mtb was evaluated. We demonstrate that wild-type Mtb but not the ∆choD mutant decreased the cytosolic IRAK4 and TRAF6 protein levels while strongly enhancing IRAK4 and TRAF6 mRNA levels in macrophages. Our data show that the TLR2 present on the surface of macrophages are involved in disturbing the signalling pathway by wild-type Mtb. Moreover, recombinant TBChoD effectively decreased the cytosolic level of TRAF6 and lowered the phosphorylation of IRAK4, which strongly confirm an involvement of cholesterol oxidase in affecting the TLR2-related pathway by Mtb. Wild-type Mtb induced an immunosuppressive response of macrophages in an IRAK4- and TRAF6-dependent manner as measured by interleukin 10 production. In conclusion, ChoD is a virulence factor that enables Mtb to disturb the TLR2-related signalling pathway in macrophages and modulate their response.

Neuromedin U is upregulated by Snail at early stages of EMT in HT29 colon cancer cells.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is considered a core process that facilitates the escape of cancer cells from the primary tumor site. The transcription factor Snail was identified as a key regulator of EMT; however, the cascade of regulatory events leading to metastasis remains unknown and new predictive markers of the process are awaited. METHODS: Gene expressions were analysed using real-time PCR, protein level by Western immunoblotting and confocal imaging. The motility of the cells was examined using time-lapse microscopy. Affymetrix GeneChip Human Genome U133 Plus 2.0 analysis was performed to identify transcriptomic changes upon Snail. Snail silencing was performed using siRNA nucleofection. NMU detection was performed by ELISA. RESULTS: HT29 cells overexpressing Snail showed changed morphology, functions and transcriptomic profile indicating EMT induction. Changes in expression of 324 genes previously correlated with cell motility were observed. Neuromedin U was the second highest upregulated gene in HT29-Snail cells. This increase was validated by real-time PCR. Additionally elevated NMU protein was detected by ELISA in cell media. CONCLUSIONS: These results show that Snail in HT29 cells regulates early phenotype conversion towards an intermediate epithelial state. We provided the first evidence that neuromedin U is associated with Snail regulatory function of metastatic induction in colon cancer cells. GENERAL SIGNIFICANCE: We described the global, early transcriptomic changes induced through Snail in HT29 colon cancer cells and suggested NMU involvement in this process.

NFAT2 regulates COX-2 expression and modulates the integrin repertoire in endothelial cells at the crossroads of angiogenesis and inflammation.

The mechanisms controlling the switch between the pro-angiogenic and pro-inflammatory states of endothelial cells are still poorly understood. In this paper, we show that: (a) COX-2 expression induced by VEGF-A is NFAT2-dependent; and (b) the integrin profile in endothelial cells induced by the pro-angiogenic VEGF-A is distinct from that brought on by the inflammatory cytokine TNF-α. Two groups of integrin subunits specifically upregulated over time by both cytokines were identified using RT-PCR and Western Immunoblotting. The first group included α4, α5, α6, and ß5 subunits that were upregulated by VEGF-A; the second group consisted of αV and ß3 induced by TNF-α. Both cytokines significantly enhanced the expression of ß1 and modulated α2 mRNA. In contrast to TNF-α, VEGF-A induction of integrin subunits depended on the activation of the calcineurin/NFAT pathway. Both calcineurin inhibitors (cyclosporineA and 11R-VIVIT) and downregulation of NFAT2 with specific siRNA decreased induction of integrin subunits. This process of induction could be increased by upregulation of NFAT2 by pBJ5-NFAT2 transfection. This suggests that NFAT2 mediates VEGF-induced upregulation of integrin subunit synthesis by providing a constant supply of newly synthesized "refreshed" mature integrin receptors, particularly α2ß1, α5ß1, α4ß1, α6ß1 and αVß5, which are involved at different stages of angiogenesis.

Nitric oxide donors reduce the invasion ability of ovarian cancer cells in vitro.

The most important factors involved in tumor metastasis and angiogenesis are metalloproteinases (MMPs), vascular endothelial growth factor, and multifunctional transforming growth factor ß1. These factors are responsible for extracellular matrix degradation, induction of vascular permeability, and enhancement of tumor cells' invasion and metastasis. Elevated expression and secretion of the above-mentioned factors are correlated with the higher aggressiveness of tumors and low patient survival for example, patients with ovarian cancer. Therefore, regulation of the expression, secretion, and activity of these factors is still considered a potent target for therapeutic intervention in cancer patients. Nitric oxide (NO) donors belong to the class of agents with multivalent targeted activities in cancer cells and are considered potential anticancer therapeutics. Our studies have shown that NO donors such as spermine/NO and diethylenetriamine/NO decrease the secretion of vascular endothelial growth factor-A from the OVCAR-3 ovarian cancer cell line, but not from the SK-OV-3 ovarian cancer cell line. The release of MMP-2 from both cell lines was reduced in a soluble guanylate cyclase-dependent manner by spermine/NO and diethylenetriamine/NO. Nevertheless, MMP-2 activity was only affected in SK-OV-3 cells. Both NO donors reduced the transmigration of the ovarian cancer cell lines. We did not observe any significant effect of spermine/NO and diethylenetriamine/NO on mRNA expression of the tested aggressiveness factors. In conclusion, our data indicated that NO donors reduced the metastatic potential of ovarian cancer cells, but its impact is rather low and requires high concentrations of donors. Moreover, both the tested cell lines differed in the susceptibility to NO donors.

Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli.

Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells.

Snail transcription factors as key regulators of chemoresistance, stemness and metastasis of ovarian cancer cells.

Ovarian cancer is one of the deadliest gynecological malignancies among women. The reason for this outcome is the frequent acquisition of cancer cell resistance to platinum-based drugs and unresponsiveness to standard therapy. It has been increasingly recognized that the ability of ovarian cancer cells to adopt more aggressive behavior (mainly through the epithelial-to-mesenchymal transition, EMT), as well as dedifferentiation into cancer stem cells, significantly affects drug resistance acquisition. Transcription factors in the Snail family have been implicated in ovarian cancer chemoresistance and metastasis. In this article, we summarize published data that reveal Snail proteins not only as key inducers of the EMT in ovarian cancer but also as crucial links between the acquisition of ovarian cancer stem properties and spheroid formation. These Snail-related characteristics significantly affect the ovarian cancer cell response to treatment and are related to the acquisition of chemoresistance.

Association of plasminogen activator inhibitor type 2 (PAI-2) with proteasome within endothelial cells activated with inflammatory stimuli.

Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling.

Plasminogen activator inhibitor type 1 interacts with alpha3 subunit of proteasome and modulates its activity.

Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting.

Matrin 3 as a key regulator of endothelial cell survival.

Matrin 3 is an integral component of nuclear matrix architecture that has been implicated in interacting with other nuclear proteins and thus modulating the activity of proximal promoters. In this study, we evaluated the contribution of this protein to proliferation of endothelial cells. To selectively modulate matrin 3 expression, we used siRNA oligonucleotides and transfection of cells with a pEGFP-N1-Mtr3. Our data indicate that downregulation of matrin 3 is responsible for reduced proliferation and leads to necrosis of endothelial cells. This conclusion is supported by observations that reducing matrin 3 expression results in (a) producing signs of necrosis detected by PI staining, LDH release, and scatter parameters in flow cytometry, (b) affecting cell cycle progression. It does not cause (c) membrane asymmetry of cells as indicated by lack of Annexin V binding as well as (d) activation of caspase 3 and cleavage of PARP. We conclude that matrin 3 plays a significant role in controlling cell growth and proliferation, probably via formation of complexes with nuclear proteins that modulate pro- and antiapoptotic signaling pathways. Thus, degradation of matrin 3 may be a switching event that induces a shift from apoptotic to necrotic death of cells.

Autocrine effects of VEGF-D on endothelial cells after transduction with AD-VEGF-D(DeltaNDeltaC).

Endothelial cells in tumor vessels display unusual characteristics in terms of survival and angiogenic properties which result from the increased expression of VEGF-D and its autocrine effect. To evaluate mechanisms by which VEGF-D leads to such abnormal phenotype, we searched for proteins with modified expression in HUVECs enriched in the recombinant mature VEGF-D (VEGFD(DeltaNDeltaC)) delivered by adenovirus. Expression of membrane proteins in endothelial cells was characterized by FACS using anti-human IT-Box-135 antibodies. HUVECs transduced with Ad-VEGF-D(DeltaNDeltaC) revealed markedly increased expression of proteins involved in adhesion and migration such as (a) integrins (alphaVbeta5, alpha2beta1, alpha5beta1, alphaMbeta2, alphaLbeta2), (b) matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), (c) components of fibrinolytic system (PAI-1, u-PAR), and (d) CD45, CD98, CD147. Interestingly, there also were numerous proteins with significantly reduced expression, particularly among surface exposed membrane proteins. Thus, it can be concluded that to induce proangiogenic phenotype and facilitate migration of HUVECs, VEGF-D(DeltaNDeltaC) not only upregulates expression of proteins known to participate in the cell-matrix interactions but also silences some membrane proteins which could interfere with this process.

Neuromedin U induces an invasive phenotype in CRC cells expressing the NMUR2 receptor.

BACKGROUND: Successful colorectal cancer (CRC) therapy often depends on the accurate identification of primary tumours with invasive potential. There is still a lack of identified pathological factors associated with disease recurrence that could help in making treatment decisions. Neuromedin U (NMU) is a secretory neuropeptide that was first isolated from the porcine spinal cord, and it has emerged as a novel factor involved in the tumorigenesis and/or metastasis of many types of cancers. Previously associated with processes leading to CRC cell invasiveness, NMU has the potential to be a marker of poor outcome, but it has not been extensively studied in CRC. METHODS: Data from The Cancer Genome Atlas (TCGA) were used to analyse NMU and NMU receptor (NMUR1 and NMUR2) expression in CRC tissues vs. normal tissues, and real-time PCR was used for NMU and NMU receptor expression analysis. NMU protein detection was performed by immunoblotting. Secreted NMU was immunoprecipitated from cell culture-conditioned media and analysed by immunoblotting and protein sequencing. DNA demethylation by 5-aza-CdR was used to analyse the regulation of NMUR1 and NMUR2 expression. NMU receptor activity was monitored by detecting calcium mobilisation in cells loaded with fluo-4, and ERK1/2 kinase activation was detected after treatment with NMU or receptor agonist. Cell migration and invasion were investigated using membrane filters. Integrin expression was evaluated by flow cytometry. RESULTS: The obtained data revealed elevated expression of NMU and NMUR2 in CRC tissue samples and variable expression in the analysed CRC cell lines. We have shown, for the first time, that NMUR2 activation induces signalling in CRC cells and that NMU increases the motility and invasiveness of NMUR2-positive CRC cells and increases prometastatic integrin receptor subunit expression. CONCLUSIONS: Our results show the ability of CRC cells to respond to NMU via activation of the NMUR2 receptor, which ultimately leads to a shift in the CRC phenotype towards a more invasive phenotype.

Snail Overexpression Alters the microRNA Content of Extracellular Vesicles Released from HT29 Colorectal Cancer Cells and Activates Pro-Inflammatory State In Vivo.

During metastasis, cancer cells undergo phenotype changes in the epithelial-mesenchymal transition (EMT) process. Extracellular vesicles (EVs) released by cancer cells are the mediators of intercellular communication and play a role in metastatic process. Knowledge of factors that influence the modifications of the pre-metastatic niche for the migrating carcinoma cells is important for prevention of metastasis. We focus here on how cancer progression is affected by EVs released from either epithelial-like HT29-cells or from cells that are in early EMT stage triggered by Snail transcription factor (HT29-Snail). We found that EVs released from HT29-Snail, as compared to HT29-pcDNA cells, have a different microRNA profile. We observed the presence of interstitial pneumonias in the lungs of mice injected with HT29-Snail cells and the percent of mice with lung inflammation was higher after injection of HT29-Snail-EVs. Incorporation of EVs released from HT29-pcDNA, but not released from HT29-Snail, leads to the increased secretion of IL-8 from macrophages. We conclude that Snail modifications of CRC cells towards more invasive phenotype also alter the microRNA cargo of released EVs. The content of cell-released EVs may serve as a biomarker that denotes the stage of CRC and EVs-specific microRNAs may be a target to prevent cancer progression.

Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment.

BACKGROUND: Haematopoiesis is a process of formation of mature blood cells from hematopoietic progenitors in bone marrow. Haematopoietic progenitors are stimulated by growth factors and cytokines to proliferate and differentiate, and they die via apoptosis when these factors are depleted. An aberrant response to growth environment may lead to haematological disorders. Bomapin (serpinb10) is a hematopoietic- and myeloid leukaemia-specific protease inhibitor with unknown function. RESULTS: We found that the majority of naturally expressed bomapin was located in the nucleus. Both the natural and recombinant bomapin had a disulfide bond which linked the only two bomapin cysteines: one located in the CD-loop and the other near the C-terminus. Computer modelling showed that the cysteines are distant in the reduced bomapin, but can easily be disulfide-linked without distortion of the overall bomapin structure. Low-level ectopic expression of bomapin in bomapin-deficient K562 cells resulted in about 90% increased cell proliferation under normal growth conditions. On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation. Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important. The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation. In contrast to the survival-promoting activity of bomapin in cells cultured under optimal growth conditions, bomapin enhanced cell apoptosis following growth factor withdrawal. CONCLUSIONS: We propose that bomapin is a redox-sensitive nuclear serpin that augments proliferation or apoptosis of leukaemia cells, depending on growth factors availability.

Erratum: Neuromedin U: A Small Peptide in the Big World of Cancer. Cancers 2019, 11, 1312.

The authors wish to make the following corrections to this paper [...].

Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail.

The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma.

Neuromedin U: A Small Peptide in the Big World of Cancer.

Neuromedin U (NMU), a neuropeptide isolated from porcine spinal cord and named because of its activity as a rat uterus smooth muscle contraction inducer, is emerging as a new player in the tumorigenesis and/or metastasis of many types of cancers. Expressed in a variety of tissues, NMU has been shown to possess many important activities in the central nervous system as well as on the periphery. Along with the main structural and functional features of NMU and its currently known receptors, we summarized a growing number of recently published data from different tissues and cells that associate NMU activity with cancer development and progression. We ask if, based on current reports, NMU can be included as a marker of these processes and/or considered as a therapeutic target.

Naturally Occurring Nervonic Acid Ester Improves Myelin Synthesis by Human Oligodendrocytes.

The dysfunction of oligodendrocytes (OLs) is regarded as one of the major causes of inefficient remyelination in multiple sclerosis, resulting gradually in disease progression. Oligodendrocytes are derived from oligodendrocyte progenitor cells (OPCs), which populate the adult central nervous system, but their physiological capability to myelin synthesis is limited. The low intake of essential lipids for sphingomyelin synthesis in the human diet may account for increased demyelination and the reduced efficiency of the remyelination process. In our study on lipid profiling in an experimental autoimmune encephalomyelitis brain, we revealed that during acute inflammation, nervonic acid synthesis is silenced, which is the effect of shifting the lipid metabolism pathway of common substrates into proinflammatory arachidonic acid production. In the experiments on the human model of maturating oligodendrocyte precursor cells (hOPCs) in vitro, we demonstrated that fish oil mixture (FOM) affected the function of hOPCs, resulting in the improved synthesis of myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein, as well as sphingomyelin. Additionally, FOM reduces proinflammatory cytokines and chemokines, and enhances fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) synthesis by hOPCs was also demonstrated. Based on these observations, we propose that the intake of FOM rich in the nervonic acid ester may improve OL function, affecting OPC maturation and limiting inflammation.

Regulation of miRNAs by Snail during epithelial-to-mesenchymal transition in HT29 colon cancer cells.

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasis and is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.