Incautious design of shRNAs for stable overexpression of miRNAs could result in generation of undesired isomiRs.

shRNA-mediated strategy of miRNA overexpression based on RNA Polymerase III (Pol III) expression cassettes is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that expression of miRNAs longer than 19 nt (e.g. 23 nt in length hsa-miR-93-5p) using such approach could be accompanied by undesired predominant generation of 5' end miRNA isoforms (5'-isomiRs). Extra U residues (up to five) added by Pol III at the 3' end of the transcribed shRNA during transcription termination could cause a shift in the Dicer cleavage position of the shRNA. This results in the formation of 5'-isomiRs, which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. We demonstrated that the commonly used qPCR method is insensitive to the formation of 5'-isomiRs and cannot be used to confirm miRNA overexpression. However, the predominant expression of 5'-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5'-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. Overall, the presented results show that structures of shRNAs for stable overexpression of miRNAs requires careful design to avoid generation of undesired 5'-isomiRs.

Spontaneous metastasis xenograft models link CD44 isoform 4 to angiogenesis, hypoxia, EMT and mitochondria-related pathways in colorectal cancer.

Hematogenous metastasis limits the survival of colorectal cancer (CRC) patients. Here, we illuminated the roles of CD44 isoforms in this process. Isoforms 3 and 4 were predominantly expressed in CRC patients. CD44 isoform 4 indicated poor outcome and correlated with epithelial-mesenchymal transition (EMT) and decreased oxidative phosphorylation (OxPhos) in patients; opposite associations were found for isoform 3. Pan-CD44 knockdown (kd) independently impaired primary tumor formation and abrogated distant metastasis in CRC xenografts. The xenograft tumors mainly expressed the clinically relevant CD44 isoforms 3 and 4. Both isoforms were enhanced in the paranecrotic, hypoxic tumor regions but were generally absent in lung metastases. Upon CD44 kd, tumor angiogenesis was increased in the paranecrotic areas, accompanied by reduced hypoxia-inducible factor-1α and CEACAM5 but increased E-cadherin expression. Mitochondrial genes and proteins were induced upon pan-CD44 kd, as were OxPhos genes. Hypoxia increased VEGF release from tumor spheres, particularly upon CD44 kd. Genes affected upon CD44 kd in xenografts specifically overlapped concordantly with genes correlating with CD44 isoform 4 (but not isoform 3) in patients, validating the clinical relevance of the used model and highlighting the metastasis-promoting role of CD44 isoform 4.

RNA-binding proteins regulating the CD44 alternative splicing.

Alternative splicing is often deregulated in cancer, and cancer-specific isoform switches are part of the oncogenic transformation of cells. Accumulating evidence indicates that isoforms of the multifunctional cell-surface glycoprotein CD44 play different roles in cancer cells as compared to normal cells. In particular, the shift of CD44 isoforms is required for epithelial to mesenchymal transition (EMT) and is crucial for the maintenance of pluripotency in normal human cells and the acquisition of cancer stem cells phenotype for malignant cells. The growing and seemingly promising use of splicing inhibitors for treating cancer and other pathologies gives hope for the prospect of using such an approach to regulate CD44 alternative splicing. This review integrates current knowledge about regulating CD44 alternative splicing by RNA-binding proteins.

Challenges in characterization of transcriptomes of extracellular vesicles and non-vesicular extracellular RNA carriers.

Since its original discovery over a decade ago, extracellular RNA (exRNA) has been found in all biological fluids. Furthermore, extracellular microRNA has been shown to be involved in communication between various cell types. Importantly, the exRNA is protected from RNases degradation by certain carriers including membrane vesicles and non-vesicular protein nanoparticles. Each type of carrier has its unique exRNA profile, which may vary depending on cell type and physiological conditions. To clarify putative mechanisms of intercellular communication mediated by exRNA, the RNA profile of each carrier has to be characterized. While current methods of biofluids fractionation are continuously improving, they fail to completely separate exRNA carriers. Likewise, most popular library preparation approaches for RNA sequencing do not allow obtaining exhaustive and unbiased data on exRNA transcriptome. In this mini review we discuss ongoing progress in the field of exRNA, with the focus on exRNA carriers, analyze the key methodological challenges and provide recommendations on how the latter could be overcome.

A novel approach for a joint analysis of isomiR and mRNA expression data reveals features of isomiR targeting in breast cancer.

A widely used procedure for selecting significant miRNA-mRNA or isomiR-mRNA pairs out of predicted interactions involves calculating the correlation between expression levels of miRNAs/isomiRs and mRNAs in a series of samples. In this manuscript, we aimed to assess the validity of this procedure by comparing isomiR-mRNA correlation profiles in sets of sequence-based predicted target mRNAs and non-target mRNAs (negative controls). Target prediction was carried out using RNA22 and TargetScan algorithms. Spearman's correlation analysis was conducted using miRNA and mRNA sequencing data of The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) project. Luminal A, luminal B, basal-like breast cancer subtypes, and adjacent normal tissue samples were analyzed separately. Using the sets of putative targets and non-targets, we introduced adjusted isomiR targeting activity (ITA)-the number of negatively correlated potential isomiR targets adjusted by the background (estimated using non-target mRNAs). We found that for most isomiRs a significant negative correlation between isomiR-mRNA expression levels appeared more often in a set of predicted targets compared to the non-targets. This trend was detected for both classical seed region binding types (8mer, 7mer-m8, 7mer-A1, 6mer) predicted by TargetScan and the non-classical ones (G:U wobbles and up to one mismatch or unpaired nucleotide within seed sequence) predicted by RNA22. Adjusted ITA distributions were similar for target sites located in 3'-UTRs and coding mRNA sequences, while 5'-UTRs had much lower scores. Finally, we observed strong cancer subtype-specific patterns of isomiR activity, highlighting the differences between breast cancer molecular subtypes and normal tissues. Surprisingly, our target prediction- and correlation-based estimates of isomiR activities were practically non-correlated with the average isomiR expression levels neither in cancerous nor in normal samples.

Specificity of viscumin revised. As probed with a printed glycan array.

Viscumin, a lectin used in anti-cancer therapy, was originally considered as ßGal recognizing protein; later, an ability to bind 6'-sialyl N-acetyllactosamine (6'SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galß1-4GlcNAc, was observed. Unexpectedly, only weak interaction was detected with parent neutral ß-galactosides including LN-LN-LN and branched (LN)2LN oligolactosamines; in the light of these data, one should not confidently classify viscumin as a ß-galactoside-binding lectin. Carrying out PGA in the presence of neutral or sulfated/sialylated glycan, together with sequential elution from lactose-sepharose and consideration of the protein structure, lead to the conclusion that two glycan-binding sites of viscumin have different specificities, one of which prefers charged sulfated and sialylated moieties.

Differences in Medium-Induced Conformational Plasticity Presumably Underlie Different Cytotoxic Activity of Ricin and Viscumin.

Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We assume that the higher conformational plasticity of RTA is favorable for the ER-mediated translocation pathway, which leads to a higher rate of toxin penetration into the cytoplasm.

Fra-2 overexpression upregulates pro-metastatic cell-adhesion molecules, promotes pulmonary metastasis, and reduces survival in a spontaneous xenograft model of human breast cancer.

PURPOSE: The transcription factor Fra-2 affects the invasive potential of breast cancer cells by dysregulating adhesion molecules in vitro. Previous results suggested that it upregulates the expression of E- and P-selectin ligands. Such selectin ligands are important members of the leukocyte adhesion cascade, which govern the adhesion and transmigration of cancer cells into the stroma of the host organ of metastasis. As so far, no in vivo data are available, this study was designed to elucidate the role of Fra-2 expression in a spontaneous breast cancer metastasis xenograft model. METHODS: The effect of Fra-2 overexpression in two stable Fra-2 overexpressing clones of the human breast cancer cell line MDA MB231 on survival and metastatic load was studied after subcutaneous injection into scid and E- and P-selectin-deficient scid mice. RESULTS: Fra-2 overexpression leads to a significantly shorter overall survival and a higher amount of spontaneous lung metastases not only in scid mice, but also in E- and P-deficient mice, indicating that it regulates not only selectin ligands, but also selectin-independent adhesion processes. CONCLUSION: Thus, Fra-2 expression influences the metastatic potential of breast cancer cells by changing the expression of adhesion molecules, resulting in increased adherence to endothelial cells in a breast cancer xenograft model.

Low expression of CD24 is associated with poor survival in colorectal cancer.

In this study we analyzed expression of CD24 in a cohort of colorectal cancer patients using immunohistochemistry staining of CD24. We found a significant association between absence or low expression of CD24 (10% of membranous and 55% of cytoplasmic staining) and shortened patient survival. Protein localization played a crucial role in the prognosis: membranous form was the major and prognostic one in primary tumors, while cytoplasmic expression was elevated in liver metastases compared to the primary tumors and contained prognostic information. Then, using The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) RNA-seq data, we showed that CD24 mRNA level was two-fold decreased in primary colorectal cancers compared to adjacent normal mucosa. Like the protein staining data, ten percent of patients with the lowest mRNA expression levels of CD24 in primary tumors had reduced survival compared to the ones with higher expression. To explain these findings mechanistically, shRNA-mediated CD24 knockdown was performed in HT-29 colorectal cancer cells. It resulted in the increase of cell migration in vitro, no changes in proliferation and apoptosis, and a slight decrease in cell invasion. As increased cell migration is a hallmark of metastasis formation, this finding corroborates the association of a decreased CD24 expression with poor prognosis. Differential gene expression analysis revealed upregulation of genes involved in cell migration in the group of patients with low CD24 expression, including integrin subunit α3 and α3, ß3 subunits of laminin 332. Further co-expression analysis identified SPI1, STAT1 and IRF1 transcription factors as putative master-regulators in this group.

HIF-Dependent NFATC1 Activation Upregulates ITGA5 and PLAUR in Intestinal Epithelium in Inflammatory Bowel Disease.

Intestinal epithelial cells exist in physiological hypoxia, leading to hypoxia-inducible factor (HIF) activation and supporting barrier function and cell metabolism of the intestinal epithelium. In contrast, pathological hypoxia is a common feature of some chronic disorders, including inflammatory bowel disease (IBD). This work was aimed at studying HIF-associated changes in the intestinal epithelium in IBD. In the first step, a list of genes responding to chemical activation of hypoxia was obtained in an in vitro intestinal cell model with RNA sequencing. Cobalt (II) chloride and oxyquinoline treatment of both undifferentiated and differentiated Caco-2 cells activate the HIF-signaling pathway according to gene set enrichment analysis. The core gene set responding to chemical hypoxia stimulation in the intestinal model included 115 upregulated and 69 downregulated genes. Of this set, protein product was detected for 32 genes, and fold changes in proteome and RNA sequencing significantly correlate. Analysis of publicly available RNA sequencing set of the intestinal epithelial cells of patients with IBD confirmed HIF-1 signaling pathway activation in sigmoid colon of patients with ulcerative colitis and terminal ileum of patients with Crohn's disease. Of the core gene set from the gut hypoxia model, expression activation of ITGA5 and PLAUR genes encoding integrin α5 and urokinase-type plasminogen activator receptor (uPAR) was detected in IBD specimens. The interaction of these molecules can activate cell migration and regenerative processes in the epithelium. Transcription factor analysis with the previously developed miRGTF tool revealed the possible role of HIF1A and NFATC1 in the regulation of ITGA5 and PLAUR gene expression. Detected genes can serve as markers of IBD progression and intestinal hypoxia.

An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation.

Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.

Knockdown of the α5 laminin chain affects differentiation of colorectal cancer cells and their sensitivity to chemotherapy.

The interaction of tumor cells with the extracellular matrix (ECM) may affect the rate of cancer progression and metastasis. One of the major components of ECM are laminins, the heterotrimeric glycoproteins consisting of α-, ß-, and γ-chains (αßγ). Laminins interact with their cell surface receptors and, thus, regulate multiple cellular processes. In this work, we demonstrate that shRNA-mediated knockdown of the α5 laminin chain results in Wnt- and mTORC1-dependent partial dedifferentiation of colorectal cancer cells. Furthermore, we showed that this dedifferentiation involved activation of ER-stress signaling, pathway promoting the sensitivity of cells to 5-fluorouracil.

Towards embedding Caco-2 model of gut interface in a microfluidic device to enable multi-organ models for systems biology.

BACKGROUND: A cancer cell line originating from human epithelial colorectal adenocarcinoma (Caco-2 cells) serves as a high capacity model for a preclinical screening of drugs. Recent need for incorporating barrier tissue into multi-organ chips calls for inclusion of Caco-2 cells into microperfused environment. RESULTS: This article describes a series of systems biology insights obtained from comparing Caco-2 models cells grown as conventional 2D layer and in a microfluidic chip. When basic electrical parameters of Caco-2 monolayers were evaluated using impedance spectrometry and MTT assays, no differences were noted. On the other hand, the microarray profiling of mRNAs and miRNAs revealed that grows on a microfluidic chip leads to the change in the production of specific miRNA, which regulate a set of genes for cell adhesion molecules (CAMs), and provide for more complete differentiation of Caco-2 monolayer. Moreover, the sets of miRNAs secreted at the apical surface of Caco-2 monolayers grown in conventional 2D culture and in microfluidic device differ. CONCLUSIONS: When integrated into a multi-tissue platform, Caco-2 cells may aid in generating insights into complex pathophysiological processes, not possible to dissect in conventional cultures.

Cumulative prognostic power of laminin genes in colorectal cancer.

BACKGROUND: Laminins are a major family of extracellular matrix proteins and the main component of basement membranes. Laminins are involved in many if not all stages of cancer progression, and expression of laminin genes has prognostic value in various types of cancer, including colorectal. Only single laminin genes or components of a single laminin trimer with significant differential expression have been regarded as potential biomarkers to date. RESULTS: Here we compared prognostic power of classifiers constructed from sets of laminin genes with that of any single laminin gene. The analysis showed that cumulative prognostic power of sets of laminin genes was higher and was achieved already with pairs and triples of the genes. Interestingly, components of the pairs and the triples did not belong to any known laminin trimer, but, taken together with the gene weights, suggested higher LAMA4/LAMA5 expression ratio in patients with poor prognosis. CONCLUSIONS: Analysis of the laminin expression profile rather than expression of the single genes or components of laminin trimers is useful for colorectal cancer prognosis in patients. High LAMA4/LAMA5 ratio is associated with increased permeability of basement membranes suggesting that basement membranes produced by colorectal tumors might be an important hindrance to their own dissemination in patients.

Selectin-independent adhesion during ovarian cancer metastasis.

PURPOSE: Ovarian cancer (OvCa) progression mainly takes place by intraperitoneal spread. Adhesion of tumor cells to the mesothelial cells which form the inner surface of the peritoneum is a crucial step in this process. Cancer cells use in principle different molecules of the leukocyte adhesion cascade to facilitate adhesion. This cascade is initiated by selectin-ligand interactions followed by integrin - extracellular matrix protein interactions. Here we address the question whether all tumor cells predominantly employ selectin-dependent leukocyte-like adhesion cascade (SDAC) or whether they use integrin mediated adhesion for OvCa progression as well. METHODS: A comparative transcriptomic analysis of the human OvCa cell lines OVCAR8 and SKOV3 was performed. Intraperitoneal xenograft model of OVCAR8 cells was used to determine whether there is a correlation between SDAC gene expression and the metastatic potential of the control cells and the cells overexpressing c-Fos. Transcriptomic analysis of OVCAR8 and SKOV3 samples was performed using microarrays. RESULTS: One-third of the protein-coding genes involved in SDAC exhibited lower expression levels in OVCAR8 than in SKOV3 cells. In contrast to SKOV3 cells, c-Fos overexpression in OVCAR8 cells did not significantly influence the expression of SDAC genes. Intraperitoneal xenograft model of OVCAR8 cells unexpectedly demonstrated that the aggressiveness of OVCAR8 tumors was not depended on the c-Fos expression level and was comparable to that of SKOV3 control tumors. Gene expression analysis of tumors suggests that SKOV3-derived tumor progression was mainly depended on SDAC. Progression of OVCAR8 tumors relied on other cell adhesion molecules that do not interact with selectins. CONCLUSIONS: High expression of c-Fos in ovarian cancer cells is not always associated with reduced metastatic potential. Low expression level of SDAC genes may not ensure low OvCa metastatic potential hence alternative adhesion mechanisms involving laminin-integrin interactions exist as well.

The Transcriptome of Type I Murine Astrocytes under Interferon-Gamma Exposure and Remyelination Stimulus.

Astrocytes are considered to be an important contributor to central nervous system (CNS) disorders, particularly multiple sclerosis. The transcriptome of these cells is greatly affected by cytokines released by lymphocytes, penetrating the blood-brain barrier-in particular, the classical pro-inflammatory cytokine interferon-gamma (IFNγ). We report here the transcriptomal profiling of astrocytes treated using IFNγ and benztropine, a putative remyelinization agent. Our findings indicate that the expression of genes involved in antigen processing and presentation in astrocytes are significantly upregulated upon IFNγ exposure, emphasizing the critical role of this cytokine in the redirection of immune response towards self-antigens. Data reported herein support previous observations that the IFNγ-induced JAK-STAT signaling pathway may be regarded as a valuable target for pharmaceutical interventions.

Receptor Mincle promotes skin allergies and is capable of recognizing cholesterol sulfate.

Sterile (noninfected) inflammation underlies the pathogenesis of many widespread diseases, such as allergies and autoimmune diseases. The evolutionarily conserved innate immune system is considered to play a key role in tissue injury recognition and the subsequent development of sterile inflammation; however, the underlying molecular mechanisms are not yet completely understood. Here, we show that cholesterol sulfate, a molecule present in relatively high concentrations in the epithelial layer of barrier tissues, is selectively recognized by Mincle (Clec4e), a C-type lectin receptor of the innate immune system that is strongly up-regulated in response to skin damage. Mincle activation by cholesterol sulfate causes the secretion of a range of proinflammatory mediators, and s.c. injection of cholesterol sulfate results in a Mincle-mediated induction of a severe local inflammatory response. In addition, our study reveals a role of Mincle as a driving component in the pathogenesis of allergic skin inflammation. In a well-established model of allergic contact dermatitis, the absence of Mincle leads to a significant suppression of the magnitude of the skin inflammatory response as assessed by changes in ear thickness, myeloid cell infiltration, and cytokine and chemokine secretion. Taken together, our results provide a deeper understanding of the fundamental mechanisms underlying sterile inflammation.

miRNome of inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is an extremely malignant form of breast cancer which can be easily misdiagnosed. Conclusive prognostic IBC molecular biomarkers which are also providing the perspectives for targeted therapy are lacking so far. The aim of this study was to reveal the IBC-specific miRNA expression profile and to evaluate its association with clinicopathological parameters. METHODS: miRNA expression profiles of 13 IBC and 17 non-IBC patients were characterized using comprehensive Affymetrix GeneChip miRNA 3.0 microarray platform. Bioinformatic analysis was used to reveal IBC-specific miRNAs, deregulated pathways and potential miRNA targets. RESULTS: 31 differentially expressed miRNAs characterize IBC and mRNAs regulated by them and their associated pathways can functionally be attributed to IBC progression. In addition, a minimal predictive set of 4 miRNAs characteristic for the IBC phenotype and associated with the TP53 mutational status in breast cancer patients was identified. CONCLUSIONS: We have characterized the complete miRNome of inflammatory breast cancer and found differentially expressed miRNAs which reliably classify the patients to IBC and non-IBC groups. We found that the mRNAs and pathways likely regulated by these miRNAs are highly relevant to cancer progression. Furthermore a minimal IBC-related predictive set of 4 miRNAs associated with the TP53 mutational status and survival for breast cancer patients was identified.

Exercise immunology meets MiRNAs.

A large body of evidence indicates modified expression of protein-coding genes in response to different kinds of physical activity. Recent years have exposed another level of regulation of cellular processes mediated by non-coding RNAs. MicroRNAs (miRNAs) are one of the largest families of non-coding RNAs. MiRNAs mediate post-transcriptional regulation of gene expression. The amount of data supporting the key role of miRNAs in the adaptation of the immune and other body systems to exercise steadily grows. MiRNAs change their expression profiles after exercise and seem to be involved in regulation of exercise-responsive genes in immune and other cell types. Here we discuss existing data and future directions in the field.

Dynamically regulated miRNA-mRNA networks revealed by exercise.

BACKGROUND: MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period. RESULTS: Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. CONCLUSIONS: We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation.