Identification of differentially expressed mRNA/lncRNA modules in acutely regorafenib-treated sorafenib-resistant Huh7 hepatocellular carcinoma cells.

The multikinase inhibitor sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), but many patients become sorafenib-resistant (SR). This study investigated the efficacy of another kinase inhibitor, regorafenib (Rego), as a second-line treatment. We produced SR HCC cells, wherein the PI3K-Akt, TNF, cAMP, and TGF-beta signaling pathways were affected. Acute Rego treatment of these cells reversed the expression of genes involved in TGF-beta signaling but further increased the expression of genes involved in PI3K-Akt signaling. Additionally, Rego reversed the expression of genes involved in nucleosome assembly and epigenetic gene expression. Weighted gene co-expression network analysis (WGCNA) revealed four differentially expressed long non-coding RNA (DElncRNA) modules that were associated with the effectiveness of Rego on SR cells. Eleven putative DElncRNAs with distinct expression patterns were identified. We associated each module with DEmRNAs of the same pattern, thus obtaining DElncRNA/DEmRNA co-expression modules. We discuss the potential significance of each module. These findings provide insights and resources for further investigation into the potential mechanisms underlying the response of SR HCC cells to Rego.

Defined Conditions Control the Morphological Dualism of Rat Primitive Extraembryonic Endoderm Stem Cells.

Rat primitive extraembryonic endoderm (pXEN) stem cell lines indefinitely preserve the characteristic features of the early extraembryonic endoderm (ExEn) in vitro, but require unknown serum factors and exhibit a hybrid (mesenchymal-epithelial) phenotype. We report two chemically defined conditions that differ by the addition of the cytokine leukemia inhibitory factor (Lif) and the ß-catenin-stabilizing drug Chir99021, and enable permanent self-renewal as mesenchymal and epithelial morphotypes, respectively. The morphotypes are interconvertible and equipotent, as shown by the formation of well-differentiated organoids. Surprisingly, the proliferation of both morphotypes requires Lif-type Gp130/Stat3 signaling (autocrine in the absence of added Lif) and noncanonical Wnt signaling (autocrine). In addition, the epithelial version requires ß-catenin for proliferation and morphology. Interestingly, the mesenchymal cells also express key epithelial markers, but those are improperly structured and/or not functional, indicating a primed state. These results provide an improved platform for studying the proliferation and plasticity of the early ExEn, which occurs in mesenchymal and epithelial forms in vivo.

Analysis of differentially expressed long non-coding RNAs in LPS-induced human HMC3 microglial cells.

BACKGROUND: Long non-coding RNAs (lncRNAs) are emerging as key modulators of inflammatory gene expression, but their roles in neuroinflammation are poorly understood. Here, we identified the inflammation-related lncRNAs and correlated mRNAs of the lipopolysaccharide (LPS)-treated human microglial cell line HMC3. We explored their potential roles and interactions using bioinformatics tools such as gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG), and weighted gene co-expression network analysis (WGCNA). RESULTS: We identified 5 differentially expressed (DE) lncRNAs, 4 of which (AC083837.1, IRF1-AS1, LINC02605, and MIR3142HG) are novel for microglia. The DElncRNAs with their correlated DEmRNAs (99 total) fell into two network modules that both were enriched with inflammation-related RNAs. However, treatment with the anti-inflammatory agent JQ1, an inhibitor of the bromodomain and extra-terminal (BET) protein BRD4, neutralized the LPS effect in only one module, showing little or even enhancing effect on the other. CONCLUSIONS: These results provide insight into, and a resource for studying, the regulation of microglia-mediated neuroinflammation and its potential therapy by small-molecule BET inhibitors.

The bromodomain inhibitor JQ1 up-regulates the long non-coding RNA MALAT1 in cultured human hepatic carcinoma cells.

The epigenetic reader, bromodomain-containing 4 (BRD4), is overexpressed in hepatocellular carcinoma (HCC), and BRD4 inhibition is considered as a new therapeutic approach. The BRD inhibitor JQ1 is known to inhibit the enrichment of BRD4 at enhancer sites. Gene network analyses have implicated long non-coding RNAs (lncRNAs) in the effects of JQ1, but the precise molecular events remain unexplored. Here, we report that in HepG2 cells, JQ1 significantly reduced various proliferation-related lncRNAs, but up-regulated the known liver tumor marker, MALAT1. Using ChIP-sequencing data, ChIP-qPCR, luciferase reporter assays, and chromatin conformation capture (3C), we characterized the MALAT1 gene locus. We found that JQ1 elicited a rearrangement of its chromatin looping conformation, which involved the putative enhancers E1, E2, E3, the gene body, and the promoter. We further found that the forkhead box protein A2 (FOXA2) binds to E2 and the promoter; suppression of FOXA2 expression resulted in MALAT1 up-regulation and increased cell proliferation. These results suggest that the inhibition of MALAT1 may improve the effect of BET inhibitors as an anti-cancer therapy and that FOXA2 would be a suitable target for that approach.

Comprehensive transcriptome profiling of BET inhibitor-treated HepG2 cells.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and poor prognosis. Emerging evidence suggests that epigenetic alterations play a crucial role in HCC, suggesting epigenetic inhibition as a promising therapeutic approach. Indeed, the bromodomain and extra-terminal (BET) inhibitors inhibit the proliferation and invasion of various cancers but still lack a strong mechanistic rationale. Here, we identified the differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) in human HCC cell line HepG2 treated with the BET inhibitors, JQ1, OTX015, or ABBV-075. We analyzed the correlation between DEmRNAs and DElncRNAs in common for the three inhibitors based on their expression profiles and performed functional annotation pathway enrichment analysis. Most of these shared DEmRNAs and DElncRNAs, including some novel transcripts, were downregulated, indicating decreased proliferation/adhesion and increased apoptosis/inflammation. Our study suggests that BET proteins play a crucial role in regulating cancer progression-related genes and provide a valuable resource for novel putative biomarkers and therapeutic targets in HCC.

Targeting of noncoding RNAs encoded by a novel MYC enhancers inhibits the proliferation of human hepatic carcinoma cells in vitro.

The proto-oncogene MYC is important for development and cell growth, however, its abnormal regulation causes cancer. Recent studies identified distinct enhancers of MYC in various cancers, but any MYC enhancer(s) in hepatocellular carcinoma (HCC) remain(s) elusive. By analyzing H3K27ac enrichment and enhancer RNA (eRNA) expression in cultured HCC cells, we identified six putative MYC enhancer regions. Amongst these, two highly active enhancers, located ~ 800 kb downstream of the MYC gene, were identified by qRT-PCR and reporter assays. We functionally confirmed these enhancers by demonstrating a significantly reduced MYC expression and cell proliferation upon CRISPR/Cas9-based deletion and/or antisense oligonucleotide (ASO)-mediated inhibition. In conclusion, we identified potential MYC enhancers of HCC and propose that the associated eRNAs may be suitable targets for HCC treatment.

Biphasic Production of Anti-ApoB100 Autoantibodies in Obese Humans and Mice.

Obesity is associated with autoimmunity, a phenomenon considered as harmful. Here we show that obese mice and humans produce IgG-type autoantibodies that specifically recognize apolipoprotein B-100 (ApoB100), its native epitope p210, and the synthetic p210 mimotope pB1. By contrast, antibodies against epitopes p45 and p240, which have been associated with atherosclerosis, were not detected in either the humans or mice. In a longitudinal analysis of high fat diet-fed mice, autoantibody production rose with increasing body weight, then decreased and plateaued at morbid obesity. Likewise, in a cross-sectional analysis of sera from 148 human volunteers spanning a wide BMI range and free of comorbidities, the immunoreactivity increased and then decreased with increasing BMI. Thus, the obesity-related ApoB100-specific natural autoantibodies characteristically showed the same epitope recognition, IgG-type, and biphasic serum levels in humans and mice. We previously reported that a pB1-based vaccine induces similar antibodies and can prevent obesity in mice. Therefore, our present results suggest that autoantibodies directed against native ApoB100 may mitigate obesity, and that the vaccination approach may be effective in humans.

Chemical-based primary human hepatocyte monolayer culture for the study of drug metabolism and hepatotoxicity: Comparison with the spheroid model.

Traditionally cultured monolayers of primary human hepatocytes (PHHs) deteriorate within days and thereby become unsuitable for drug-related studies. PHH spheroids (3D PHHs) maintain liver functions for weeks, but are considerably more demanding. Recently, a chemical-based approach (5C PHHs) succeeded in long-term culture of hepatocyte monolayers, but it remains unclear whether the drug-related functions are preserved. To clarify this, we compared the 5C and 3D PHHs in terms of gene expression analysis, proteomic analysis, functionality (basal and induced activities of representative CYP450 enzymes and urea and albumin secretions), survival in culture, and sensitivity to representative drugs. In all comparisons, which spanned culture durations of up to 4 weeks, the 5C PHHs performed at least as well as the 3D PHHs. Hence, the novel 5C PHH monolayer format combines the convenience of the traditional monolayer format with the functionality and maintainability of the spheroid format. Our results suggest that 5C PHH monolayers can be used more conveniently and efficiently for high-throughput drug screening, preclinical drug safety evaluations, and mechanistic studies.

Hypoxanthine phosphoribosyltransferase (HPRT)-deficiency is associated with impaired fertility in the female rat.

The purine hypoxanthine plays important role in regulating oocyte maturation and early embryonic development. The enzyme hypoxanthine phosphoribosyltransferase (HPRT) recycles hypoxanthine to generate substrates for nucleotide synthesis and key metabolites, and here we show that HPRT deficiency in the rat disrupts early embryonic development and causes infertility in females.

Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells.

BACKGROUND: Toll-like receptor 4 (TLR4) ligands such as lipopolysaccharide (LPS) activate immunomodulatory functions and the migration of human mesenchymal stromal cells (hMSCs). Here, we study the migration-related gene expression of LPS-stimulated hMSCs and the role and regulation of one of the upregulated genes, encoding the interferon-induced transmembrane protein 1 (IFITM1). METHODS: Gene expression profiles were determined by whole-transcriptome analysis (RNA-seq) and quantitative real-time PCR (qRT-PCR). Bioinformatics approaches were used to perform network and pathway analyses. The cell migration-related genes were identified with an in vitro wound healing assay. RNA interference (RNAi) was used to suppress the IFITM1 gene expression. The IFITM1 gene enhancer was analyzed by chromatin immunoprecipitation (ChIP) sequencing, ChIP-to-PCR, luciferase reporter assays, and qRT-PCR for enhancer RNAs (eRNAs). RESULTS: RNA-seq confirmed IFITM1 as an LPS-stimulated gene, and RNAi demonstrated its importance for the LPS-stimulated migration. LPS treatment increased the eRNA expression in enhancer region R2 (2 kb upstream) of the IFITM1 gene and enriched R2 for H3K27ac. Bioinformatics implicated the transcription factors NF-κB and IRF1, ChIP assays revealed their binding to R2, and chemical inhibition of NF-κB and RNAi directed against IRF1 prevented R2 eRNA and IFITM1 gene expression. CONCLUSIONS: Increased expression of the IFITM1 gene is required for LPS-stimulated hMSC migration. We described several underlying changes in the IFITM1 gene enhancer, most notably the NF-κB-mediated activation of enhancer region R2.

Basal-type lumenogenesis in extraembryonic endoderm stem cells models the early visceral endoderm.

Cultured rat primitive extraembryonic endoderm (pXEN) cells easily form free-floating multicellular vesicles de novo, exemplifying a poorly studied type of morphogenesis. Here, we reveal the underlying mechanism and the identity of the vesicles. We resolve the morphogenesis into vacuolization, vesiculation and maturation, and define the molecular characteristics and requirements of each step. Vacuolization is fueled by macropinocytosis and occurs by default if not blocked by high cell density or matrix proteins. Fine-tuned cell-cell contact then forms nascent three-cell vesicles with vacuole-derived lumina. In maturation, the vesicles complete epithelialization, expand via mitosis and continued fluid uptake, and differentiate further. The mature vesicles consist of a simple squamous epithelium with an apical-outside/basal-inside polarity that we trace back to the single cell stage. The polarity and gene expression pattern of the vesicles are similar to those of the early visceral endoderm. pXEN cells provide a useful in vitro model for study of matrix-independent, basal-type lumenogenesis and the physiology of the visceral endoderm.This article has an associated First Person interview with the first author of the paper.

Forkhead box O1 (FOXO1) controls the migratory response of Toll-like receptor (TLR3)-stimulated human mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) can potently regulate the functions of immune cells and are being investigated for the management of inflammatory diseases. Toll-like receptor 3 (TLR3)-stimulated human MSCs (hMSCs) exhibit increased migration and chemotaxis within and toward damaged tissues. However, the regulatory mechanisms underlying these migratory activities are unclear. Therefore, we analyzed the migration capability and gene expression profiles of TLR3-stimulated hMSCs using RNA-Seq, wound healing, and transwell cell migration assay. Along with increased cell migration, the TLR3 stimulation also increased the expression of cytokines, chemokines, and cell migration-related genes. The promoter regions of the latter showed an enrichment of putative motifs for binding the transcription factors forkhead box O1 (FOXO1), FOXO3, NF-κB (NF-κB1), and RELA proto-oncogene and NF-κB subunit. Of note, FOXO1 inhibition by the FOXO1-selective inhibitor AS1842856 significantly reduced both migration and the expression of migration-related genes. In summary, our results indicate that TLR3 stimulation induces hMSC migration through the expression of FOXO1-activated genes.

Transcriptome analysis shows ambiguous phenotypes of murine primitive endoderm-related stem cell lines.

Primitive endoderm (PrE)-related cell lines (XEN, pXEN and nEnd cells) show key features of the PrE. By transcriptome analysis, we show: (a) Compared to embryonic stem cells, PrE-related cell lines are less in vivo like, although early nEnd cells are most similar to the PrE. (b) These cell lines show post-PrE features of parietal (XEN and pXEN cells) or visceral (nEnd cells) endoderm, likely driven by Tgf-ß and Wnt/Activin signaling, respectively. (c) pXEN and nEnd cell lines additionally show pre-PrE features. Hence, neither pXEN nor nEnd cell cultures represent a distinct in vivo entity. Rather, their properties are compatible with mixed and hybrid phenotypes. Our findings indicate that pre-PrE, PrE and early post-PrE phenotypes result from different niches, which need to be better understood to derive cell lines that distinctly represent the early stages of the extraembryonic endoderm.

Inflammatory mediators ATP and S100A12 activate the NLRP3 inflammasome to induce MUC5AC production in airway epithelial cells.

Danger-associated molecular patterns (DAMPs) play a proinflammatory role in the pathogenesis of airway obstructive diseases such as severe asthma and chronic obstructive pulmonary disease. The NLRP3 inflammasome is a cytosolic multiprotein platform that activates the caspase-1 pathway in response to inflammatory stimuli such as DAMPs. ATP and S100 proteins are newly identified DAMPs that accumulate in inflamed airways. We previously demonstrated that S100A8, S100A9, and S100A12 induce production and secretion of MUC5AC, a major mucin in the conducting airway mucosa. The purpose of this study was to determine the involvement of NLRP3 inflammasome in, and the contribution of ATP to, S100 protein-induced MUC5AC production by NCI-H292 mucoepidermoid carcinoma cells. Stimulation with either S100A12 or ATP led to MUC5AC production at comparable levels. Simultaneous treatment with both stimuli resulted in additive increases in NLRP3, active caspase-1, IL-1ß, NLRP3/caspase-1 colocalization, and MUC5AC. NLRP3 siRNA or inhibitors of NF-κB, NLRP3 inflammasome oligomerization, or caspase-1 nearly completely inhibited ATP- and S100A12-mediated MUC5AC production. Furthermore, S100A12-as well as ATP-mediated MUC5AC production was almost equally blunted by both nonspecific and specific antagonists of the purinergic receptor P2X7, a principal receptor mediating NLRP3 inflammasome activation by ATP. Thus, these two danger signals contribute to MUC5AC production in airway epithelial cells through overlapping signaling pathways for NLRP3 inflammasome activation.

Isolation of primitive mouse extraembryonic endoderm (pXEN) stem cell lines.

Mouse blastocysts contain the committed precursors of the extraembryonic endoderm (ExEn), which express the key transcription factor Oct4, depend on LIF/LIF-like factor-driven Jak/Stat signaling, and initially exhibit lineage plasticity. Previously described rat blastocyst-derived ExEn precursor-like cell lines (XENP cells/HypoSCs) also show these features, but equivalent mouse blastocyst-derived cell lines are lacking. We now present mouse blastocyst-derived cell lines, named primitive XEN (pXEN) cells, which share these and additional characteristics with the XENP cells/HypoSCs, but not with previously known mouse blastocyst-derived XEN cell lines. Otherwise, pXEN cells are highly similar to XEN cells by morphology, lineage-intrinsic differentiation potential, and multi-gene expression profile, although the pXEN cell profile correlates better with the blastocyst stage. Finally, we show that pXEN cells easily convert into XEN-like cells but not vice versa. The findings indicate that (i) pXEN cells are more representative than XEN cells of the blastocyst stage; (ii) mouse pXEN, rather than XEN, cells are homologs of rat XENP cells/HypoSCs, which we propose to call rat pXEN cells.

An ApoB100-mimetic vaccine prevents obesity and liver steatosis in ApoE-/- mice.

BACKGROUND: Recently, a peptide vaccine (B4T) was developed that prevents high fat diet (HFD)-induced obesity and liver steatosis in wild type mice and appears to target an epitope present in ApoB100 but not ApoB48. Here, we ask whether B4T remains effective in ApoE knockout (ApoE-ko) mice, which exhibit a greatly increased ApoB48/ApoB100 ratio and develop atherosclerosis under HFD. METHODS: HFD-fed male ApoE-ko mice were injected with B4T or vehicle 3 times between 5 and 15 weeks of age. Until 45 weeks of age, they were regularly weighed and antibody titers determined. In the end, adiposity and organ histologies were examined. RESULTS: We find that in the ApoE-ko mice, B4T prevents HFD-induced body weight increases (p<0.01) to a comparable degree as previously shown in wild type mice. Also, liver steatosis was prevented as previously shown in wild type mice. By contrast, atherosclerotic plaque formation was not prevented in any of the vaccinated mice studied, in line with the observation that antibody production paralleled the weight reduction but largely preceded atherogenesis. CONCLUSION: The findings demonstrate effectiveness of B4T despite the increased ApoB48/B100 ratio, but argue against an effect on de novo plaque formation. At least under the current vaccination schedule, the obesity- and atherosclerosis-related roles of ApoB appear to be dissociable.

GPNMB promotes proliferation of developing eosinophils.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane protein that is expressed in a wide variety of cell types, including haematopoietic lineages. We previously demonstrated that GPNMB is one of the most highly expressed genes at an early and intermediate stage of eosinophil development. We herein examined GPNMB expression and its possible functional effect using cord blood (CB) CD34+ haematopoietic stem cells differentiating toward eosinophils during a 24-day culture period. Western blot and confocal microscopy analyses showed that GPNMB reached its highest levels at day 12 with most GPNMB-positive cells also expressing major basic protein 1 (MBP1), an eosinophil granule protein. GPNMB declined thereafter, but was still present at an appreciable level at day 24, the time when CB eosinophils most abundantly expressed MBP1 and were thus considered fully differentiated. When the developing CB cells were cultured in the presence of a blocking anti-GPNMB antibody, cell proliferation was significantly reduced. In agreement, ectopic expression of GPNMB in heterologous cells resulted in a significant increase in cell proliferation, while small interfering RNA of GPNMB inhibited the GPNMB-mediated proliferation. Thus, GPNMB is expressed in a temporal manner during eosinophil development and delivers a proliferative signal upon activation.

Transcriptome sequencing wide functional analysis of human mesenchymal stem cells in response to TLR4 ligand.

Due to their multipotentiality and immunomodulation, human mesenchymal stem cells (hMSCs) are widely studied for the treatment of degenerative and inflammatory diseases. Transplantation of hMSCs to damaged tissue is a promising approach for tissue regeneration. However, the physiological mechanisms and regulatory processes of MSC trafficking to injured tissue are largely unexplored. Here, we evaluated the gene expression profile and migratory potential of hMSCs upon stimulation with the TLR4 ligand lipopolysaccharide (LPS). Using RNA sequencing, we identified unique induction patterns of interferon stimulated genes, cytokines and chemokines involved in chemotaxis and homing. The -950 to +50 bp regions of many of these LPS-responsive genes were enriched with putative binding motifs for the transcription factors (TFs) interferon regulatory factor (IRF1) and nuclear factor kappa B (NF-κB1, REL), which were also induced by LPS along with other TFs. Chromatin immunoprecipitation assays showed that IRF1 bound within their target genes promoter region. In addition, IRF1 attenuation significantly down-regulated interferon stimulated genes as well as key cytokines. Furthermore, using pharmacological inhibitors, we showed that the NF-κB and phosphatidylinositol 3-kinase (PI3K) pathways regulate the migratory and cytokines/chemokines response to LPS. These unprecedented data suggest that IRF1 and NF-κB orchestrate the TLR4-primed immunomodulatory response of hMSCs and that this response also involves the PI3K pathway.

Olig2 is expressed late in human eosinophil development and controls Siglec-8 expression.

Oligodendrocyte transcription factor 2, a basic helix-loop-helix transcription factor that binds to E-box motifs, is known to have a key role in determining lineage specification of oligodendrocytes and motor neurons. In the present study, we report that oligodendrocyte transcription factor 2 is expressed in human eosinophils and involved in transcriptional activation of the gene encoding sialic acid binding immunoglobulin-like lectin 8 (Siglec-8), a late eosinophil-differentiation marker known to exert eosinophil apoptosis. When cord blood CD34+ hematopoietic stem cells differentiated toward eosinophils during a 24-d culture period, oligodendrocyte transcription factor 2 protein was expressed in cord blood eosinophils on d 24, a time when cord blood eosinophils are considered fully differentiated, whereas it was not detectable on d 18 or at earlier time points. Oligodendrocyte transcription factor 2 protein was also abundantly expressed in human peripheral-blood eosinophils but not in neutrophils, monocytes, lymphocytes, or cord blood mast cells. RNA sequencing analysis showed that numerous genes, especially those encoding eosinophil surface molecules, were highly up-regulated along with OLIG2 Among the genes examined, SIGLEC-8 messenger RNA and protein were markedly down-regulated in parallel with OLIG2 by an oligodendrocyte transcription factor 2 small interfering RNA or a short hairpin RNA, as evidenced by real-time polymerase chain reaction, fluorescence-activated cell sorting, and Western blot analyses. In reporter gene and chromatin immunoprecipitation experiments, an E-box in the first intron was found to stimulate SIGLEC-8 gene transcription and to bind oligodendrocyte transcription factor 2. Hence, at least one important aspect of eosinophil differentiation is regulated by oligodendrocyte transcription factor 2, a transcription factor that has not previously been reported, to our knowledge, in normal granulocytes.

An apolipoprotein B100 mimotope prevents obesity in mice.

Although apolipoprotein B100 (ApoB100) plays a key role in peripheral fat deposition, it is not considered a suitable therapeutic target in obesity. In the present study we describe a novel ApoB100 mimotope, peptide pB1, and the use of pB1-based vaccine-like formulations (BVFs) against high-fat diet (HFD)-induced obesity. In HFD- compared with chow-fed adolescent mice, BVFs reduced the 3-month body-weight gains attributable to increased dietary fat by 44-65%, and prevented mesenteric fat accumulation and liver steatosis. The body-weight reductions paralleled the titres of pB1-reactive immunoglobulin G (IgG) antibodies, and pB1-reactive antibodies specifically recognized native ApoB100 and a synthetic peptide from the C-terminal half of ApoB100. In cultured 3T3L1 adipocytes, anti-pB1 antibodies increased lipolysis and inhibited low-density lipoprotein (LDL) uptake. In cultured RAW 264.7 macrophages, the same antibodies enhanced LDL uptake (without causing foam cell formation). These findings make ApoB100 a promising target for an immunization strategy against HFD-induced obesity.