uPAR (PLAUR) Marks Two Intra-Tumoral Subtypes of Glioblastoma: Insights from Single-Cell RNA Sequencing.

Urokinase plasminogen activator receptor (uPAR) encoded by the PLAUR gene is known as a clinical marker for cell invasiveness in glioblastoma multiforme (GBM). It is additionally implicated in various processes, including angiogenesis and inflammation within the tumor microenvironment. However, there has not been a comprehensive study that depicts the overall functions and molecular cooperators of PLAUR with respect to intra-tumoral subtypes of GBM. Using single-cell RNA sequencing data from 37 GBM patients, we identified PLAUR as a marker gene for two distinct subtypes in GBM. One subtype is featured by inflammatory activities and the other subtype is marked by ECM remodeling processes. Using the whole-transcriptome data from single cells, we are able to uncover the molecular cooperators of PLAUR for both subtypes without presuming biological pathways. Two protein networks comprise the molecular context of PLAUR, with each of the two subtypes characterized by a different dominant network. We concluded that targeting PLAUR directly influences the mechanisms represented by these two protein networks, regardless of the subtype of the targeted cell.

Quantitative Evaluation of Stem-like Markers of Human Glioblastoma Using Single-Cell RNA Sequencing Datasets.

Targeting glioblastoma (GBM) stem-like cells (GSCs) is a common interest in both the laboratory investigation and clinical treatment of GBM. Most of the currently applied GBM stem-like markers lack validation and comparison with common standards regarding their efficiency and feasibility in various targeting methods. Using single-cell RNA sequencing datasets from 37 GBM patients, we obtained a large pool of 2173 GBM stem-like marker candidates. To evaluate and select these candidates quantitatively, we characterized the efficiency of the candidate markers in targeting the GBM stem-like cells by their frequencies and significance of being the stem-like cluster markers. This was followed by further selection based on either their differential expression in GBM stem-like cells compared with normal brain cells or their relative expression level compared with other expressed genes. The cellular location of the translated protein was also considered. Different combinations of selection criteria highlight different markers for different application scenarios. By comparing the commonly used GSCs marker CD133 (PROM1) with markers selected by our method regarding their universality, significance, and abundance, we revealed the limitations of CD133 as a GBM stem-like marker. Overall, we propose BCAN, PTPRZ1, SOX4, etc. for laboratory-based assays with samples free of normal cells. For in vivo targeting applications that require high efficiency in targeting the stem-like subtype, the ability to distinguish GSCs from normal brain cells, and a high expression level, we recommend the intracellular marker TUBB3 and the surface markers PTPRS and GPR56.

In vivo detection of urokinase-type plasminogen activator receptor (uPAR) expression in arterial atherogenesis using [64Cu]Cu-DOTA-AE105 positron emission tomography (PET).

BACKGROUND AND AIMS: Urokinase-type plasminogen activator receptor (uPAR) is associated with extracellular matrix (ECM) degradation and cancer aggressiveness. Its role in arterial atherogenesis as a molecular imaging target is not well-established. The aim of this study was to non-invasively visualize uPAR expression in atherosclerosis by a novel uPAR-targeting positron emission tomography (PET) tracer [64Cu]Cu-DOTA-AE105. METHODS: We used molecular biology to investigate uPAR expression by analyzing human atherosclerotic plaques and cultured cells. A retrospective analysis was performed on patients, who underwent combined PET/CT (n = 10) to measure [64Cu]Cu-DOTA-AE105 uptake in five large arteries, divided into a high and low-risk group based on coronary artery calcium score (CAC score). RESULTS: The in vitro assay for THP-1 monocytes displayed a significantly upregulated uPAR expression upon stimulation (5.2-fold upregulation, p < 0.0001 by a one-way ANOVA followed by Tukey's test) by single-cell flowcytometric analysis. Freshly excised human atherosclerotic plaques underwent flow cytometric and microarray analyses manifesting 73.9 ± 2.9% of mononuclear phagocyte system (MPS) cells expressing uPAR and had a greater than 7-fold higher gene expression of plasminogen activator urokinase receptor (PLAUR, p = 0.002), integrin subunit alpha X (ITGAX, p = 0.0008), and cluster of differentiation 163 (CD163, p < 0.0001). The tissue-to-background ratios (TBRmax) in five large arteries showed a higher [64Cu]Cu-DOTA-AE105 uptake in the group with high CAC score compared to the group with low CAC score (2.4 ± 0.1 vs 1.7 ± 0.1, p = 0.057), significantly higher in the ascending aorta (2.7 ± 0.1 vs 2.0 ± 0.1, p = 0.038) and the abdominal aorta (3.2 ± 0.2 vs 2.0 ± 0.2, p = 0.038) by a non-parametric Mann-Whitney test. CONCLUSIONS: uPAR is abundantly expressed by MPS cells in atherosclerotic plaques and can be visualized by the novel PET tracer [64Cu]Cu-DOTA-AE105 that may non-invasively detect extracellular matrix remodeling during atherogenesis.

The Initial Cardiac Tissue Response to Cryopreserved Allogeneic Adipose Tissue-Derived Mesenchymal Stromal Cells in Rats with Chronic Ischemic Cardiomyopathy.

Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2+ and CD38+ macrophages (p = 0.047 and p = 0.021), as well as in the CD4/CD8 lymphocyte ratio (p = 0.021), were found in the ASC group compared to the saline group. This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.

Somatostatin Analogue Treatment Primarily Induce miRNA Expression Changes and Up-Regulates Growth Inhibitory miR-7 and miR-148a in Neuroendocrine Cells.

Somatostatin (SST) analogues are used to control the proliferation and symptoms of neuroendocrine tumors (NETs). MicroRNAs (miRNA) are small non-coding RNAs that modulate posttranscriptional gene expression. We wanted to characterize the miRNAs operating under the control of SST to elucidate to what extent they mediate STT actions. NCI-H727 carcinoid cell line was treated with either a chimeric SST/dopamine analogue; a SST or dopamine analogue for proliferation assays and for identifying differentially expressed miRNAs using miRNA microarray. The miRNAs induced by SST analogue treatment are investigated in carcinoid cell lines NCI-H727 and CNDT2 using in situ hybridization, qPCR and proliferation assays. SST analogues inhibited the growth of carcinoid cells more potently compared to the dopamine analogue. Principal Component Analysis (PCA) of the samples based on miRNA expression clearly separated the samples based on treatment. Two miRNAs which were highly induced by SST analogues, miR-7 and miR-148a, were shown to inhibit the proliferation of NCI-H727 and CNDT2 cells. SST analogues also produced a general up-regulation of the let-7 family members. SST analogues control and induce distinct miRNA expression patterns among which miR-7 and miR-148a both have growth inhibitory properties.

Down-Regulation of miR-129-5p and the let-7 Family in Neuroendocrine Tumors and Metastases Leads to Up-Regulation of Their Targets Egr1, G3bp1, Hmga2 and Bach1.

Expression of miRNAs in Neuroendocrine Neoplasms (NEN) is poorly characterized. We therefore wanted to examine the miRNA expression in Neuroendocrine Tumors (NETs), and identify their targets and importance in NET carcinogenesis. miRNA expression in six NEN primary tumors, six NEN metastases and four normal intestinal tissues was characterized using miRNA arrays, and validated by in-situ hybridization and qPCR. Among the down-regulated miRNAs miR-129-5p and the let-7f/let-7 family, were selected for further characterization. Transfection of miR-129-5p inhibited growth of a pulmonary and an intestinal carcinoid cell line. Analysis of mRNA expression changes identified EGR1 and G3BP1 as miR-129-5p targets. They were validated by luciferase assay and western blotting, and found robustly expressed in NETs by immunohistochemistry. Knockdown of EGR1 and G3BP1 mimicked the growth inhibition induced by miR-129-5p. let-7 overexpression inhibited growth of carcinoid cell lines, and let-7 inhibition increased protein content of the transcription factor BACH1 and its targets MMP1 and HMGA2, all known to promote bone metastases. Immunohistochemistry analysis revealed that let-7 targets are highly expressed in NETs and metastases. We found down-regulation of miR-129-5p and the let-7 family, and identified new neuroendocrine specific targets for these miRNAs, which contributes to the growth and metastatic potential of these tumors.

Alternative polyadenylation of tumor suppressor genes in small intestinal neuroendocrine tumors.

The tumorigenesis of small intestinal neuroendocrine tumors (SI-NETs) is poorly understood. Recent studies have associated alternative polyadenylation (APA) with proliferation, cell transformation, and cancer. Polyadenylation is the process in which the pre-messenger RNA is cleaved at a polyA site and a polyA tail is added. Genes with two or more polyA sites can undergo APA. This produces two or more distinct mRNA isoforms with different 3' untranslated regions. Additionally, APA can also produce mRNAs containing different 3'-terminal coding regions. Therefore, APA alters both the repertoire and the expression level of proteins. Here, we used high-throughput sequencing data to map polyA sites and characterize polyadenylation genome-wide in three SI-NETs and a reference sample. In the tumors, 16 genes showed significant changes of APA pattern, which lead to either the 3' truncation of mRNA coding regions or 3' untranslated regions. Among these, 11 genes had been previously associated with cancer, with 4 genes being known tumor suppressors: DCC, PDZD2, MAGI1, and DACT2. We validated the APA in three out of three cases with quantitative real-time-PCR. Our findings suggest that changes of APA pattern in these 16 genes could be involved in the tumorigenesis of SI-NETs. Furthermore, they also point to APA as a new target for both diagnostic and treatment of SI-NETs. The identified genes with APA specific to the SI-NETs could be further tested as diagnostic markers and drug targets for disease prevention and treatment.

Characterization of miRNA expression in human degenerative lumbar disks.

BACKGROUND DATA: microRNAs (miRNAs) are short ∼22 nucleotide RNA sequences that regulate messengerRNA translation. miRNAs have shown to play a role in synthesis of inflammatory mediators. Since inflammation play a role in intervertebral disk (IVD) degeneration, the objective was to isolate miRNA from human lumbar intervertebral disks and subsequently characterize the difference in miRNA expression between the annulus fibrosus (AF) and nucleus pulposus (NP). METHODS: Fourteen patients undergoing anterior interbody fusion for degenerative disk disease of the lumbar spine were included. During surgery biopsies from the intervertebral disks were obtained and immediately placed in RNAlater. The RNAlater was decanted and the samples frozen at -80˚C until RNA extraction. This was performed using the Trizol method. Global miRNA expression analysis was performed using the Affymetrix GeneChip® miRNA array. RESULTS: We developed a method allowing the extraction of miRNA from human intervertebral disks usually yielding 1-4 µg of total RNA pr. 100 mg of disk. Twenty-seven miRNAs had a higher expression in the AF and 10 had the highest expression in the NP. Among the top 15 signaling pathways most likely to be controlled by these miRNAs were the transforming growth factor ß (TGFß), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) epidermal growth factor (EGF), and actin cytoskeletal pathway. CONCLUSION: We have demonstrated the presence of miRNA in the human IVD. The miRNA expression differs from muscle tissue and there are differences between the miRNA expressed in the NP and AF. The miRNAs identified control signaling pathways important for maintenance of the IVD. Future studies may determine the importance of miRNA in the development of IVD disease.

Human native lipoprotein-induced de novo DNA methylation is associated with repression of inflammatory genes in THP-1 macrophages.

BACKGROUND: We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, i.e. de novo DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20) hypermethylation in THP-1 macrophages. Here, we: 1) ask what gene expression changes accompany these epigenetic responses; 2) test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages. RESULTS: Native lipoprotein-induced de novo DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as de novo DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1) surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2) independent of the Dicer/micro-RNA pathway. CONCLUSIONS: Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a de novo DNA methyltransferase independently of canonical de novo enzymes, and show proof of principle that de novo DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.

Interferon-γ inhibits ghrelin expression and secretion via a somatostatin-mediated mechanism.

AIM: To investigate if and how the proinflammatory cytokine interferon γ (IFNγ) affects ghrelin expression in mice. METHODS: The plasma concentration of ghrelin, and gastric ghrelin and somatostatin expression, were examined in wild-type mice and mice infected with Helicobacter pylori (H. pylori). Furthermore, ghrelin expression was examined in two achlorhydric mouse models with varying degrees of gastritis due to bacterial overgrowth. To study the effect of IFNγ alone, mice were given a subcutaneous infusion of IFNγ for 7 d. Finally, the influence of IFNγ and somatostatin on the ghrelin promoter was characterized. RESULTS: H. pylori infection was associated with a 50% reduction in ghrelin expression and plasma concentration. Suppression of ghrelin expression was inversely correlated with gastric inflammation in achlorhdyric mouse models. Subcutaneous infusion of IFNγ suppressed fundic ghrelin mRNA expression and plasma ghrelin concentrations. Finally, we showed that the ghrelin promoter operates under the control of somatostatin but not under that of IFNγ. CONCLUSION: Gastric infection and inflammation is associated with increased IFNγ expression and reduced ghrelin expression. IFNγ does not directly control ghrelin expression but inhibits it indirectly via somatostatin.

Apolipoprotein B epitopes are present in nuclear preparations of human and mouse cells.

We previously showed that a native very low-density lipoprotein (VLDL)- and low-density lipoprotein (LDL)-rich mix induces global changes in DNA methylation in cultured THP-1 human macrophages. The exact molecular mechanisms for this response are not yet known. Previous studies showed that apolipoprotein B (ApoB) or its fragments can be localized in nuclei following cellular uptake, thus suggesting that ApoB may have a chromatin-modifying activity. To verify this hypothesis, we assessed whether ApoB epitopes are detected in THP-1 and mouse cell nuclei. Using a combination of immunoblotting, immunocytochemistry and chromatin immunoprecipitation, we showed that ApoB epitopes are present in THP-1 cell and mouse monocyte/macrophage nuclear fractions, but are perinuclear rather than intranuclear. Our results are not consistent with a direct chromatin-ApoB interaction as an underlying mechanism for the observed epigenetic responses to lipoproteins in THP-1 cells.

Chromatin modification by lipids and lipoprotein components: an initiating event in atherogenesis?

PURPOSE OF REVIEW: This review examines recent evidence proposing that lipids and lipoproteins can act as nuclear factors regulating chromatin structure. These novel data broaden our understanding of the mechanisms by which lipoproteins can affect basic biological phenomena such as transcription, genome stability, and cell differentiation. Furthermore, they provide novel insights into the mechanisms of diseases associated with abnormal lipid levels, such as atherosclerosis and diabetes. RECENT FINDINGS: Data consistent with a role for lipids and lipoprotein components as nuclear factors, as well as initiators of cytoplasmic signalling events resulting in chromatin modification, have been published in the past year. In particular, new insights into the mechanisms of interaction between chromatin and small lipid molecules such as short-chain fatty acids and cholesterol, and endogenous lipid peroxidation products have been obtained. Furthermore, it has been shown that hyperlipidaemic lipoprotein profiles are associated with aberrant DNA methylation patterns at early stages of atherosclerosis in mice and in cultured human macrophages, suggesting that a rearrangement of DNA methylation patterns is among early molecular changes associated with atherogenesis. SUMMARY: The findings described here are prompting efforts to understand further how lipids and lipoprotein components can affect gene expression in normal and pathological cell behaviour through regulation of the chromatin structure. It is possible that novel candidate therapeutic tools will emerge from these studies.