MicroRNA-30b Is Both Necessary and Sufficient for Interleukin-21 Receptor-Mediated Angiogenesis in Experimental Peripheral Arterial Disease

The interleukin-21 receptor (IL-21R) can be upregulated in endothelial cells (EC) from ischemic muscles in mice following hind-limb ischemia (HLI), an experimental peripheral arterial disease (PAD) model, blocking this ligand–receptor pathway-impaired STAT3 activation, angiogenesis, and perfusion recovery. We sought to identify mRNA and microRNA transcripts that were differentially regulated following HLI, based on the ischemic muscle having intact, or reduced, IL-21/IL21R signaling. In this comparison, 200 mRNAs were differentially expressed but only six microRNA (miR)/miR clusters (and among these only miR-30b) were upregulated in EC isolated from ischemic muscle. Next, myoglobin-overexpressing transgenic (MgTG) C57BL/6 mice examined following HLI and IL-21 overexpression displayed greater angiogenesis, better perfusion recovery, and less tissue necrosis, with increased miR-30b expression. In EC cultured under hypoxia serum starvation, knock-down of miR-30b reduced, while overexpression of miR-30b increased IL-21-mediated EC survival and angiogenesis. In Il21r−/− mice following HLI, miR-30b overexpression vs. control improved perfusion recovery, with a reduction of suppressor of cytokine signaling 3, a miR-30b target and negative regulator of STAT3. Together, miR-30b appears both necessary and sufficient for IL21/IL-21R-mediated angiogenesis and may present a new therapeutic option to treat PAD if the IL21R is not available for activation.

The Influence of Angiotensin Peptides on Survival and Motility of Human High-Grade Serous Ovarian Cancer Cells in Serum Starvation Conditions

High-grade serous ovarian carcinoma (HGSOC) is the most frequent and malignant form of ovarian cancer. A local renin–angiotensin system (RAS) has been found in the ovary, and changes in selected components of this system were observed in pathological states and also in ovarian cancer. In the present study, we examined the effect of three peptides, Ang-(1-7), Ang-(1-9) and Ang-(3-7), on proliferation and motility of the OVPA8 cell line, a new well-defined and preclinical model of HGSOC. We confirmed the presence of mRNA for all angiotensin receptors in the tested cells. Furthermore, our findings indicate that all tested angiotensin peptides increased the metabolic serum in the medium by activation of cell defense mechanisms such as nuclear factor kappaB signaling pathway andapoptosis. Moreover, tested angiotensin peptides intensified serum starvation-induced cell cycle arrest at the G0/G1 phase. In the case of Ang-(3-7), a significant decrease in the number of Ki67 positive cells (Ki67+) and reduced percentage of activated ERK1/2 levels in ovarian cancer cells were additionally reported. The angiotensin-induced effect of the accumulation of cells in the G0/G1 phase was not observed in OVPA8 cells growing on the medium with 10% FBS. Moreover, in the case of Ang-(3-7), the tendency was quite the opposite. Ang-(1-7) but not Ang-(1-9) or Ang-(3-7) increased the mobility of reluctant-to-migrate OVAP8 cells cultured in the serum-free medium. In any cases, the changes in the expression of VIM and HIF1A gene, associated with epithelial–mesenchymal transition (EMT), were not observed. In conclusion, we speculate that the adaptation to starvation in nutrient-deprived tumors can be modulated by peptides from the renin–angiotensin system. The influence of angiotensin peptides on cancer cells is highly dependent on the availability of growth factors and nutrients.

The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.

Vitronectin acts as a key regulator of adhesion and migration in umbilical cord-derived MSCs under different stress conditions

Mesenchymal stem cell (MSC)-based cellular therapy gets compromised as adverse microenvironmental conditions like nutrient deprivation, ischemia, hypoxia affect migration and engraftment, in addition to viability, of MSCs at the target site post transplantation. To improve the treatment efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under such microenvironmental stress conditions. In our study, we observed that Wharton's jelly-MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion with reduction in cellular migration under serum starvation. The changes in adhesion and migration characteristics were accompanied by extensive stress fibre formation and altered ECM gene expression with notable induction in vitronectin (VTN) expression and reduction in MMP-1 expression. Molecular and phenotypic correlative studies advocated the possible role of VTN and not MMP-1, in regulating adhesion and migration of WJ-MSCs. NF-kb was found to be the positive regulator of VTN expression while ERK pathway regulated it negatively. Further investigation with inhibition of these signalling pathways or VTN knockdown studies under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in migration. VTN knockdown under serum starvation also led to reduction in actin stress fibre along with reversal in expression of several ECM genes. Additionally, VTN induction being absent in hypoxia-treated WJ-MSCs, the hypoxic cells showed no significant change in the adhesion and migration properties. However, when VTN expression was induced under hypoxia by ERK pathway inhibition, similar increase in cell spread area and adhesion was observed. Our study thus highlights VTN as a factor which is induced under serum starvation stress and possibly affects the adhesion and migration properties of WJ-MSCs.

STEM-08. ST6Gal1-MEDIATED SILALYLATION IS CRITICAL FOR GLIOBLASTOMA GROWTH

Although altered cell surface glycosylation was one of the earliest modifications observed in neoplastic progression, this facet of cancer cell biology has received meager attention, particularly in brain tumors. Among the various glycosyltransferases present in human cells, golgi sialyltransferase ST6Gal1 [beta-galactoside alpha-2,6-sialyltransferase 1] adds sialic acid residues in α2-6 linkage to membrane-bound and secreted N-glycans. ST6Gal1 is known to be pro-tumorigenic in epithelial cancers where it can promote epithelial to mesenchymal transformation, tumor-initiating cell (TIC) phenotypes, and survival of cells exposed to stressors such as chemo- and radiotherapy, hypoxia, or serum starvation. However, roles for this potent TIC regulator have not been well explored in GBM as experiments in standard cell lines suggested ST6Gal1 was epigenetically silenced. To explore our hypothesis that ST6Gal1-mediated α2,6 sialylation is elevated in Brain Tumor Initiating Cells (BTICs) and promotes GBM growth, we utilized GBM patient-derived xenografts (PDXs). ST6Gal1 is expressed in GBM PDX tissue sections and elevated in stem-like BTICs in comparison to differentiated GBM cells or astrocytes. Knockdown of ST6Gal1 in BTICs decreased growth and neurosphere formation capacity in vitro, suggesting that ST6Gal1 regulates BTIC maintenance. Similarly, cells isolated directly from PDXs that were sorted for high and low expression of α2,6 sialylation showed that α2,6 sialylationhigh GBM PDX have elevated neurosphere formation capacity and growth. Further, immunocompromised mice injected with sorted α2,6 sialylationhigh PDX cells had significantly lower survival compared to mice injected with α2,6 sialylationlow cells. Using proteomic analysis of ST6Gal1 KD vs NT PDX, we identified novel regulators of cancer stem cell biology directly modulated by ST6Gal1. As we identified a small subset of IDHwt GBMs with ST6Gal1 and SOX2 amplification, we are generating a novel gliomagenesis model with conditional ST6Gal1 overexpression. Together, our data strongly implicates ST6Gal1 as a regulator of GBM BTIC maintenance and GBM growth.

Targeting NOX4 Disrupts the Resistance of Papillary Thyroid Carcinoma to Chemotherapeutic Drugs and Lenvatinib

Background: Advanced differentiated thyroid cancer cells are subjected to extreme nutritional starvation which contributes to develop resistance to treatments; however, the underlying mechanism remains unclear.Methods: We used 0.5% serum to mimic starvation during cell culture. A CCK8 assay, cell death Detection ELISAPLUS kit, PI staining were measured to determine cell viability, cell apoptosis and cell cycle respectively in BCPAP cells and TPC-1 cells expressing shRNA against NOX4. The cells were then treated with etoposide and doxorubicin, two chemotherapeutic drugs, as well as lenvatinib to determine the role of NOX4 in resistance. Lenvatinib-resistant BCPAP cells (LRBCs) were also established to confirm the role. Finally, GLX351322, a chemical inhibitor targeting NOX4, was used to inhibit NOX4-derived ROS and detect the the contribution of NOX4 to resistance in vitro and in vivo. Results: NADPH oxidase 4 (NOX4) is highly expressed under serum starvation in BCPAP or TPC-1 cells. NOX4 knockdown impairs cell viability, increases cell apoptosis, extends G1 phase in cell cycle and modulates the level of energy-associated metabolites in starved cells. When these starved cells or Lenvatinib-resistant BCPAP cells (LRBCs) are treated with chemotherapeutic drugs or Lenvatinib, NOX4 knockdown inhibits cell viability and aggravates cell apoptosis depending on NOX4-derived ROS production. GLX351322, a NOX4-derived ROS inhibitor, has a significantly inhibitory effect on cell growth in vitro and the growth of BPCPA-derived even LRBCs-derived xenografts in vivo.Conclusions: These findings highlight NOX4 and NOX4-derived ROS as a potential therapeutic target in resistance of PTC patients.

Stress response in Entamoeba histolytica is associated with robust processing of tRNA to tRNA-halves

AbstracttRNA-derived fragments have been reported in many different organisms and have diverse cellular roles such as regulating gene expression, inhibiting protein translation, silencing transposable elements and modulating cell proliferation. In particular tRNA halves, a class of tRNA fragments produced by the cleavage of tRNAs in the anti-codon loop, have been widely reported to accumulate under stress and regulate translation in cells. Here we report the presence of tRNA-derived fragments in Entamoeba with tRNA halves being the most abundant. We further established that tRNA halves accumulate in the parasites upon different stress stimuli such as oxidative stress, heat shock, and serum starvation. We also observed differential expression of tRNA halves during developmental changes of trophozoite to cyst conversion with various tRNA halves accumulating during early encystation. In contrast to other systems, the stress response does not appear to be mediated by a few specific tRNA halves as multiple tRNAs appear to be processed during the various stresses. Furthermore, we identified some tRNA-derived fragments are associated with Entamoeba Argonaute proteins, EhAgo2-2, and EhAgo2-3, which have a preference for different tRNA-derived fragment species. Finally, we show that tRNA halves are packaged inside extracellular vesicles secreted by amoeba. The ubiquitous presence of tRNA-derived fragments, their association with the Argonaute proteins, and the accumulation of tRNA halves during multiple different stresses including encystation suggest a nuanced level of gene expression regulation mediated by different tRNA-derived fragments in Entamoeba.ImportancetRNA-derived fragments are small RNAs formed by the cleavage of tRNAs at specific positions. These have been reported in many organisms to modulate gene expression and thus regulate various cell functions. In the present study, we report for the first time the presence of tRNA-derived fragments in Entamoeba. tRNA-derived fragments were identified by bioinformatics analyses of small RNA sequencing datasets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during developmental process of encystation. We also found that shorter tRNA-derived fragments are bound to Entamoeba Argonaute proteins, indicating that they may have a potential role in the Argonaute-mediated RNA-interference pathway which mediates robust gene silencing in Entamoeba. Our results suggest that tRNA-derived fragments in Entamoeba have a possible role in regulating gene expression during environmental stress.

Identification of new reference genes with stable expression patterns for gene expression studies using human cancer and normal cell lines

Reverse transcription—quantitative real-time PCR (RT-qPCR) is a ubiquitously used method in biological research, however, finding appropriate reference genes for normalization is challenging. We aimed to identify genes characterized with low expression variability among human cancer and normal cell lines. For this purpose, we investigated the expression of 12 candidate reference genes in 13 widely used human cancer cell lines (HeLa, MCF-7, A-549, K-562, HL-60(TB), HT-29, MDA-MB-231, HCT 116, U-937, SH-SY5Y, U-251MG, MOLT-4 and RPMI-8226) and, in addition, 7 normal cell lines (HEK293, MRC-5, HUVEC/TERT2, HMEC, HFF-1, HUES 9, XCL-1). In our set of genes, we included SNW1 and CNOT4 as novel candidate reference genes based on the RNA HPA cell line gene data from The Human Protein Atlas. HNRNPL and PCBP1 were also included along with the „classical” reference genes ACTB, GAPDH, IPO8, PPIA, PUM1, RPL30, TBP and UBC. Results were evaluated using GeNorm, NormFiner, BestKeeper and the Comparative ΔCt methods. In conclusion, we propose IPO8, PUM1, HNRNPL, SNW1 and CNOT4 as stable reference genes for comparing gene expression between different cell lines. CNOT4 was also the most stable gene upon serum starvation.