Jun mRNA Research Articles

JUN mRNA translation regulation is mediated by multiple 5' UTR and start codon features.

Regulation of mRNA translation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5' untranslated region (5' UTR) and with the 5' -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5' UTR is nearly one kilobase in length, and has a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Here we find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5' UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Our results reveal additional layers of regulation for JUN translation and show the potential of JUN as a model transcript for understanding multiple interacting modes of translation regulation.

Identification of aging-related genes in diagnosing osteoarthritis via integrating bioinformatics analysis and machine learning.

Osteoarthritis (OA) is one of the main causes of pain and disability in the world, it may be caused by many factors. Aging plays a significant role in the onset and progression of OA. However, the mechanisms underlying it remain unknown. Our research aimed to uncover the role of aging-related genes in the progression of OA. In Human OA datasets and aging-related genes were obtained from the GEO database and the HAGR website, respectively. Bioinformatics methods including Gene Ontology (GO), Kyoto Encyclopedia of Genes Genomes (KEGG) pathway enrichment, and Protein-protein interaction (PPI) network analysis were used to analyze differentially expressed aging-related genes (DEARGs) in the normal control group and the OA group. And then weighted gene coexpression network analysis (WGCNA), the least absolute shrinkage and selection operator (LASSO) regression, and the Random Forest (RF) machine learning algorithms were used to find the hub genes. Four overlapping hub genes: HMGB2, CDKN1A, JUN, and DDIT3 were identified. According to the nomogram model and receiver operating characteristic (ROC) curve analysis, four hub DEARGs had good diagnostic value in distinguishing normal from OA. Furthermore, the qRT-PCR test demonstrated that HMGB2, CDKN1A, JUN, and DDIT3 mRNA expression levels were lower in OA group than in normal group. Finally, these four-hub aging-related genes may help us understand the underlying mechanism of aging in osteoarthritis and could be used as possible diagnostic and therapeutic targets.

Integrative metabolomic and network pharmacological analysis reveals potential mechanisms of Cardamine circaeoides Hook.f. & Thomson in alleviating potassium oxonate-induced asymptomatic hyperuricemia in rats.

Cardamine circaeoides Hook.f. & Thomson (CC), a herb of the genus Cardamine (family Brassicaceae), has a rich historical usage in China for both culinary and medicinal purposes. It is distinguished by its remarkable ability to hyperaccumulate selenium (Se). CC has demonstrated efficacy in the prevention of metabolic disorders. However, investigations into the effects of CC on asymptomatic hyperuricemia remain scarce. The objective of this study is to elucidate the mechanism by which CC aqueous extract (CCE) exerts its anti-hyperuricemic effects on asymptomatic hyperuricemic rats induced by potassium oxonate (PO) by integrating metabolomics and network pharmacological analysis. Asymptomatic hyperuricemia was induced by feeding rats with PO (1000mg/kg) and CCE (0.75, 1.5, or 3g/kg) once daily for 30days. Various parameters, including body weight, uric acid (UA) levels, histopathology of renal tissue, and inflammatory factors (IL-1β, IL-6, IL-8, and TNF-α) were assessed. Subsequently, metabolomic analysis of kidney tissues was conducted to explore the effects of CCE on renal metabolites and the related pathways. Furthermore, network pharmacology was employed to explicate the mechanism of action of CCE components identified through UPLC-Q-TOF-MS analysis. Finally, metabolomic and network-pharmacology analyses were performed to predict crucial genes dysregulated in the disease model and rescued by CCE, which were then subjected to verification by RT-qPCR. The findings revealed that CCE significantly inhibited the UA levels from the 21stday to the 30thday. Moreover, CCE exhibited significant inhibition of IL-1β, IL-6, IL-8, and TNF-α levels in renal tissues. The dysregulation of 18 metabolites and the tyrosine, pyrimidine, cysteine, methionine, sphingolipid, and histidine metabolism pathways was prevented by CCE treatment. A joint analysis of targets predicted using the network pharmacology approach and the differential metabolites found in metabolics predicted 8 genes as potential targets of CCE, and 3 of them (PNP gene, JUN gene, and ADA gene) were verified at the mRNA level by RT-qPCR. We conclude that CCE has anti-hyperuricemia effects and alleviates renal inflammation in a rat model of hyperuricemia, and these efficacies are associated with the reversal of increased ADA, PNP, and JUN mRNA expression in renal tissues.

Altering the Cleaving Effector in Chimeric Molecules that Target RNA Enhances Cellular Selectivity.

Small molecules that target RNA and effect their cleavage are useful chemical probes and potential lead medicines. In this study, we investigate factors affecting degradation of two cancer-associated RNA targets, the mRNA that encodes the transcription factor JUN (c-Jun) and the hairpin precursor to microRNA-372 (pre-miR-372). The two RNA targets harbor the same small-molecule binding site juxtaposed with different neighboring structures. Specifically, pre-miR-372 has AU pairs and contiguous purines on one strand near the small-molecule binding site, making it an ideal substrate for oxidative cleavage via the direct degrader bleomycin A5. In contrast, while JUN mRNA has a similar number of AU pairs near the small-molecule binding site, it lacks contiguous purine nucleotides. Instead, it contains unpaired pyrimidine nucleotides, which are preferred substrates for RNase L, a ribonuclease that can be recruited to RNA with heterobifunctional ribonuclease targeting chimeras (RiboTACs). We hypothesized that structural features surrounding the binding site could be leveraged to program selective small-molecule degradation by alteration of the cleaving module. Indeed, the bleomycin degrader cleaves pre-miR-372 in gastric cancer cells but not JUN mRNA. Conversely, the RiboTAC cleaves JUN mRNA but not pre-miR-372. Thus, the selection of the appropriate cleaving effector moiety for an RNA-binding small molecule can be leveraged to cleave an RNA selectively in a predictable manner, which could have broad implications.

Bioinformatics and network pharmacology identify promotional effects and potential mechanisms of ethanol on esophageal squamous cell carcinoma and experimental validation

Ethanol is an important risk factor for esophageal squamous cell carcinoma (ESCC); however, the molecular mechanisms behind how ethanol promotes ESCC development remain poorly understood. In this study, ethanol-ESCC-associated target genes were constructed and screened using network pharmacology and subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and bioinformatics analysis. A mouse ethanol-exposed esophageal cancer model was constructed with 4-nitroquinoline-1-oxide (4-NQO) to assess its survival and tumor lesion status, and the mechanism of ethanol-promoted ESCC lesions was verified by qRT–PCR and Western blotting. The results showed that 126 ethanol-ESCC crossover genes were obtained, which were significantly enriched in the PI3K/AKT signaling pathway. Bioinformatics results showed that the target genes TNF, IL6, IL1β and JUN were highly expressed in esophageal tumor samples and positively correlated with tumor proliferation and apoptosis genes, and the genetic information of these genes was mutated to different degrees. Animal model experiments showed that ethanol decreased the survival rate and aggravated the occurrence of esophageal cancer in mice. qRT–PCR showed that ethanol promoted the expression of TNF, IL6, IL1β and JUN mRNA in mouse esophageal tumor tissues, and Western blotting showed that ethanol promoted p-PI3K and p-AKT protein expression in mouse esophageal tumor tissues. In conclusion, ethanol promotes esophageal carcinogenesis by increasing the expression of TNF, IL6, IL1β and JUN and activating the PI3K/AKT signaling pathway.

Programming inactive RNA-binding small molecules into bioactive degraders

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure–activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.

Bioinformatics analysis of ferroptosis in spinal cord injury.

Ferroptosis plays a key role in aggravating the progression of spinal cord injury (SCI), but the specific mechanism remains unknown. In this study, we constructed a rat model of T10 SCI using a modified Allen method. We identified 48, 44, and 27 ferroptosis genes that were differentially expressed at 1, 3, and 7 days after SCI induction. Compared with the sham group and other SCI subgroups, the subgroup at 1 day after SCI showed increased expression of the ferroptosis marker acyl-CoA synthetase long-chain family member 4 and the oxidative stress marker malondialdehyde in the injured spinal cord while glutathione in the injured spinal cord was lower. These findings with our bioinformatics results suggested that 1 day after SCI was the important period of ferroptosis progression. Bioinformatics analysis identified the following top ten hub ferroptosis genes in the subgroup at 1 day after SCI: STAT3, JUN, TLR4, ATF3, HMOX1, MAPK1, MAPK9, PTGS2, VEGFA, and RELA. Real-time polymerase chain reaction on rat spinal cord tissue confirmed that STAT3, JUN, TLR4, ATF3, HMOX1, PTGS2, and RELA mRNA levels were up-regulated and VEGFA, MAPK1 and MAPK9 mRNA levels were down-regulated. Ten potential compounds were predicted using the DSigDB database as potential drugs or molecules targeting ferroptosis to repair SCI. We also constructed a ferroptosis-related mRNA-miRNA-lncRNA network in SCI that included 66 lncRNAs, 10 miRNAs, and 12 genes. Our results help further the understanding of the mechanism underlying ferroptosis in SCI.

M6A RNA methylation regulates the transcription factors JUN and JUNB in TGF-β-induced epithelial–mesenchymal transition of lung cancer cells

N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3'UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta-induced EMT of lung cancer cells.

Obesity and exercise training alter inflammatory pathway skeletal muscle small extracellular vesicle microRNAs.

New Findings What is the central question of this study? Is 1 week of exercise training sufficient to reduce local and systemic inflammation?Do obesity and short‐term concurrent aerobic and resistance exercise training alter skeletal muscle extracellular vesicle (EV) contents? What is the main finding and its importance? Obesity alters skeletal muscle small EV microRNAs targeting inflammatory and growth pathways. Exercise training alters skeletal muscle small EV microRNAs targeting inflammatory pathways, indicative of reduced inflammation. Our findings provide support for the hypotheses that EVs play a vital role in intercellular communication during health and disease and that EVs mediate many of the beneficial effects of exercise. Obesity is associated with chronic inflammation characterized by increased levels of inflammatory cytokines, whereas exercise training reduces inflammation. Small extracellular vesicles (EVs; 30–150 nm) participate in cell‐to‐cell communication in part through microRNA (miRNA) post‐transcriptional regulation of mRNA. We examined whether obesity and concurrent aerobic and resistance exercise training alter skeletal muscle EV miRNA content and inflammatory signalling. Vastus lateralis biopsies were obtained from sedentary individuals with (OB) and without obesity (LN). Before and after 7 days of concurrent aerobic and resistance training, muscle‐derived small EV miRNAs and whole‐muscle mRNAs were measured. Pathway analysis revealed that obesity alters small EV miRNAs that target inflammatory (SERPINF1, death receptor and Gαi) and growth pathways (Wnt/β‐catenin, PTEN, PI3K/AKT and IGF‐1). In addition, exercise training alters small EV miRNAs in an anti‐inflammatory manner, targeting the IL‐10, IL‐8, Toll‐like receptor and nuclear factor‐κB signalling pathways. In whole muscle, IL‐8 mRNA was reduced by 50% and Jun mRNA by 25% after exercise training, consistent with the anti‐inflammatory effects of exercise on skeletal muscle. Obesity and 7 days of concurrent exercise training differentially alter skeletal muscle‐derived small EV miRNA contents targeting inflammatory and anabolic pathways.

Expression and purification of recombinant human CCL5 and its biological characterization.

C-C motif chemokine ligand 5 (CCL5) is crucial in the tumor microenvironment. It has been previously reported to act as a key role in tumor invasion and metastasis. However, the function of exogenous CCL5 in ovarian cancer has not been well-characterized. The present study attempted to express and purify recombinant CCL5 protein and investigate the exogenous CCL5 in ovarian cancer cell proliferation. The human CCL5 was amplified and inserted into the pET-30a vectors for prokaryotic expression in Escherichia coli BL21. Soluble His-CCL5 was successfully expressed with 0.1 mmol/L of isopropyl-β-D-1-tiogalactopiranoside at 25 ℃ and purified by affinity chromatography. Additionally, methyl thiazolyl tetrazolium (MTT) assay demonstrated that CCL5 promotes ovarian cancer cell proliferation; increases the phosphorylation levels of extracellular-signal-regulated kinase and mitogen-activated protein kinase/ERK kinase, and increases the mRNA levels of Jun, NF-κB2, Nras, Relb, and Traf2. Furthermore, treatment with the MEK inhibitor reduced the Jun, NF-κB2, and Traf2 mRNA levels, indicating that exogenous CCL5 increased ovarian cancer cell proliferation, through MEK/ERK pathway activation, and Jun, NF-κB2, and Traf2 expression. The present study provided primary data for further studies to discover more CCL5 functions in ovarian cancer.

ВЛИЯНИЕ ФИТОХИМИЧЕСКИХ ЭКСТРАКТОВ НА ЭКСПРЕССИЮ ГЕНОВ NFE2L2, JUN, SOD1 В КЛЕТКАХ ЧЕЛОВЕКА

Investigations of the effects of phitochemical compounds from extracts of pomegranate, grape seeds and garlic on expression of NFE2L2, JUN, SOD1 in cultivated cells of the human blood showed that transcription of SOD1 increased in the presence of pomegranate and grape seed extracts. Level of mRNA in NFE2L2 and JUN rose briskly only under the influence of a high concentration of the grape seed extract. Level of JUN mRNA correlated with NFE2L2 mRNA. Garlic extract had no effect on transcription of NFE2L2, JUN, SOD1. Activation of the transcription of all 3 genes under study by the grape seeds extract can contribute to controlling the free radical processes in human cells.

Prognostic role of EGR1 in breast cancer: a systematic review

EGR1 (early growth response 1) is dysregulated in many cancers and exhibits both tumor suppressor and promoter activities, making it an appealing target for cancer therapy. Here, we used a systematic multiomics analysis to review the expression of EGR1 and its role in regulating clinical outcomes in breast cancer (BC). EGR1 expression, its promoter methylation, and protein expression pattern were assessed using various publicly available tools. COSMIC-based somatic mutations and cBioPortal-based copy number alterations were analyzed, and the prognostic roles of EGR1 in BC were determined using Prognoscan and Kaplan-Meier Plotter. We also used bc-GenEx-Miner to investigate the EGR1 co-expression profile. EGR1 was more often downregulated in BC tissues than in normal breast tissue, and its knockdown was positively correlated with poor survival. Low EGR1 expression levels were also associated with increased risk of ER+, PR+, and HER2-BCs. High positive correlations were observed among EGR1, DUSP1, FOS, FOSB, CYR61, and JUN mRNA expression in BC tissue. This systematic review suggested that EGR1 expression may serve as a prognostic marker for BC patients and that clinicopathological parameters influence its prognostic utility. In addition to EGR1, DUSP1, FOS, FOSB, CYR61, and JUN can jointly be considered prognostic indicators for BC.

Endothelial METTL3 (Methyltransferase-Like 3) Inhibits Fibrinolysis by Promoting PAI-1 (Plasminogen Activator Inhibitor-1) Expression Through Enhancing Jun Proto-Oncogene N6-Methyladenosine Modification.

METTL3 (methyltransferase-like protein 3)-mediated N6-methyladenosine modification is the most abundant RNA modification on eukaryote mRNAs and plays a crucial role in diverse physiological and pathological processes. However, whether N6-methyladenosine modification has function in thrombosis is unknown. This study aims to determine the role of METTL3 in the endothelial cells-mediated thrombosis. Approach and Results: RNA-sequencing and real-time quantitative PCR revealed that the expression of PAI-1 (plasminogen activator inhibitor-1) was downregulated in METTL3 knockdown human umbilical vein endothelial cells. In vitro experiments showed that METTL3 suppressed fibrinolysis. Mechanically, RNA methylation sequencing and meRIP-quantitative real-time PCR showed that METTL3 catalyzed N6-methyladenosine modification on 3' UTR of JUN mRNA. Western blotting analysis showed that METTL3 promoted JUN protein expression. Chromatin immunoprecipitation analysis demonstrated that JUN bound to the PAI-1 promoter in human umbilical vein endothelial cells. Furthermore, mice challenged with lipopolysaccharide resulted in higher METTL3 expression in vessels. Endothelial-specific knockdown of Mettl3 decreased expression of active PAI-1 in plasma and attenuated fibrin deposition in livers and lungs during endotoxemia. Our study reveals that METTL3-mediated N6-methyladenosine modification plays a crucial role in fibrinolysis and is an underlying target for the therapy of thrombotic disorders.

Regulation of Jun and Fos AP-1 transcription factors by JNK MAPKs signaling cascade in areca nut extract treated KB cells

The edible endosperm of Areca catechu is recognized as a potent carcinogenic agent either consumed alone or in combination with tobacco. Habitual chewing of areca nut leads to orally potential malignant disorders which are highly effective in malignant transformation and thereby lead to oral carcinogenesis. Human buccal epithelial KB carcinoma cells were used as an experimental cell system to inspect the mechanistic act of aqueous extract of areca nut on biochemical status and their implications on transcriptional activation of cancer signaling cascade that could possibly trigger numerous oncogenic players and finally decides the cells fate. Extract treated cells showed reduced viability with altered balance between oxidants and antioxidants which lead to redox status and which is known to distort various biological processes within the cell system. Results of RT-PCR demonstrated decreased expression of BCl2, cell cycle regulators along with Activator Protein −1 (AP-1) components. While Bax, p16 and p21 mRNAs showed increased expression in extract treated KB cells. Likewise, the translational levels of proliferation cell nuclear antigen (PCNA), tumor suppressor p53, retinoblastoma (Rb) and cyclin dependent kinase 4 (CDK4) were decreased along with AP-1 subunits (c-Jun/c-Fos) with increased protein levels of p21 in extract treated KB cells. Further, the downstream activation and regulation of AP-1 transcription factors could be through stress activated c-Jun – N terminal Kinase (JNK) Mitogen Activated Protein Kinases (MAPKs) which downregulated both Jun and Fos mRNA transcripts in areca nut extract exposed KB cells. Thus, outcome of the study provides insights into mechanistic path of pathogenesis of areca related disorders. Further, it could aid in designing new therapeutic modalities that specific targets these oncogenic players and help in disease management.

The Pharmacological IGF1R-IRS1/2 Inhibitor NT157 Presents Multiple Anti-Neoplastic Effects in Myeloproliferative Neoplasms

Introduction: The recurrent V617F mutation in JAK2 plays an important role in the pathogenesis of BCR-ABL1 negative myeloproliferative neoplasms (MPN). Absence of complete clinical response to ruxolitinib, a JAK1/2 inhibitor, highlights the need to target other signaling pathways that contribute to JAK2. Recent evidence indicates that the IGF1R/IRS pathway is a potential target in MPN: (i) insulin receptor substrate 2 (IRS2) cooperates with malignant transformation induced by JAK2V617F, and (ii) increased IGF1 signaling induced by AIRAPL down-regulation causes MPN in mice. NT157 is a synthetic compound designed as an IRS1/2 inhibitor, which presented anti-neoplastic effects in solid tumors. Aims: The objective herein was tocharacterize the molecular and cellular effects of NT157 treatment, combined or not with ruxolitinib, in MPN JAK2V617F. Methods: HEL and SET2 cell lines (both carrying JAK2V617F mutation) were treated or not with vehicle, NT157 and/or ruxolitinib and submitted to evaluation of cell viability (MTT assay), apoptosis (annexin V/PI and caspase cleavage), proliferation (Ki-67), clonogenicity (colony forming assay), cell cycle (PI), gene expression (PCR array and qPCR) and protein expression/activation (Western blot). Primary cells from polycythemia vera (PV) patients (n=3) were exposed to NT157 (3.2 µM) treatment and evaluated for erythropoietin-independent colony formation. Statistical analysis was performed using ANOVA or Student's t test. P -value <0.05 was considered statistically significant. Results: NT157 treatment significantly decreased cell viability, clonogenicity and cell proliferation (all p <0.05). NT157 induced apoptosis at doses equal or higher than 0.8 μM as verified by flow cytometry analysis of annexin V/PI (p ˂0.001) and confirmed by increased levels of cleaved caspase 3, 8 and 9. The percentage of apoptotic cells (annexin V positive) identified for untreated cells and cells exposed to NT157 0.8 μM was respectively, 14 and 56% after 24 hours of treatment; 11 and 60% after 48 hours, 6 and 40% after 72 hours (all p ˂0.001). Cell cycle analysis was performed 24 hours after NT157 exposition and revealed cell cycle arrest in G2/M, associated with decrease in G0/G1 population (p ˂0.001). In a molecular scenario, NT157 treatment strongly reduced IRS1 and IRS2 expression, and STAT3, STAT5 and ERK1/2 phosphorylation in a dose dependent manner. PCR array analyzes revealed that NT157 treatment modulated 23 oncogenes and tumor suppressor genes. Based on the cellular effects observed after NT157 treatment, we selected six genes, three repressed oncogenes and three upregulated apoptotic-related genes, to be validated in a larger number of experiments. Upon NT157 exposition, CCND1 (cyclin D1), MYB and WT1 mRNA levels were reduced, while CDKN1A (p21), FOS and JUN mRNA levels increased (all p ˂0.05). The combined treatment of NT157 plus ruxolitinib did not present potentializing effects when compared to the monotherapies. In contrast, the combined treatment prevented, at least in part, the cytotoxic effects of NT157. In HEL cells, NT157 0.8 µM monotherapy promoted enhanced apoptosis rate compared to the combined treatment, using NT157 0.8 plus ruxolitinib 0.3 µM (52% vs. 38%, respectively [ p ˂0.05]). Ruxolitinib also prevented NT157 effect on cell cycle progression, causing a reduction in G2/M arrest when compared to NT157 monotherapy (p <0.05). In primary hematopoietic cells from PV patients, NT157 exposition reduced erythropoietin-independent colony formation by 49 % (patient #1), 12% (patient #2) and 23% (patient #3); the mean reduction of erythroid colony number for all PV samples was 29% (p <0.05). Conclusions: IGF1R-IRS1/2 pharmacological inhibitor NT157 displayed remarkable anti-neoplastic effects in JAK2V617F cells lines and MPN primary cells. Our exploratory approaches provided new insights about molecular mechanisms of NT157. Our findings indicate that inhibition of IGF1R/IRS signaling, using NT157 monotherapy, emerges as a novel therapeutic opportunity for MPN JAK2V617F. DisclosuresNo relevant conflicts of interest to declare.

Amniotic fluid mesenchymal stem cells repair mouse corneal cold injury by promoting mRNA N4-acetylcytidine modification and ETV4/JUN/CCND2 signal axis activation

Severe corneal injury is one of the main causes of loss of visual function. Mesenchymal stem cells (MSCs) have the ability to repair damaged cells in vivo. The present study aimed to explore whether MSCs could function as a cell therapy tool to replace traditional methods to treat corneal injury. CD44 + /CD105 + mesenchymal stem cells isolated from mouse amniotic fluid (mAF-MSCs) were injected into mice after cryoinjury to induce corneal endothelial cell injury. Histopathological assays indicated that mAF-MSCs could promote the growth of corneal epithelial cells, reduce keratitis, and repair the corneal damage caused by low temperature. cDNA microarray analysis revealed that the mAF-MSCs affected the expression patterns of mRNAs related to cell proliferation and differentiation pathways in the mice after transplantation. The results of quantitative real-time PCR and western blotting revealed that NAT12, NAT10, and the ETV4/JUN/CCND2 signaling axis were elevated significantly in the mAF-MSC-transplantation group, compared with those in the phosphate-buffered saline-treated groups. High performance liquid chromatography–mass spectroscopy results revealed that mAF-MSCs could promote mRNA N4-acetylcytidine (ac4C) modification and high expression of N-acetyltransferase in the eyeballs. RNA immunoprecipitation-PCR results showed that a specific product comprising Vegfa, Klf4, Ccnd2, Jun, and Etv4 mRNA specific coding region sites could be amplified using PCR from complexes formed in mAF-MSC-transplanted samples cross-linked with anti-ac4C antibodies. Thus, mouse amniotic fluid MSCs could repair the mouse corneal cold injury by promoting the ETV4/JUN/CCND2 signal axis activation and improving its stability by stimulating N4-acetylcytidine modification of their mRNAs.

Eukaryotic Initiation Factor 3 Interactions with 5′ and 3′ RNA structures of the Barley Yellow Dwarf Virus Facilitate a Non‐Canonical Translation Mechanism

Barley Yellow Dwarf Virus (BYDV) is a positive strand RNA plant virus that translates without using a 5′ 7‐methylguanosine cap or a 3′ poly‐adenosine tail that are required for the canonical translation mechanism used by most mRNA. BYDV’s non‐canonical translation relies on RNA structures in the 5′ and 3′ untranslated regions (UTRs) that recruit eukaryotic initiation factors (eIFs) and ribosomes. Using a combination of biophysical, biochemical and chemical probing techniques, we have measured direct binding of eukaryotic initiation factors (eIFs) to BYVD 5′ and 3′ UTRs and probed binding sites. Our data show that one of the initiation factors, eIF3, can bind tightly, specifically, and simultaneously to structures in both 5′ and 3′ UTRs. This suggests that eIF3’s mechanistic role includes stabilization of the long‐distance RNA‐RNA interaction and the transfer of 3′‐bound factors/complexes to the 5′ UTR. Further work with RNA chemical probing has identified regions of the BYDV UTRs that interact with eIF3. This data highlights commonalities between BYDV structures and structures found in other viruses (e.g. Hepatitis C virus) and cellular mRNAs (e.g. c_JUN mRNA).Support or Funding InformationNSF MCB‐1902054 to Dixie J. Goss, NSF IGERT‐0965983 to Hunter College

Stress responses in the chemistry and mRNA abundance of the peripheral blood in the American alligator.

To monitor physiological and toxicological conditions in an endangered species, noninvasive to minimally invasive sampling methods are needed. We analyzed peripheral blood cells to determine if we could monitor some physiological responses of the American alligator following capture stress. Juvenile American alligators were restrained for 16 h to examine the stress response in plasma and blood cells. Plasma corticosterone concentrations were increased by restraint as were plasma concentrations of aspartate aminotransferase (AST), creatine kinase (CK), uric acid, and glucose; a sexually dimorphic response was seen in AST and CK concentrations. The lapse time of restraint was associated with altered messenger RNA (mRNA) levels of the glucocorticoid receptor (GCR) in red blood cells and JUN proto-oncogene in both white and red blood cells. A two-way cluster analysis revealed that two major clusters of factors were associated with the responses seen: (a) mRNA levels of GCR and heat-shock proteins in both blood cells were associated with plasma corticosterone concentration, whereas (b) androgen receptors and JUN mRNA levels in both blood cells were associated with cloacal temperature and body composition. Blood cells appear to be an excellent source to examine the cellular stress response to steroid hormone signals in mRNA levels. We propose that this approach, using blood cells, could provide essential insights into the molecular responses associated with stress in reptiles as well as many other nontraditional model species, including endangered species.

Elevation of Intracellular Na+ Contributes to Expression of Early Response Genes Triggered by Endothelial Cell Shrinkage.

Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.

Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori.

In this study, phosphorylation of c-Jun N-terminal kinase (JNK) by the prothoracicotropic hormone (PTTH) was investigated in prothoracic glands (PGs) of the silkworm, Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC) and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin) that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126) of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and an inhibitor (LY294002) of phosphoinositide 3-kinase (PI3K) failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant (N-acetylcysteine, NAC) or diphenylene iodonium (DPI), PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.