Post-transcriptional Mechanisms Research Articles

MiR-277 regulates the phase of circadian activity-rest rhythm in Drosophila melanogaster.

Circadian clocks temporally organize behaviour and physiology of organisms with a rhythmicity of about 24h. In Drosophila, the circadian clock is composed of mainly four clock genes: period (per), timeless (tim), Clock (Clk) and cycle (cyc) which constitutes the transcription-translation feedback loop. The circadian clock is further regulated via post-transcriptional and post-translational mechanisms among which microRNAs (miRNAs) are well known post-transcriptional regulatory molecules. Here, we identified and characterized the role of miRNA-277 (miR-277) expressed in the clock neurons in regulating the circadian rhythm. Downregulation of miR-277 in the pacemaker neurons expressing circadian neuropeptide, pigment dispersing factor (PDF) advanced the phase of the morning activity peak under 12h light: 12h dark cycles (LD) at lower light intensities and these flies exhibited less robust rhythms compared to the controls under constant darkness. In addition, downregulation of miR-277 in the PDF expressing neurons abolished the Clk gene transcript oscillation under LD. Our study points to the potential role of miR-277 in fine tuning the Clk expression and in maintaining the phase of the circadian rhythm in Drosophila.

Targeting IGF2BP3 Enhances Anti-Leukemic Effects of Menin-MLL Inhibition in MLL-AF4 Leukemia

Background: MLL-rearranged leukemias are a clinically challenging and biologically unique subtype of leukemias associated with poor prognosis. While novel therapeutic strategies have been primarily directed at epigenetic dysregulation, post-transcriptional gene regulatory mechanisms have emerged as important mediators in leukemogenesis and have the unexplored potential to be potent combinatorial therapeutic targets. Previously, we found that the RNA-binding protein IGF2BP3 is a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Methods: We studied the combined effects of targeting IGF2BP3 and the Menin-MLL interaction in MLL-AF4 driven leukemia in vitro and in vivo, using genetic inhibition through CRISPR-Cas9 mediated deletion of Igf2bp3 and pharmacologic inhibition of the Menin-MLL interaction with commercially available inhibitors MI-503, MI-463, and MI-538. In vitro, we tested the human B-cell acute lymphoblastic leukemia cell lines, SEM, RS4;11 and NALM6, and MLL-Af4-transformed murine hematopoietic stem and progenitor cells (herein referred to as MLL-Af4 Lin-), derived from bone marrow of Cas9 mice. Results: Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the negative effects of Menin-MLL inhibition on leukemic cell growth, colony formation and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and Menin-MLL inhibition led to increased differentiation in vitro and in vivo in functional readouts and by gene expression analyses. Both MI-503 treatment and IGF2BP3 knockdown in MLL-Af4 Lin- cells showed a shift towards more differentiated colony morphologies in colony formation assays, decreased expression of c-Kit and increased expression of maturation markers by flow cytometry, and morphologic changes consistent with increased differentiation. To gain insight into these phenotypic findings, we examined gene expression from MLL-Af4 Lin- cells with IGF2BP3 knockdown and treated with MI-503. Metascape analysis revealed significant enrichment in pathways involved in cell differentiation and activation, particularly in leukocytes. This was observed in both I3KO and MI-503 treated cells, with significant overlap in shared differentially expressed genes in both conditions. Next, we looked at the overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 knockdown with targets identified from MLL-AF4 ChIP and IGF2BP3 CLIP in CD11b+ and MLL-Af4 Lin- cells. We found significant overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 CLIP targets, suggesting that IGF2BP3 directly regulates genes that are affected by Menin-MLL inhibition. Furthermore, we saw overlap between differentially expressed genes with IGF2BP3 knockdown and MLL-AF4 ChIP targets identified in SEM and RS4;11 cells. These patterns highlight the interaction between the MLL-AF4 and IGF2BP3 transcriptomes and suggest a mechanism for the synergistic inhibition of leukemia by targeting both MLL-AF4 and IGF2BP3. To examine the combined effects of IGF2BP3 knockdown and Menin-MLL inhibition on leukemic engraftment and survival in vivo, we transplanted MLL-Af4 Lin- cells, depleted (or non-depleted) for IGF2BP3 and treated with MI-503 at 0.5 μM for 5 days, to initiate leukemia. We observed a significant decrease in peripheral blood leukemic engraftment with MI-503 treatment and IGF2BP3 knockdown. IGF2BP3 knockdown had a greater effect on survival and attenuating disease than MI-503 alone and showed enhanced anti-leukemic effects in combination. Conclusions: Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4 mediated leukemogenesis and is a potent therapeutic target and suggests a novel combinatorial approach to targeting leukemia at both the transcriptional and post-transcriptional level.

The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells

Lipid metabolism is the key to ferroptosis susceptibility. However, little is known about the underlying mechanisms in osteosarcoma cells. Functional restriction of bromodomain-containing protein 4 (BRD4) reduced the susceptibility to erastin-induced ferroptosis of osteosarcoma cells both in vitro and in vivo. Mechanically, BRD4 controls the splicing efficiency of the RNA precursor (pre-mACSL3) of ACSL3 (ACSL3) by recruiting serinerich/threonine protein kinase 2 (SRPK2) to assemble the splicing catalytic platform. Moreover, the AMP-binding domain of ACSL3 significantly influences arachidonic acid synthesis and thus determines the susceptibility to erastin-induced ferroptosis. Overall, we found a BRD4-mediated pre-mACSL3 splicing influences erastin-induced ferroptosis by affecting arachidonic acid synthesis in osteosarcoma cells. Data in this study fills some of the gap in understanding the post-transcriptional regulatory mechanisms of ACSL3 and provides new insights into the mechanisms of lipid metabolism regulation and its effect on susceptibility to ferroptosis in osteosarcoma cells.

Anti-Inflammatory Properties of Eugenol in Lipopolysaccharide-Induced Macrophages and Its Role in Preventing β-Cell Dedifferentiation and Loss Induced by High Glucose-High Lipid Conditions.

Inflammation is a natural immune response to injury, infection, or tissue damage. It plays a crucial role in maintaining overall health and promoting healing. However, when inflammation becomes chronic and uncontrolled, it can contribute to the development of various inflammatory conditions, including type 2 diabetes. In type 2 diabetes, pancreatic β-cells have to overwork and the continuous impact of a high glucose, high lipid (HG-HL) diet contributes to their loss and dedifferentiation. This study aimed to investigate the anti-inflammatory effects of eugenol and its impact on the loss and dedifferentiation of β-cells. THP-1 macrophages were pretreated with eugenol for one hour and then exposed to lipopolysaccharide (LPS) for three hours to induce inflammation. Additionally, the second phase of NLRP3 inflammasome activation was induced by incubating the LPS-stimulated cells with adenosine triphosphate (ATP) for 30 min. The results showed that eugenol reduced the expression of proinflammatory genes, such as IL-1β, IL-6 and cyclooxygenase-2 (COX-2), potentially by inhibiting the activation of transcription factors NF-κB and TYK2. Eugenol also demonstrated inhibitory effects on the levels of NLRP3 mRNA and protein and Pannexin-1 (PANX-1) activation, eventually impacting the assembly of the NLRP3 inflammasome and the production of mature IL-1β. Additionally, eugenol reduced the elevated levels of adenosine deaminase acting on RNA 1 (ADAR1) transcript, suggesting its role in post-transcriptional mechanisms that regulate inflammatory responses. Furthermore, eugenol effectively decreased the loss of β-cells in response to HG-HL, likely by mitigating apoptosis. It also showed promise in suppressing HG-HL-induced β-cell dedifferentiation by restoring β-cell-specific biomarkers. Further research on eugenol and its mechanisms of action could lead to the development of therapeutic interventions for inflammatory disorders and the preservation of β-cell function in the context of type 2 diabetes.

Spt5 C-terminal repeat domain phosphorylation and length negatively regulate heterochromatin through distinct mechanisms.

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomyces pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Prf1/Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that length and phosphorylation of the Spt5 CTD repeat array have distinct regulatory effects on transcription.

Competing endogenous RNAs in head and neck squamous cell carcinoma: a review.

Our understanding of RNA biology has evolved with recent advances in research from it being a non-functional product to molecules of the genome with specific regulatory functions. Competitive endogenous RNA (ceRNA), which has gained prominence over time as an essential part of post-transcriptional regulatory mechanism, is one such example. The ceRNA biology hypothesis states that coding RNA and non-coding RNA co-regulate each other using microRNA (miRNA) response elements. The ceRNA components include long non-coding RNAs, pseudogene and circular RNAs that exert their effect by interacting with miRNA and regulate the expression level of its target genes. Emerging evidence has revealed that the dysregulation of the ceRNA network is attributed to the pathogenesis of various cancers, including the head and neck squamous cell carcinoma (HNSCC). This is the most prevalent cancer developed from the mucosal epithelium in the lip, oral cavity, larynx and pharynx. Although many efforts have been made to comprehend the cause and subsequent treatment of HNSCC, the morbidity and mortality rate remains high. Hence, there is an urgent need to understand the holistic progression of HNSCC, mediated by ceRNA, that can have immense relevance in identifying novel biomarkers with a defined therapeutic intervention. In this review, we have made an effort to highlight the ceRNA biology hypothesis with a focus on its involvement in the progression of HNSCC. For the identification of such ceRNAs, we have additionally highlighted a number of databases and tools.

Co-expression analysis suggests lncRNA-mRNA interactions enhance antiviral immune response during acute Chikungunya fever in whole blood of pediatric patients.

Chikungunya virus is an arbovirus that causes the neglected tropical disease chikungunya fever, common in tropical areas worldwide. There is evidence that arboviruses alter host transcriptome and modulate immune response; this modulation may involve transcriptional and post-transcriptional control mechanisms mediated by long non-coding RNAs (lncRNAs). Herein, we employed bioinformatic analysis to evaluate co-expression of lncRNAs and their putative target mRNAs in whole blood during natural Chikungunya infection in adolescent boys. Sequencing data from GSE99992 was uploaded to the Galaxy web server, where data was aligned with HISAT2, gene counts were estimated with HTSeq-count, and differential expression was run with DESeq2. After gene classification with Biomart, Pearson's correlation was applied to identify potential interactions between lncRNAs and mRNAs, which were later classified into cis and trans according to genomic location (FEELnc) and binding potential (LncTar), respectively. We identified 1,975 mRNAs and 793 lncRNAs that were differentially expressed between the acute and convalescent stages of infection in the blood. Of the co-expressed lncRNAs and mRNAs, 357 potentially interact in trans and 9 in cis; their target mRNAs enriched pathways related to immune response and viral infections. Out of 52 enriched KEGG pathways, the RIG-I like receptor signaling is enriched by the highest number of target mRNAs. This pathway starts with the recognition of viral pathogens, leading to innate immune response mediated by the production of IFN-I and inflammatory cytokines. Our findings indicate that alterations in lncRNA expression in adolescent boys, induced by acute Chikungunya infection, potentially modulate mRNAs that contribute to antiviral immune responses.

RNA binding protein AUF1/HNRNPD regulates nuclear export, stability and translation of SNCA transcripts.

Alpha-synuclein (SNCA) accumulation plays a central role in the pathogenesis of Parkinson's disease. Determining and interfering with the mechanisms that control SNCA expression is one approach to limiting disease progression. Currently, most of our understanding of SNCA regulation is protein-based. Post-transcriptional mechanisms directly regulating SNCA mRNA expression via its 3' untranslated region (3'UTR) were investigated here. Mass spectrometry of proteins pulled down from murine brain lysates using a biotinylated SNCA 3'UTR revealed multiple RNA-binding proteins, of which HNRNPD/AUF1 was chosen for further analysis. AUF1 bound both proximal and distal regions of the SNCA 3'UTR, but not the 5'UTR or CDS. In the nucleus, AUF1 attenuated SNCA pre-mRNA maturation and was indispensable for the export of SNCA transcripts. AUF1 destabilized SNCA transcripts in the cytosol, primarily those with shorter 3'UTRs, independently of microRNAs by recruiting the CNOT1-CNOT7 deadenylase complex to trim the polyA tail. Furthermore, AUF1 inhibited SNCA mRNA binding to ribosomes. These data identify AUF1 as a multi-tasking protein regulating maturation, nucleocytoplasmic shuttling, stability and translation of SNCA transcripts.

Identifying Macrophage-Related Genes in Ulcerative Colitis Using Weighted Coexpression Network Analysis and Machine Learning.

Ulcerative colitis (UC) is an inflammatory bowel disease of unknown cause that typically affects the colon and rectum. Innate intestinal immunity, including macrophages, plays a significant role in the pathological development of UC. Using the CIBERSORT algorithm, we observed elevated levels of 22 types of immune cell infiltrates, as well as increased M1 and decreased M2 macrophages in UC compared to normal colonic mucosa. Weighted gene coexpression network analysis (WGCNA) was used to identify modules associated with macrophages and UC, resulting in the identification of 52 macrophage-related genes (MRGs) that were enriched in macrophages at single-cell resolution. Consensus clustering based on these 52 MRGs divided the integrated UC cohorts into three subtypes. Machine learning algorithms were used to identify ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), sodium- and chloride-dependent neutral and basic amino acid transporter B(0+) (SLC6A14), and 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) in the training set, and their diagnostic value was validated in independent validation sets. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) revealed the main biological effects, and that interleukin-17 was one of several signaling pathways enriched by the three genes. We also constructed a competitive endogenous RNA (CeRNA) network reflecting a potential posttranscriptional regulatory mechanism. Expression of diagnostic markers was validated in vivo and in biospecimens, and our immunohistochemistry (IHC) results confirmed that HMGCS2 gradually decreased during the transformation of UC to colorectal cancer. In conclusion, ENPP1, SLC6A14, and HMGCS2 are associated with macrophages and the progression of UC pathogenesis and have good diagnostic value for patients with UC.

Novel Insights into the Molecular Mechanisms Governing Embryonic Epicardium Formation.

The embryonic epicardium originates from the proepicardium, an extracardiac primordium constituted by a cluster of mesothelial cells. In early embryos, the embryonic epicardium is characterized by a squamous cell epithelium resting on the myocardium surface. Subsequently, it invades the subepicardial space and thereafter the embryonic myocardium by means of an epithelial-mesenchymal transition. Within the myocardium, epicardial-derived cells present multilineage potential, later differentiating into smooth muscle cells and contributing both to coronary vasculature and cardiac fibroblasts in the mature heart. Over the last decades, we have progressively increased our understanding of those cellular and molecular mechanisms driving proepicardial/embryonic epicardium formation. This study provides a state-of-the-art review of the transcriptional and emerging post-transcriptional mechanisms involved in the formation and differentiation of the embryonic epicardium.

The m6A reader IGF2BP3 preserves NOTCH3 mRNA stability to sustain Notch3 signaling and promote tumor metastasis in nasopharyngeal carcinoma

Metastasis remains the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC), in which sustained activation of the Notch signaling plays a critical role. N6-Methyladenosine (m6A)-mediated post-transcriptional regulation is involved in fine-tuning the Notch signaling output; however, the post-transcriptional mechanisms underlying NPC metastasis remain poorly understood. In the present study, we report that insulin-like growth factor 2 mRNA-binding proteins 3 (IGF2BP3) serves as a key m6A reader in NPC. IGF2BP3 expression was significantly upregulated in metastatic NPC and correlated with poor prognosis in patients with NPC. IGF2BP3 overexpression promoted, while IGF2BP3 downregulation inhibited tumor metastasis and the stemness phenotype of NPC cells in vitro and in vivo. Mechanistically, IGF2BP3 maintains NOTCH3 mRNA stability via suppression of CCR4-NOT complex-mediated deadenylation in an m6A-dependent manner, which sustains Notch3 signaling activation and increases the transcription of stemness-associated downstream genes, eventually promoting tumor metastasis. Our findings highlight the pro-metastatic function of the IGF2BP3/Notch3 axis and revealed the precise role of IGF2BP3 in post-transcriptional regulation of NOTCH3, suggesting IGF2BP3 as a novel prognostic biomarker and potential therapeutic target in NPC metastasis.

Comprehensive Profiling of Alternative Splicing and Alternative Polyadenylation during Fruit Ripening in Watermelon (Citrullus lanatus).

Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for watermelon fruit ripening have not been comprehensively studied. In this study, we conducted PacBio single-molecule long-read sequencing to identify genome-wide alternative splicing (AS), alternative polyadenylation (APA) and long non-coding RNAs (lncRNAs) in watermelon fruit. In total, 6,921,295 error-corrected and mapped full-length non-chimeric (FLNC) reads were obtained. Notably, more than 42,285 distinct splicing isoforms were derived from 5,891,183 intron-containing full-length FLNC reads, including a large number of AS events associated with fruit ripening. In addition, we characterized 21,506 polyadenylation sites from 11,611 genes, 8703 of which have APA sites. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that fructose and mannose metabolism, starch and sucrose metabolism and carotenoid biosynthesis were both enriched in genes undergoing AS and APA. These results suggest that post-transcriptional regulation might potentially have a key role in regulation of fruit ripening in watermelon. Taken together, our comprehensive PacBio long-read sequencing results offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of watermelon fruit ripening.

Transcriptional Analysis of lncRNA and Target Genes Induced by Influenza A Virus Infection in MDCK Cells.

The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

Extracellular vesicles could be a putative posttranscriptional regulatory mechanism that shapes intracellular RNA levels in Plasmodium falciparum

Plasmodium falciparum secretes extracellular vesicles (PfEVs) that contain parasite-derived RNA. However, the significance of the secreted RNA remains unexplored. Here, we compare secreted and intracellular RNA from asexual cultures of six P. falciparum lines. We find that secretion of RNA via extracellular vesicles is not only periodic throughout the asexual intraerythrocytic developmental cycle but is also highly conserved across P. falciparum isolates. We further demonstrate that the phases of RNA secreted via extracellular vesicles are discernibly shifted compared to those of the intracellular RNA within the secreting whole parasite. Finally, transcripts of genes with no known function during the asexual intraerythrocytic developmental cycle are enriched in PfEVs compared to the whole parasite. We conclude that the secretion of extracellular vesicles could be a putative posttranscriptional RNA regulation mechanism that is part of or synergise the classic RNA decay processes to maintain intracellular RNA levels in P. falciparum.

Hantavirus: an overview and advancements in therapeutic approaches for infection.

Hantaviruses are a significant and emerging global public health threat, impacting more than 200,000 individuals worldwide each year. The single-stranded RNA viruses belong to the Hantaviridae family and are responsible for causing two acute febrile diseases in humans: Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Currently, there are no licensed treatments or vaccines available globally for HTNV infection. Various candidate drugs have shown efficacy in increasing survival rates during the early stages of HTNV infection. Some of these drugs include lactoferrin, ribavirin, ETAR, favipiravir and vandetanib. Immunotherapy utilizing neutralizing antibodies (NAbs) generated from Hantavirus convalescent patients show efficacy against HTNV. Monoclonal antibodies such as MIB22 and JL16 have demonstrated effectiveness in protecting against HTNV infection. The development of vaccines and antivirals, used independently and/or in combination, is critical for elucidating hantaviral infections and the impact on public health. RNA interference (RNAi) arised as an emerging antiviral therapy, is a highly specific degrades RNA, with post-transcriptional mechanism using eukaryotic cells platform. That has demonstrated efficacy against a wide range of viruses, both in vitro and in vivo. Recent antiviral methods involve using small interfering RNA (siRNA) and other, immune-based therapies to target specific gene segments (S, M, or L) of the Hantavirus. This therapeutic approach enhances viral RNA clearance through the RNA interference process in Vero E6 cells or human lung microvascular endothelial cells. However, the use of siRNAs faces challenges due to their low biological stability and limited in vivo targeting ability. Despite their successful inhibition of Hantavirus replication in host cells, their antiviral efficacy may be hindered. In the current review, we focus on advances in therapeutic strategies, as antiviral medications, immune-based therapies and vaccine candidates aimed at enhancing the body's ability to control the progression of Hantavirus infections, with the potential to reduce the risk of severe disease.

Regulation of Cholesterol Transporters by Nuclear Receptors

Atherosclerosis is a pathological condition characterized by the accumulation of plaques in the arteries, leading to cardiovascular diseases. The deposition of cholesterol in peripheral cells increases the risk of atherosclerosis. Reverse cholesterol transport (RCT) is essential to reduce the risk of atherosclerosis because it removes excessive cholesterol from the peripheral tissues. ATP-binding cassette transporters such as ABCA1, ABCG1, ABCG5, and ABCG8 are involved in the efflux of cholesterol. The upregulation of these ABC transporters enhances RCT, thereby promoting the removal of excess cholesterol from the body. The expression and activity of ABC transporters are regulated by transcriptional and post-transcriptional mechanisms, as well as by post-translational modifications. In this review, the regulation of ABC transporters by nuclear receptors such as farnesoid X receptor, liver X receptor, retinoid X receptor, retinoic acid receptor, and peroxisome proliferator-activated receptors is discussed. Pharmacological and natural compounds serving as agonists for the nuclear receptors have been identified to elevate the mRNA levels of the transporters. Consequently, it is anticipated that these compounds will attenuate the development of atherosclerosis through stimulation of the ABC transporters, thereby enhancing RCT and fecal cholesterol excretion. Understanding these regulatory processes can aid in the development of therapeutic approaches to prevent atherosclerosis.

Differential gene expression in a subpopulation of Phaeodactylum tricornutum with enhanced growth and carotenoid production after FACS-mediated selection

Fluorescence-Activated Cell Sorting (FACS) is a powerful method with many applications in microalgal research, especially for screening and selection of cells with improved phenotypes. However, the technology requires review of gene expression changes responsible for enhanced phenotypes in sorted populations. Phaeodactylum tricornutum cells were sorted using FACS with excitation/emission parameters targeted to favouring the industrially-relevant carotenoid fucoxanthin. The resulting cultures showed significantly higher growth rate (1.10 ×), biomass (1.30 ×), chlorophyll a levels (1.22 ×) and fucoxanthin content (1.28 ×) relative to the wild-type strain. RNA-seq was used to elucidate the underlying molecular-level regulatory changes associated with these traits and represents the first study do so on FACS-sorted microalgal cultures. Transcriptome analysis corroborated evidence of increased chlorophyll a and fucoxanthin, showing enrichment for the genes/pathways for tetrapyrrole biosynthesis and for suites of genes directly related to photosynthesis. Only three genes were upregulated in the MEP (non-mevalonate) pathway to carotenoid biosynthesis pathway, suggesting either a strong influence of IDI, CRTISO5 and ZEP1 on fucoxanthin biosynthesis or a post-transcriptional or post-translational mechanism for the observed increase in fucoxanthin content.

THU475 Ovarian Tumor-cell Glucocorticoid Receptor Activity Modulates Cytokine Secretion Promoting Recruitment And Activation of Immunosuppressive Cells Into The Tumor Microenvironment

Abstract Disclosure: M. Taya: None. E. LeBlanc: None. L. Bennett: None. S.D. Conzen: None. Elevated levels of immunosuppressive myeloid-derived suppressor cells (MDSCs) circulate in the peripheral blood or infiltrate both tumors and ascites in ovarian cancer patients, however the cause for this observed phenotype is unknown. MDSCs contribute to tumor immune tolerance primarily via inhibition of cytotoxic T-cells or by activation of immunosuppressive T-regulatory cells. We previously showed that high tumor cell glucocorticoid receptor (GR) expression is associated with a relatively poor prognosis in ovarian cancer patients. Additionally, using primary tumor data retrieved from the ovarian cancer TCGA dataset, we show high tumor GR expression is associated with significantly higher expression of Foxp3, a marker of immunosuppressive T-regulatory cells, as well as CD33, a marker of immunosuppressive myeloid cells, further suggesting an immunosuppressive phenotype in ovarian cancer patients. We thus hypothesize that tumor-intrinsic GR activation in ovarian cancer is associated with increased MDSC infiltration into the tumor microenvironment partly due to the modulation of tumor cell cytokine secretion. To determine whether tumor-cell GR activation modulates the cytokine secretome, we previously showed that GR activation in ovarian cancer cells upregulates secretion of immunomodulatory factors including G-CSF, TGFb and CXCL5. Moreover, co-treatment of cells with a selective GR modulator (SGRM) reverses this effect. Preliminary gene expression data indicate GR mediated cytokine secretion and signaling may involve post-transcriptional or post-translational mechanisms. Considering that elevated levels of these cytokines are necessary for the production and recruitment of MDSCs, these data highlight the relevance of this evident glucocorticoid receptor-cytokine circuit in ovarian cancer cells. Furthermore, we show that this tumor-cell cytokine secretome has the capacity to promote differentiation of MDSCs from precursor myeloid cells in healthy PBMCs. Importantly, measuring expression of activation markers such as Arginase-1 and NOS2 illustrated that these MDSCs are in their functional state. To our knowledge, this is the first evidence that a nuclear receptor in ovarian cancer tumor cells is actively driving MDSC differentiation. Finally, employing ovarian cancer xenograft models as well as a syngeneic ID8 tumor model, treatment with SGRMs demonstrated reduced levels of circulating MDSCs in the peripheral blood as well as tumor infiltrated MDSCs. Using neutralizing antibodies for cytokines, we will now identify key mechanisms, specifically, which targetable immunomodulatory factors are contributing to immune suppression. Based on the insight provided by these data, we propose to determine whether targeting ovarian tumor cell-intrinsic GR activation and resulting MDSC infiltration contributes to improved anti-tumor immune response. Presentation: Thursday, June 15, 2023

OR18-02 Mammary Insulin Sensitivity Increases At The Onset Of Lactation And Promotes Metabolic Pathways Supporting Breast Milk Synthesis

Abstract Disclosure: X. Meng: None. H. Rostom: None. A. Fry: None. T. Elajnaf: None. F. Hannan: None. The initiation of lactation during postpartum days 1-4 is hormonally regulated and critical for successful breastfeeding. Insulin represents a key lactogenic hormone as evidenced by women with diabetes mellitus who have delayed lactation onset. However, the role of insulin in lactation and its influence on mammary cells are unclear. We utilised clinical and cellular approaches to investigate this, and recruited n=38 healthy pregnant women (age range 26-42 years) following informed consent and measured serum insulin at 36 weeks’ gestation and during post-partum days 1-4 (1). Serum insulin progressively decreased from 36 weeks’ gestation to day 4 post-partum (244±33pmol/L vs 37±4pmol/L, p&amp;lt;0.0001). Moreover, reverse transcription-quantitative PCR (RT-qPCR) of lactating human mammary epithelial cells (HMECs) isolated from breast milk showed a &amp;gt;20-fold increase in insulin receptor expression compared to non-lactating HMECs (p&amp;lt;0.01, n=4). These findings indicated an increase in both systemic and mammary insulin sensitivity at lactation onset. We hypothesised that insulin promotes lactation by influencing mammary metabolism and used cultured HMECs to evaluate this. Stimulation of HMECs with 10nM insulin caused a &amp;gt;7-fold increase (p=0.001, n=5) in the phosphorylation of Akt, a signalling protein required for initiating lactation. Akt influences oxidative phosphorylation and glycolysis, and we assessed these processes by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. Cultured HMECs treated with 10nM insulin for ≤18hrs showed increased OCR (22±0.9 vs. 17±0.4pmol O2/min/106 cells for control HMECs, p&amp;lt;0.05, n=4) and ECAR (0.23±0.003 vs. 0.18±0.002mpH/min/106 cells for control HMECs, p&amp;lt;0.001, n=4), consistent with increased oxidative phosphorylation and glycolysis. HMECs treated with 10nM insulin for 8hrs had significant upregulation of genes encoding glycolytic enzymes, namely hexokinase 2 (&amp;gt;4-fold increase, p&amp;lt;0.0001, n=4) and pyruvate kinase M1/2 (1.4-fold increase, p&amp;lt;0.05, n=4). However, RT-qPCR analysis of HMECs showed that insulin did not increase expression of genes mediating oxidative phosphorylation, suggesting an Akt-mediated post-transcriptional mechanism. In summary, increased mammary insulin sensitivity together with insulin-stimulated oxidative phosphorylation and glycolysis may support mammary function and milk component synthesis at the onset of lactation. Reference: (1) Rostom, H. et al. Protocol for an observational study investigating hormones triggering the onset of sustained lactation: the INSIGHT study. BMJ Open 12, e062478. Presentation: Saturday, June 17, 2023

DUSP1/MKP-1 represents another piece in the P2X7R intracellular signaling puzzle in cerebellar cells: our last journey with Mª Teresa along the purinergic pathways of Eden

The P2X7 receptor (P2X7R) stands out within the purinergic family as it has exclusive pharmacological and regulatory features, and it fulfills distinct roles depending on the type of stimulation and cellular environment. Tonic activation of P2X7R promotes cell proliferation, whereas sustained activation is associated with cell death. Yet strikingly, prolonged P2X7R activation in rat cerebellar granule neurons and astrocytes does not affect cell survival. The intracellular pathways activated by P2X7Rs involve proteins like MAPKs, ERK1/2 and p38, and interactions with growth factor receptors could explain their behavior in populations of rat cerebellar cells. In this study, we set out to characterize the intracellular mechanisms through which P2X7Rs and Trk receptors, EGFR (epidermal growth factor receptor) and BDNFR (brain-derived neurotrophic factor receptor), regulate the dual-specificity phosphatase DUSP1. In cerebellar astrocytes, the regulation of DUSP1 expression by P2X7R depends on ERK and p38 activation. EGFR stimulation can also induce DUSP1 expression, albeit less strongly than P2X7R. Conversely, EGF was virtually ineffective in regulating DUSP1 in granule neurons, a cell type in which BDNF is the main regulator of DUSP1 expression and P2X7R only induces a mild response. Indeed, the regulation of DUSP1 elicited by BDNF reflects the balance between both transcriptional and post-transcriptional mechanisms. Importantly, when the regulation of DUSP1 expression is compromised, the viability of both astrocytes and neurons is impaired, suggesting this phosphatase is essential to maintain proper cell cytoarchitecture and functioning.