Human lncRNAs Research Articles

Extracellular vesicle-incorporated long noncoding RNA PUNISHER is increased in coronary artery disease and regulates endothelial cell function

Abstract Aims Long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators of cardiovascular disease. Circulating lncRNAs can be incorporated into extracellular vesicles (EVs). Our aim was to study the expression pattern of circulating EV-incorporated lncRNAs in patients with and without coronary artery disease (CAD). The regulation of the lncRNA PUNISHER was further assessed as an intercellular messenger in endothelial cell (EC) biology. Methods and results Circulating EVs were isolated from the plasma of patients using ultracentrifugation. Electron microscopy, western blot, and NTA were used to determine the size and origin of the EVs. PCR-based human lncRNA array analysis revealed that certain EV-lncRNAs are significantly different in patients with CAD compared to patients without CAD. To validate the lncRNA array results, 60 patients with (n=30) or without (n=30) CAD were prospectively studied. Four atherosclerosis-related lncRNAs (PUNISHER, GAS5, MALAT1, and H19) were quantified in the circulating EVs by using real-time quantitative PCR (RT-qPCR). Among these, PUNISHER (p=0.002) and GAS5 (0.02) were significantly increased in patients with CAD, compared to non-CAD patients. Analysis of RNA degradation revealed that circulating PUNISHER is mainly incorporated within EVs. In vitro, atherosclerotic stimuli consisting of oxLDL or TNF-a treatment caused an upregulation of PUNISHER levels in human coronary artery endothelial cells (HCAEC) and in the corresponding EVs. Labeling of EVs followed by RT-qPCR demonstrated that functional PUNISHER was transported into the recipient ECs, which accelerated cell migration, proliferation, and tube formation. Mechanistically, the RNA-binding protein hnRNPK was identified by an RNA immunoprecipitation (RIP) assay as an interaction partner of PUNISHER, regulating its loading into small EVs. Knockdown of PUNISHER abrogated the EV-mediated effects on EC migration, proliferation, and tube formation. PCR-based gene profiling showed that the expression of VEGF RNA was significantly increased in ECs by treatment with EVs. Protein stability and RNA-immunoprecipitation followed by RT-qPCR analysis indicated that the PUNISHER-hnRNPK axis regulates the stability and binding of VEGFA mRNA to hnRNPK. Knockdown of PUNISHER in EVs abrogated the EV-mediated promotion of VEGFA gene- and protein expression. Conclusion The circulating lncRNA PUNISHER is increased in CAD patients. Intercellular transfer of EV-incorporated PUNISHER promotes a pro-angiogenic phenotype via a VEGFA-dependent mechanism.

Calcific aortic valve disease-associated long noncoding RNA H19 promotes the osteogenic transition of valvular interstitial cells via the JAG1/NOTCH1 axis

Abstract Background Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease causing tremendous suffering globally. The osteogenic differentiation of the valvular interstitial cells (VICs) is a key process in the pathogenesis of CAVD leading to calcification and stiffening. Here, we focus on the contribution of long noncoding RNA H19 in the osteogenic differentiation of VICs promoting CAVD. Methods and results A human lncRNA array performed in AVS patients with coronary artery disease (CAD) revealed the differential expression of lncRNA H19. For the experiments in this study, loss of function (LOF) was performed in primary VICs isolated from the aortic valves of patients with CAVD, and commercially-available VICs. Therefore, H19 expression was effectively downregulated via RNAi. Subsequent gene expression analysis via RT-qPCR revealed that the expression of calcification markers RUNX2 and BMP2, as well as the expression of the H19 downstream targets JAG1 and NOTCH1 were unchanged after the H19 knockdown alone. Then, the VICs were additionally treated with osteogenic medium (OM) and procalcifying medium (PCM) for 7 d to induce the calcification of the cells. In the OM, inorganic phosphate is generated in an alkaline phosphatase (ALPL)-dependent manner and integrated into newly formed hydroxyapatite crystals, thus promoting cellular calcification. PCM contains inorganic phosphate to form hydroxyapatite crystals and is therefore not ALPL-dependent. Gene expression analysis via RT-qPCR and protein analysis via immunofluorescence revealed that the calcification markers BMP2 and RUNX2 were upregulated after H19 knockdown and calcification induction. This indicates that H19 regulates the expression of calcification makers promoting cellular calcification. Furthermore, RT-qPCR and immunoblotting revealed the differential expression of several potential H19 downstream targets, namely JAG1, NOTCH1, and STAT3. The analysis showed that the potential downstream targets were upregulated after H19 knockdown and calcification treatment hinting at the involvement of the NOTCH1 or STAT3 pathway in the pathogenesis of CAVD. Moreover, an Alizarin Red S staining of calcified nodules revealed reduced calcification after H19 knockdown and calcification treatment for 21 d. This demonstrates that H19 also regulates a late-stage event in the cellular calcification of VICs. Conclusion Our study showed that H19 regulates calcification marker expression and the expression of potential downstream targets JAG1, NOTCH1, and STAT3 both in healthy and AVS patient VICs. This hints at the involvement of the H19/JAG1/NOTCH1 pathway in the calcification of VICs, which might play a critical role in the pathogenesis of CAVD.

Syntenic lncRNA locus exhibits DNA regulatory functions with sequence evolution

Syntenic long non-coding RNAs (lncRNAs) often show limited sequence conservation across species, prompting concern in the field. This study delves into functional signatures of syntenic lncRNAs between humans and zebrafish. Syntenic lncRNAs are highly expressed in zebrafish, with ∼90 % located near protein-coding genes, either in sense or antisense orientation. During early zebrafish development and in human embryonic stem cells (H1-hESC), syntenic lncRNA loci are enriched with cis-regulatory repressor signatures, influencing the expression of development-associated genes. In later zebrafish developmental stages and specific human cell lines, these syntenic lncRNA loci function as enhancers or transcription start sites (TSS) for protein-coding genes. Analysis of transposable elements (TEs) in syntenic lncRNA sequences revealed intriguing patterns: human lncRNAs are enriched in simple repeat elements, while their zebrafish counterparts show enrichment in LTR elements. This sequence evolution likely arises from post-rearrangement mutations that enhance DNA elements or cis-regulatory functions. It may also contribute to vertebrate innovation by creating novel transcription factor binding sites within the locus. This study highlights the conserved functionality of syntenic lncRNA loci through DNA elements, emphasizing their conserved roles across species despite sequence divergence.

The lncRNA SNHG26 drives the inflammatory-to-proliferative state transition of keratinocyte progenitor cells during wound healing

The cell transition from an inflammatory phase to a subsequent proliferative phase is crucial for wound healing, yet the driving mechanism remains unclear. By profiling lncRNA expression changes during human skin wound healing and screening lncRNA functions, we identify SNHG26 as a pivotal regulator in keratinocyte progenitors underpinning this phase transition. Snhg26-deficient mice exhibit impaired wound repair characterized by delayed re-epithelization accompanied by exacerbated inflammation. Single-cell transcriptome analysis combined with gain-of-function and loss-of-function of SNHG26 in vitro and ex vivo reveals its specific role in facilitating inflammatory-to-proliferative state transition of keratinocyte progenitors. A mechanistic study unravels that SNHG26 interacts with and relocates the transcription factor ILF2 from inflammatory genomic loci, such as JUN, IL6, IL8, and CCL20, to the genomic locus of LAMB3. Collectively, our findings suggest that lncRNAs play cardinal roles in expediting tissue repair and regeneration and may constitute an invaluable reservoir of therapeutic targets in reparative medicine.

Genome-wide profiling of long non-coding RNA following ozone exposure: A randomized, controlled exposure trial

BackgroundExposure to ambient ozone has been associated with extrapulmonary health, but the underlying mechanisms remain to be understood. LncRNAs are involved in the regulation of gene expression, but their regulatory mechanisms in ozone-related health effects are scarcely explored. ObjectiveTo investigate genome-wide lncRNA changes after short-term ozone exposure and their regulatory roles in ozone exposure and gene expression. MethodWe conducted a randomized, crossover, controlled exposure trial in 32 healthy college students in Shanghai, China. Each participant received both 200-ppb ozone exposure and filtered air exposure for 2 h in a random order with a 14-day washout period. Blood samples were collected after each exposure and used for lncRNA sequencing. Differentially expressed lncRNAs between the two exposures were identified using orthogonal partial least squares discriminant analysis and linear regression analysis. LncRNAs-targeted mRNAs were mapped and subjected to enrichment analyses. We also constructed lncRNA-miRNA-mRNA networks. ResultsA total of 90 lncRNAs were differentially expressed after exposure to ozone, with 49 up-regulated and 41 down-regulated. Enrichment analyses suggested that these dysregulated lncRNAs were involved in a variety of biological processes, including those related to oxidative stress, inflammation response, and cell proliferation, development, and differentiation. Multiple pathways such as IL-17 signaling, NF-kB signaling, and Rho GTPases signaling were also enriched. Furthermore, the lncRNA-miRNA-mRNA network revealed that specific lncRNAs may regulate the expression of inflammation- and angiogenesis-related genes by interacting with miRNAs, such as NEAT1/hsa-miR-500a-3p/SIGLEC8, NEAT1/hsa-miR-6835–3p/SLC16A14, OIP5-AS1/miR-183–5p/EGR1, and SNHG25/hsa-miR-663a/FOSB axes. ConclusionThis study characterized a thorough profile of human lncRNAs following short-term ozone exposure and suggested the regulatory roles of these lncRNAs in ozone-induced inflammatory responses and angiogenesis, providing novel epigenetic insights into the mechanisms of the health effects of ozone exposure.

A host immune-related LncRNA and mRNA signature as a discriminant classifier for bacterial from non-bacterial sepsis in children

BackgroundThe variations in non-coding RNA alterations and the host immune response between patients with bacterial and non-bacterial sepsis, along with their clinical characteristics, are largely unknown. MethodsThe landscape of long non-coding RNA (lncRNA) and mRNA in whole blood cells from pediatric patients with bacterial sepsis or non-bacterial sepsis were characterized using an Arraystar human LncRNA microarray. Weighted correlation network analysis (WGCNA) were conducted to identify immune-related LncRNA-mRNA signatures. Least absolute shrinkage and selection operator (Lasso) regression and Ridge regression analysis were employed to develop a specific LncRNA-mRNA signature, serving as a discriminant classifier for bacterial and non-bacterial sepsis in children. ResultsA total of 33 differentially expressed lncRNAs and 52 mRNAs were identified in pediatric patients with either bacterial sepsis or non-bacterial sepsis. Among these, 69 lncRNAs and mRNAs were pinpointed using WGCNA and found to be significantly correlated with clinical parameters. Further intersection analysis identified 12 lncRNAs and 16 mRNAs as immune-related signature for discerning bacterial infections in children with sepsis. Additionaly, the lncRNA-mRNA co-expression network highlighted the key lncRNAs (AC090159.1 and AC080129.2) and mRNAs (S100A8 and TCF7L2) as an infection score model. Lasso regression analysis revealed that this infection score model achieved an area under the received operating curve (AUROC) of 0.96 in the training set and 0.86 in the validation set. Ultimately, the expression levels of these 4 key lncRNAs and mRNAs showed significant correlation with CRP or PCT levels. ConclusionThe machine learning model, developed utilizing key lncRNAs (AC090159.1 and AC080129.2) and mRNAs (S100A8 and TCF7L2), demonstrates robust discrimination and calibration capabilities for distinguishing between bacterial and non-bacterial sepsis in children.

Induction of Invasive Basal Phenotype in Triple-Negative Breast Cancers by Long Noncoding RNA BORG.

Long noncoding RNAs (lncRNAs) are known to play key roles in breast cancers; however, detailed mechanistic studies of lncRNA function have not been conducted in large cohorts of breast cancer tumors, nor has inter-donor and inter-subtype variability been taken into consideration for these analyses. Here we provide the first identification and annotation of the human BORG lncRNA gene. Using multiple tumor cohorts of human breast cancers, we show that while BORG expression is strongly induced in breast tumors as compared to normal breast tissues, the extent of BORG induction varies widely between breast cancer subtypes and even between different tumors within the same subtype. Elevated levels of BORG in breast tumors are associated with the acquisition of core cancer aggression pathways, including those associated with basal tumor and pluripotency phenotypes and with epithelial-mesenchymal transition (EMT) programs. While a subset of BORG-associated pathways was present in high BORG-expressing tumors across all breast cancer subtypes, many were specific to tumors categorized as triple-negative breast cancers. Finally, we show that genes induced by heterologous expression of BORG in murine models of TNBC both in vitro and in vivo strongly overlap with those associated with high BORG expression levels in human TNBC tumors. Our findings implicate human BORG as a novel driver of the highly aggressive basal TNBC tumor phenotype.

Antisense Oligonucleotide Embedded Context Responsive Nanoparticles Derived from Synthetic Ionizable Lipids for lncRNA Targeted Therapy of Breast Cancer.

The long noncoding RNAs (lncRNA) are primarily associated with several essential gene regulations but are also connected to cancer metabolism and progression. HOTAIR and MALAT1 are two such lncRNAs that are detected in malignancies of various origins and are responsible for the poor prognosis of cancer patients. Due to these factors, the lncRNAs have emerged as prime targets for the development of anticancer therapeutics. However, nonviral delivery of lncRNA-targeted antisense oligonucleotides (ASOs) still remains a critical challenge while maintaining their structural and functional integrity. Herein, we have designed and synthesized a new series of ionizable lipids with variations in their head groups to prepare lipid nanoparticle (LNP) formulation along with cholesterol-based twin cationic lipid and amphiphilic zwitterionic lipid. The context responsiveness of these formulations in delivering the ASOs has been thoroughly investigated by various bioanalytical techniques, and an optimum formulation has been identified. The LNPs are utilized to deliver the ASOs targeting HOTAIR lncRNA in human cancer cell lines and MALAT1 lncRNA in mouse models. This study thus standardizes an advanced nanomaterial system for nonviral gene delivery that has been validated by a considerable reduction in the target lncRNA level under in vitro and a significant reduction in tumor volume under in vivo settings.

Abstract We106: Long Non-Coding RNA LIPTER Regulates Lipid Metabolism of Human Cardiomyocytes and Preserves Cardiac Function

Background: Recently, long non-coding RNAs (lncRNAs) have emerged as novel human-specific (i.e. non-conserved) regulators of cardiovascular development and disease, which possess diverse functions through interacting with other molecules, including DNA, RNA, protein, and lipid. We identified a human lncRNA, LIPTER, which mediates lipid droplet (LD) transport to regulate lipid metabilism of human cardiomyocytes (CMs). Mechanistically, LIPTER binds phospholipids PA and PI4P on the LD membranes and the MYH10 protein, connecting LDs to the cytoskeleton and facilitating LD transport to mitochondria. LIPTER deficiencies led to prominent LD accumulation, mitochondrial dysfunction, and death of human iPS cell-derived CMs. Since downregulation of LIPTER and enahnced LD accumulation are associated with cardiac dysfunction and heart failure in patients with metabolic disorders, such as obesity and diabetes mellitus, we thereby seek to explore the preclinical potential of LIPTER for the therapy of human metabolic disorder associated cardiac dysfunction and heart failure. Hypothesis: We hypothesize that transgenic overexpression of the human lncRNA LIPTER in mouse heart could attenuate metabolic syndrome-associated myocardium lipid accumulation and cardiac dysfunction, as well as prevent isoproterenol-induced heart failure through increasing the fatty acid oxidation (FAO) of mouse heart. Goals: To evaluate the preclinical potential of human lncRNA LIPTER for the therapy of cardiac dysfunction and heart failure by using mouse cardiac dysfunction and heart failure models. Methods: We established a LIPTER transgenic overexpression mouse line (LIPTER Tg ). Both WT and LIPTER Tg mice were fed with high fat diet for 10 months to induce cardiac lipid accumulation and dysfunction. Additionally, heart failure was induced by the chronic isoproterenol administration. After treatment, mice were subjected to histological and molecular analyses. Results: We found LIPTER Tg significantly enhanced FAO of mouse heart, reduced cardiac lipid accumulation and fibrosis, and alleviated cardiac dysfunction of high-fat-diet fed mice, as well as preserved cardiac function of mice post isoproterenol administration. Conclusions: We unveil a crucial role of human lncRNA LIPTER in regulating lipid metabolism of human CMs and testify its clinical potential for treating cardiac dysfunction and heart failure.

CBIL-VHPLI: a model for predicting viral-host protein-lncRNA interactions based on machine learning and transfer learning

Virus‒host protein‒lncRNA interaction (VHPLI) predictions are critical for decoding the molecular mechanisms of viral pathogens and host immune processes. Although VHPLI interactions have been predicted in both plants and animals, they have not been extensively studied in viruses. For the first time, we propose a new deep learning-based approach that consists mainly of a convolutional neural network and bidirectional long and short-term memory network modules in combination with transfer learning named CBIL‒VHPLI to predict viral–host protein‒lncRNA interactions. The models were first trained on large and diverse datasets (including plants, animals, etc.). Protein sequence features were extracted using a k-mer method combined with the one-hot encoding and composition–transition–distribution (CTD) methods, and lncRNA sequence features were extracted using a k-mer method combined with the one-hot encoding and Z curve methods. The results obtained on three independent external validation datasets showed that the pre-trained CBIL‒VHPLI model performed the best with an accuracy of approximately 0.9. Pretraining was followed by conducting transfer learning on a viral protein–human lncRNA dataset, and the fine-tuning results showed that the accuracy of CBIL‒VHPLI was 0.946, which was significantly greater than that of the previous models. The final case study results showed that CBIL‒VHPLI achieved a prediction reproducibility rate of 91.6% for the RIP-Seq experimental screening results. This model was then used to predict the interactions between human lncRNA PIK3CD-AS2 and the nonstructural protein 1 (NS1) of the H5N1 virus, and RNA pull-down experiments were used to prove the prediction readiness of the model in terms of prediction. The source code of CBIL‒VHPLI and the datasets used in this work are available at https://github.com/Liu-Lab-Lnu/CBIL-VHPLI for academic usage.

CyCoNP lncRNA establishes cis and trans RNA-RNA interactions to supervise neuron physiology.

The combination of morphogenetic and transcription factors together with the synergic aid of noncoding RNAs and their cognate RNA binding proteins contribute to shape motor neurons (MN) identity. Here, we extend the noncoding perspective of human MN, by detailing the molecular and biological activity of CyCoNP (as Cytoplasmic Coordinator of Neural Progenitors) a highly expressed and MN-enriched human lncRNA. Through in silico prediction, in vivo RNA purification and loss of function experiments followed by RNA-sequencing, we found that CyCoNP sustains a specific neuron differentiation program, required for the physiology of both neuroblastoma cells and hiPSC-derived MN, which mainly involves miR-4492 and NCAM1 mRNA. We propose a novel lncRNA-mediated 'dual mode' of action, in which CyCoNP acts in trans as a classical RNA sponge by sequestering miR-4492 from its pro-neuronal targets, including NCAM1 mRNA, and at the same time it plays an additional role in cis by interactingwith NCAM1 mRNA and regulating the availability and localization of the miR-4492 in its proximity. These data highlight novel insights into the noncoding RNA-mediated control of human neuron physiology and point out the importance of lncRNA-mediated interactions for the spatial distribution of regulatory molecules.

Human lncRNAs harbor conserved modules embedded in different sequence contexts

We analyzed the structure of human long non-coding RNA (lncRNAs) genes to investigate whether the non-coding transcriptome is organized in modular domains, as is the case for protein-coding genes. To this aim, we compared all known human lncRNA exons and identified 340 pairs of exons with high sequence and/or secondary structure similarity but embedded in a dissimilar sequence context. We grouped these pairs in 106 clusters based on their reciprocal similarities. These shared modules are highly conserved between humans and the four great ape species, display evidence of purifying selection and likely arose as a result of recent segmental duplications. Our analysis contributes to the understanding of the mechanisms driving the evolution of the non-coding genome and suggests additional strategies towards deciphering the functional complexity of this class of molecules.

An Integrative Transcriptome Subtraction Strategy to Identify Human lncRNAs That Specifically Play a Role in Activation of Human Hepatic Stellate Cells.

Fibrotic liver features excessive deposition of extracellular matrix (ECM), primarily produced from "activated" hepatic stellate cells (HSCs). While targeting human HSCs (hHSCs) in fibrosis therapeutics shows promise, the overall understanding of hHSC activation remains limited, in part because it is very challenging to define the role of human long non-coding RNAs (lncRNAs) in hHSC activation. To address this challenge, we identified another cell type that acts via a diverse gene network to promote fibrogenesis. Then, we identified the lncRNAs that were differentially regulated in activated hHSCs and the other profibrotic cell. Next, we conducted concurrent analysis to identify those lncRNAs that were specifically involved in fibrogenesis. We tested and confirmed that transdifferentiation of vascular smooth muscle cells (VSMCs) represents such a process. By overlapping TGFβ-regulated lncRNAs in multiple sets of hHSCs and VSMCs, we identified a highly selected list of lncRNA candidates that could specifically play a role in hHSC activation. We experimentally characterized one human lncRNA, named CARMN, which was significantly regulated by TGFβ in all conditions above. CARMN knockdown significantly reduced the expression levels of a panel of marker genes for hHSC activation, as well as the levels of ECM deposition and hHSC migration. Conversely, gain of function of CARMN using CRISPR activation (CRISPR-a) yielded the completely opposite effects. Taken together, our work addresses a bottleneck in identifying human lncRNAs that specifically play a role in hHSC activation and provides a framework to effectively select human lncRNAs with significant pathophysiological role.

Mitochondrial sequencing identifies long noncoding RNA features that promote binding to PNPase.

Extranuclear localization of long noncoding RNAs (lncRNAs) is poorly understood. Based on machine learning evaluations, we propose a lncRNA-mitochondrial interaction pathway where polynucleotide phosphorylase (PNPase), through domains that provide specificity for primary sequence and secondary structure, binds nuclear-encoded lncRNAs to facilitate mitochondrial import. Using FVB/NJ mouse and human cardiac tissues, RNA from isolated subcellular compartments (cytoplasmic and mitochondrial) and cross-linked immunoprecipitate (CLIP) with PNPase within the mitochondrion were sequenced on the Illumina HiSeq and MiSeq, respectively. lncRNA sequence and structure were evaluated through supervised [classification and regression trees (CART) and support vector machines (SVM)] machine learning algorithms. In HL-1 cells, quantitative PCR of PNPase CLIP knockout mutants (KH and S1) was performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. One hundred twelve (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most abundant. Most noncoding RNAs binding PNPase were lncRNAs, including Malat1. lncRNA fragments bound to PNPase compared against randomly generated sequences of similar length showed stratification with SVM and CART algorithms. The lncRNAs bound to PNPase were used to create a criterion for binding, with experimental validation revealing increased binding affinity of RNA designed to bind PNPase compared to control RNA. The binding of lncRNAs to PNPase was decreased through the knockout of RNA binding domains KH and S1. In conclusion, sequence and secondary structural features identified by machine learning enhance the likelihood of nuclear-encoded lncRNAs binding to PNPase and undergoing import into the mitochondrion.NEW & NOTEWORTHY Long noncoding RNAs (lncRNAs) are relatively novel RNAs with increasingly prominent roles in regulating genetic expression, mainly in the nucleus but more recently in regions such as the mitochondrion. This study explores how lncRNAs interact with polynucleotide phosphorylase (PNPase), a protein that regulates RNA import into the mitochondrion. Machine learning identified several RNA structural features that improved lncRNA binding to PNPase, which may be useful in targeting RNA therapeutics to the mitochondrion.

GP-HTNLoc: A graph prototype head-tail network-based model for multi-label subcellular localization prediction of ncRNAs

Numerous research results demonstrated that understanding the subcellular localization of non-coding RNAs (ncRNAs) is pivotal in elucidating their roles and regulatory mechanisms in cells. Despite the existence of over ten computational models dedicated to predicting the subcellular localization of ncRNAs, a majority of these models are designed solely for single-label prediction. In reality, ncRNAs often exhibit localization across multiple subcellular compartments. Furthermore, the existing multi-label localization prediction models are insufficient in addressing the challenges posed by the scarcity of training samples and class imbalance in ncRNA dataset. To address these limitations, this study proposes a novel multi-label localization prediction model for ncRNAs, named GP-HTNLoc. To mitigate class imbalance, GP-HTNLoc adopts separate training approaches for head and tail location labels. Additionally, GP-HTNLoc introduces a pioneering graph prototype module to enhance its performance in small-sample, multi-label scenarios. The experimental results based on 10-fold cross-validation on benchmark datasets demonstrate that GP-HTNLoc achieves competitive predictive performance. The average results from 10 rounds of testing on an independent dataset show that GP-HTNLoc outperforms the best existing models on the human lncRNA, human snoRNA, and human miRNA subsets, with average precision improvements of 31.5%, 14.2%, and 5.6%, respectively, reaching 0.685, 0.632, and 0.704. A user-friendly online GP-HTNLoc server is accessible at https://56s8y85390.goho.co.

Abstract 1049: Long Noncoding RNA Regulation Of Inflammation In Atherosclerosis

Atherosclerosis is a chronic inflammatory disease driven by cholesterol deposition in the artery wall. Resolution of atherosclerotic inflammation and plaque regression are important clinical goals. As long noncoding RNAs (lncRNAs) are key regulators of gene expression, there is an intense interest in understanding their roles in atherosclerosis and their potential as therapeutic targets. To identify lncRNAs regulated during plaque progression and regression, we performed single cell RNA-sequencing of CD45 + immune cells isolated from aortic arch plaques of Ldlr –/– mice under conditions of plaque progression (16 wks Western diet; WD) or regression (16 wks WD + 4 wks apoB ASO). We screened for lncRNAs acting in cis to modify the expression of proximal protein-coding genes and identified Caslr ( C tsc a nti s ense l nc R NA) , a novel lncRNA transcribed from the opposite strand of Cathepsin C gene ( Ctsc ) encoding a pro-atherosclerotic lysosomal protease. Caslr–Ctsc pair is upregulated in macrophages of progressing plaques and in response to pro-inflammatory stimuli ( i.e . LPS, IL-6) in vitro , and conversely downregulated during atherosclerosis regression and in response to anti-inflammatory stimulus ( i.e. IL-4). LncRNA knockdown approaches combined with transcriptomic analyses and functional assays showed that Caslr regulates Ctsc expression and associated inflammatory responses, including NF-κB signaling and inflammasome-dependent IL-1β release. RNA purification strategies combined with mass-spectrometry revealed that Caslr interacts with spliceosome-associated proteins to coordinate splicing of Ctsc mRNA. Notably, Caslr shares functional homology with a human lncRNA, CASLR, proximal to the CTSC gene, which regulates CTSC expression and protease activity. Human CASLR and CTSC are highly expressed in the blood of hyperlipidemic patients and their expression is coordinately decreased upon aggressive cholesterol lowering by anti-PCSK9 treatment. Collectively, our data identify Caslr as an evolutionary conserved lncRNA acting in cis to drive the expression of cathepsin C with downstream consequences on inflammatory signaling in plaque macrophages, and suggest that targeting this lncRNA may be beneficial in treating atherosclerosis.

Abstract 3050: The Multifaceted Roles Of Long Noncoding Rna CHROMR In Cardiovascular Disease

The last decade revealed powerful roles for long noncoding RNA (lncRNA) in chromatin organization and orchestration of gene expression in healthy and pathological states, including atherosclerotic cardiovascular disease (ACVD). Yet, fewer than 10% of human lncRNAs have been characterized, representing unique opportunity for discoveries in the pathogenesis and treatment of ACVD. We previously showed that the primate-specific lncRNA CHROMR (CHolesterol-induced Regulator Of Metabolism RNA) is induced by cholesterol excess to restrict the availability of functionally related microRNAs that normally repress cholesterol efflux and HDL biogenesis. Using single-cell RNA-seq analysis of human carotid plaques from the NYU ATHERO-IN study, we now show that CHROMR is robustly expressed in myeloid cells within carotid plaques and strongly correlates with expression of interferon (IFN) stimulated genes (ISGs). Previously we established that CHROMR functions in the nucleus to license the expression of interferon (IFN) stimulated genes (ISGs). CHROMR acts through sequestering IFN regulatory factor-2 binding protein 2, part of the IRF2 repressor complex that antagonizes transcriptional activation of ISGs. While this coordinated regulation of lipid metabolism and IFN responses by CHROMR is beneficial in accelerating an inflammatory response upon viral infection, it has detrimental effects in the context of ACVD. As such, therapeutic inhibition of CHROMR holds potential to reduce plaque inflammation. Therefore, we treated human atherosclerotic 3D carotid vascular explants with GapmeRs to deplete CHROMR and subsequently measured the cellular response upon inflammatory stimulus. We found that Gap CHROMR reduced CHROMR levels and blunted pro-atherogenic cytokine/chemokine secretion, whereas lentiviral delivery of CHROMR increased cytokine/chemokine secretion when compared to controls. Collectively, our findings underscore the merit of investigating CHROMR, as well as other lncRNAs, to decipher novel regulatory mechanisms that govern lipid metabolism and inflammation in humans.

Emerging roles of long non-coding RNAs in human epilepsy.

Genome-scale biological studies conducted in the post-genomic era have revealed that two-thirds of human genes do not encode proteins. Most functional non-coding RNA transcripts in humans are products of long non-coding RNA (lncRNA) genes, an abundant but still poorly understood class of human genes. As a result of their fundamental and multitasking regulatory roles, lncRNAs are associated with a wide range of human diseases, including neurological disorders. Approximately 40% of lncRNAs are specifically expressed in the brain, and many of them exhibit distinct spatiotemporal patterns of expression. Comparative genomics approaches have determined that 65%-75% of human lncRNA genes are primate-specific and hence can be posited as a contributing potential cause of the higher-order complexity of primates, including human, brains relative to those of other mammals. Although lncRNAs present important mechanistic examples of epileptogenic functions, the human/primate specificity of lncRNAs questions their relevance in rodent models. Here, we present an in-depth review that supports the contention that human lncRNAs are direct contributors to the etiology and pathogenesis of human epilepsy, as a means to accelerate the integration of lncRNAs into clinical practice as potential diagnostic biomarkers and therapeutic targets. Meta-analytically, the major finding of our review is the commonality of lncRNAs in epilepsy and cancer pathogenesis through mitogen-activated protein kinase (MAPK)-related pathways. In addition, neuroinflammation may be a relevant part of the common pathophysiology of cancer and epilepsy. LncRNAs affect neuroinflammation-related signaling pathways such as nuclear factor kappa- light- chain- enhancer of activated B cells (NF-κB), Notch, and phosphatidylinositol 3- kinase/ protein kinase B (Akt) (PI3K/AKT), with the NF-κB pathway being the most common. Besides the controversy over lncRNA research in non-primate models, whether neuroinflammation is triggered by injury and/or central nervous system (CNS) toxicity during epilepsy modeling in animals or is a direct consequence of epilepsy pathophysiology needs to be considered meticulously in future studies.

Exosomal LINC00853 promotes progression of gastric cancer via the MAP17/PDZK1/AKT signaling pathway

Although rare, there is ongoing research into biomarkers that predict the onset and recurrence of gastric cancer, particularly focusing on substances found in exosomes. Long non-coding RNAs (lncRNAs) have garnered attention for their potential in diagnosing gastric cancer.This study investigates the role of lncRNAs in gastric cancer, focusing on their presence in exosomes as potential biomarkers for the disease's onset and recurrence. We utilized the ArrayStar Human LncRNA array 2.0 to analyze lncRNA expression in tissues from early-stage gastric cancer patients. Our analysis highlighted LINC00853, which was significantly upregulated in cancer tissues and implicated in promoting epithelial-mesenchymal transition via the MAP17/PDZK1/AKT pathway. Functional studies on AGS and MKN74 gastric cancer cell lines demonstrated that LINC00853 facilitates cell proliferation, invasion, and migration. Additionally, RNA immunoprecipitation and electrophoretic mobility shift assays confirmed LINC00853 interaction with MAP17. Importantly, LINC00853 was also detected in exosomes from both patient samples and cell lines, and its downregulation led to decreased tumorigenicity in AGS cells. These findings suggest that both cellular and exosomal LINC00853 contribute to gastric cancer pathogenesis and may serve as valuable biomarkers for the disease.

Abstract 2988: TUG1 exosomal lncRNA sponges let-7 miRNA family members and advances prostate cancer

Abstract Exosomal long-non coding RNAs (lncRNAs) play an important role in cancer progression and have potential prognostic biomarker potential in several cancers including prostate cancer (PCa). Taurine-upregulated gene 1 (TUG1) is an oncogenic lncRNA overexpressed in PCa. This exosomal lncRNA has been reported to promote PCa migration, invasion and Epithelial-Mesenchymal Transition (EMT). However, TUG1 PCa prognostic mechanisms remain to be elucidated. This study aimed to profile differentially expressed lncRNAs in PCa high-grade tumor cells (PC-3) cells compared to low-grade PCa tumor cells (LNCaP). This was achieved by using a 384-well plate of PCa lncRNA gene panel. A human lncRNA database, LncSEA and other bioinformatics tools were used for annotation and enrichment analysis. RT-qPCR was used to confirm the gene array data. Up or down-regulation of ±2 and p <0.05 were considered significant. Forty two (42%) percent and 14% of lncRNAs were shown to be up and downregulated, respectively. Notably, TUG1 was shown to be upregulated in PC-3 cells from PCR array and RT-qPCR results. Bioinformatics analysis revealed that TUG1 is involved in several cancer hallmarks including apoptosis evasion, migration, invasion and EMT. Furthermore, lncRNA/miRNA complexes regulate a series of oncogenic signaling pathways and this study demonstrated that TUG1 sponges the let-7 miRNA family members (a highly conserved tumor suppressor miRNA family) and promotes PCa progression. Precise mechanisms of TUG1/let-7 axis in PCa prognosis need to be largely elucidated as this may hold potential to understanding lncRNA/miRNA-mediated PCa progression and thus be targeted for novel therapeutics. Citation Format: Rahaba Makgotso Marima, Mandisa Mbeje, Jeyalakshmi Kandhavelu, Clement Penny, Zodwa Dlamini. TUG1 exosomal lncRNA sponges let-7 miRNA family members and advances prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2988.