RNA Expression Patterns Research Articles

Genome-Wide Identification of the Soybean AlkB Homologue Gene Family and Functional Characterization of GmALKBH10Bs as RNA m6A Demethylases and Expression Patterns under Abiotic Stress.

Soybean (Glycine max (L.) Merr) is one of the most important crops worldwide, but its yield is vulnerable to abiotic stresses. In Arabidopsis, the AlkB homologue (ALKBH) family genes plays a crucial role in plant development and stress response. However, the identification and functions of its homologous genes in soybean remain obscured. Here, we identified a total of 22 ALKBH genes in soybean and classified them into seven subfamilies according to phylogenetic analysis. Gene duplication events among the family members and gene structure, conserved domains, and motifs of all candidate genes were analyzed. By comparing the changes in the m6A levels on mRNA from hair roots between soybean seedlings harboring the empty vector and those harboring the GmALKBH10B protein, we demonstrated that all four GmALKBH10B proteins are bona fide m6A RNA demethylases in vivo. Subcellular localization and expression patterns of the GmALKBH10B revealed that they might be functionally redundant. Furthermore, an analysis of cis-elements coupled with gene expression data demonstrated that GmALKBH10B subfamily genes, including GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4, are likely involved in the cis-elements' response to various environmental stimuli. In summary, our study is the first to report the genome-wide identification of GmALKBH family genes in soybean and to determine the function of GmALKBH10B proteins as m6A RNA demethylases, providing insights into GmALKBH10B genes in response to abiotic stresses.

Quantum imaging-based nanobiosensors: Pioneering point-of-care approach for early diagnosis of environmental-linked breast cancer

Early detection is paramount for successful treatment outcomes in cancer diagnosis. Among women across the globe, breast cancer (BC) ranks as one of the deadliest forms of cancer. Prolonged exposure to numerous environmental pollutants has been linked to epigenetic reprogramming, which entails changes in the expression patterns of non-coding RNAs. These alterations have been strongly linked to an increased risk of developing BC. Women are confronted with hazardous smoke from polluting stoves and fuels for longer as they often perform home duties such as cooking. Inefficient combustion emits black carbon (sooty particles), which enters the bloodstream and is strongly connected to an elevated risk of BC. The use of several analytical methods, such as real-time polymerase chain reaction, microarray, and sequencing, has numerous disadvantages, such as high expenses, limitations in sensitivity, and lack of accuracy. However, the emergence of quantum dots (QDs), nanoscale semiconductor particles with unique optical properties, and the development of quantum imaging-based sensors offer a glimpse into the future of medical technology. These sensors have the potential to completely change the medical field by offering highly precise, non-invasive, and reliable techniques for early diagnosis. Our article delves into the intricacies of QDs imaging-based sensors, their applications in BC detection, and their transformative impact on improving patient care. In recent years, the confluence of quantum science and diagnostic imaging has opened new avenues for BC diagnosis. The present state of quantum imaging-based BC diagnosis sensors is examined in this article, along with potential future developments with the help of artificial intelligence.

Competition for nutrient niches within the apple blossom microbiota antagonizes the initiation of fire blight infection.

Changes in the plant microbiota composition are intimately associated with the health of the plant, but factors controlling the microbial community in flowers are poorly understood. In this study, we used apple flowers and fire blight as a model system to investigate the effects of floral microbiota and microbial competition on disease development and suppression. To compare changes in microbial flora with the RNA expression patterns of plants, the flower samples were collected in three different flowering stages (Bud, Popcorn, and Full-bloom). Using advanced sequencing technology, we analyzed the data and conducted both in vitro and in vivo experiments to validate our findings. Our results show that the Erwinia amylovora use arabinogalactan, which is secreted on the flowers, for early colonization of apple flowers. Pantoea agglomerans was more competitive for arabinogalactan than E. amylovora. Additionally, P. agglomerans suppressed the expression of virulence factors of E. amylovora by using arabinose, which is a major component of arabinogalactan, which induces virulence gene expression. The present data provide new insights into developing control strategies for diverse plant diseases, including fire blight, by highlighting the importance of nutrients in disease development or suppression.

Development of an agroinfiltration-based transient hairpin RNA expression system in pak choi leaves (Brassica rapa ssp. chinensis) for RNA interference against Liriomyza sativae

The vegetable leafminer (Liriomyza sativae) is a devastating invasive pest of many vegetable crops and horticultural plants worldwide, causing serious economic loss. Conventional control strategy against this pest mainly relies on the synthetic chemical pesticides, but widespread use of insecticides easily causes insecticide resistance development and is harmful to beneficial organisms and environment. In this context, a more environmentally friendly pest management strategy based on RNA interference (RNAi) has emerged as a powerful tool to control of insect pests. Here we report a successful oral RNAi in L. sativae after feeding on pak choi (Brassica rapa ssp. chinensis) that transiently express hairpin RNAs targeting vital genes in this pest. First, potentially lethal genes are identified by searching an L. sativae transcriptome for orthologs of the widely used V-ATPase A and actin genes, then expression levels are assessed during different life stages and in different adult tissues. Interestingly, the highest expression levels for V-ATPase A are observed in the adult heads (males and females) and for actin in the abdomens of adult females. We also assessed expression patterns of the target hairpin RNAs in pak choi leaves and found that they reach peak levels 72 h post agroinfiltration. RNAi-mediated knockdown of each target was then assessed by letting adult L. sativae feed on agroinfiltrated pak choi leaves. Relative transcript levels of each target gene exhibit significant reductions over the feeding time, and adversely affect survival of adult L. sativae at 24 h post infestation in genetically unmodified pak choi plants. These results demonstrate that the agroinfiltration-mediated RNAi system has potential for advancing innovative environmentally safe pest management strategies for the control of leaf-mining species.

Translation efficiency covariation across cell types is a conserved organizing principle of mammalian transcriptomes.

Characterization of shared patterns of RNA expression between genes across conditions has led to the discovery of regulatory networks and novel biological functions. However, it is unclear if such coordination extends to translation, a critical step in gene expression. Here, we uniformly analyzed 3,819 ribosome profiling datasets from 117 human and 94 mouse tissues and cell lines. We introduce the concept of Translation Efficiency Covariation (TEC), identifying coordinated translation patterns across cell types. We nominate potential mechanisms driving shared patterns of translation regulation. TEC is conserved across human and mouse cells and helps uncover gene functions. Moreover, our observations indicate that proteins that physically interact are highly enriched for positive covariation at both translational and transcriptional levels. Our findings establish translational covariation as a conserved organizing principle of mammalian transcriptomes.

Identifying the keyhub genes linked with lung squamous cell carcinoma by examining the differentially expressed and survival genes.

Lung Squamous Cell Carcinoma is characterised by significant alterations in RNA expression patterns, and a lack of early symptoms and diagnosis results in poor survival rates. Our study aimed to identify the hub genes involved in LUSC by differential expression analysis and their influence on overall survival rates in patients. Thus, identifying genes with the potential to serve as biomarkers and therapeutic targets. RNA sequence data for LUSC was obtained from TCGA and analysed using R Studio. Survival analysis was performed on DE genes. PPI network and hub gene analysis was performed on survival-relevant genes. Enrichment analysis was conducted on the PPI network to elucidate the functional roles of hub genes. Our analysis identified 2774 DEGs in LUSC patient datasets. Survival analysis revealed 511 genes with a significant impact on patient survival. Among these, 20 hub genes-FN1, ACTB, HGF, PDGFRB, PTEN, SNAI1, TGFBR1, ESR1, SERPINE1, THBS1, PDGFRA, VWF, BMP2, LEP, VTN, PXN, ABL1, ITGA3 and ANXA5-were found to have lower expression levels associated with better patient survival, whereas high expression of SOX2 correlated with longer survival. Enrichment analysis indicated that these hub genes are involved in critical cellular and cancer-related pathways. Our study has identified six key hub genes that are differentially expressed and exhibit significant influence over LUSC patient survival outcomes. Further, in vitro and in vivo studies must be conducted on the key genes for their utilisation as therapeutic targets and biomarkers in LUSC.

Exon-Skipping–Based Subtyping of Colorectal Cancers

The identification of colorectal cancer (CRC) molecular subtypes has prognostic and potentially diagnostic value for patients, yet reliable subtyping remains unavailable in the clinic. The current consensus molecular subtype (CMS) classification in CRCs is based on complex RNA expression patterns quantified at the gene level. The clinical application of these methods, however, is challenging due to high uncertainty of single-sample classification and associated costs. Alternative splicing, which strongly contributes to transcriptome diversity, has rarely been used for tissue type classification. Here, we present an AS-based CRC subtyping framework sensitive to differential exon use that can be adapted for clinical application. Unsupervised clustering was used to measure the strength of association between different categories of alternative splicing and CMS. To build a classifier, the ground truth for CMS labels was derived from expression data quantified at the gene level. Feature selection was achieved through bootstrapping and L1-penalized estimation. The resulting feature space was used to construct a subtype prediction framework applicable to single and multiple samples. The performance of the models was evaluated on unseen CRCs from 2 independent sources (Indivumed, n= 129; The Cancer Genome Atlas, n= 99). We developed a CRC subtype identifier based on 29 exon-skipping events that accurately classifies unseen tumors and enables more precise differentiation of subtypes characterized by distinct biological and prognostic features as compared to classifiers based on gene expression. Here, we demonstrate that a small number of exon-skipping events can reliably classify CRC subtypes using individual patient specimens in a manner suitable to clinical application.

Identification of a basement membrane-related gene signature for predicting prognosis, immune infiltration, and drug sensitivity in colorectal cancer.

Colorectal cancer (CRC) is the most common malignancy affecting the gastrointestinal tract. Extensive research indicates that basement membranes (BMs) may play a crucial role in the initiation and progression of the disease. Data on the RNA expression patterns and clinicopathological information of patients with CRC were sourced from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. A BM-linked risk signature for the prediction of overall survival (OS) was formulated using univariate Cox regression and combined machine learning techniques. Survival outcomes, functional pathways, the tumor microenvironment (TME), and responses to both immunotherapy and chemotherapy within varying risk classifications were also investigated. The expression trends of the model genes were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and the Human Protein Atlas (HPA) database. A nine-gene risk signature containing UNC5C, TINAG, TIMP1, SPOCK3, MMP1, AGRN, UNC5A, ADAMTS4, and ITGA7 was constructed for the prediction of outcomes in patients with CRC. The expression profiles of these candidate genes were verified using RT-PCR and the HPA database and were found to be consistent with the findings on differential gene expression in the TCGA dataset. The validity of the signature was confirmed using the GEO cohort. The patients were stratified into different risk groups according to differences in clinicopathological characteristics, TME features, enrichment functions, and drug sensitivities. Lastly, the prognostic nomogram model based on the risk score was found to be effective in identifying high-risk patients and predicting OS. A basement membrane-related risk signature was constructed and found to be effective for predicting the prognosis of patients with CRC.

Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture

BackgroundThe interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce.ResultsHere, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5’untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5’UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction.ConclusionWe adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

Molecular characterization, spatiotemporal expression, and background adaptation regulation of tyrosinase in loach (Misgurnus anguillicaudatus).

The Poyang Lake region is home to large-blackspot loaches (LBL), small-blackspot loaches (SBL), and non-blackspot loaches (NBL), Misgurnus anguillicaudatus. To investigate the impact of tyrosinase on spot development, the complementary DNAs (cDNA) of tyrosinase in M. anguillicaudatus (designated as Matyr) were cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The full-length cDNA for Matyr was 2020 bp, and the open-reading frame comprised 1617 bp, encoding a predicted protein with 538 amino acids. Phylogenetic studies revealed that MaTyr was first grouped with Tyr of Triplophysa tibetana and Leptobotia taeniops, and then Tyr of other cyprinid fish. The quantitative reverse-transcription-PCR results show that Matyr was highly expressed in the muscle, caudal fin, and dorsal skin. The Matyr gene's messenger RNA expression pattern steadily increased from the fertilized ovum period to the somitogenesis period, and from the muscle effect stage to 6 days after fertilization, it considerably increased (p < 0.01). The Matyr hybridization signals with similar location could be found in all developmental stages of three kinds of loaches using whole-mount in situ hybridization (WISH) technology and were the strongest during the organ development period and melanin formation period. Dot hybridization signals in LBLs rapidly spread to the back of the body beginning at the period when the eyes first formed melanin, and their dimensions were larger than those of NBLs during the same time period. The body color of loaches could change reversibly with black/white background adaptation. The α-msh, mitfa, and tyr are mainly expressed in loaches adapted with a black background. Tyr gene could be involved in the development of blackspots and body color polymorphism, and contribute to organ development in the loach.

Characteristics of the Vasa Gene in Silurus asotus and Its Expression Response to Letrozole Treatment.

The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I-V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus.

Investigating tRNA-Derived Small RNA Expression Patterns and Bioinformatics Analysis in Epicardial Fat of Atrial Fibrillation Patients

Abstract: The intricate mechanisms underlying atrial fibrillation (AF) remain elusive. It is proposed that epicardial adipose tissue (EAT) may play a role in arrhythmias by releasing bioactive molecules, including exosomes containing tRNA-derived small RNAs (tsRNAs). Although tsRNAs are known to significantly influence cellular functions, their relationship with AF remains unexplored. To investigate this, we conducted RNA sequencing on EAT samples from 6 AF patients and 6 sinus rhythm control subjects. Our analysis identified 146 upregulated and 126 downregulated tsRNAs in AF. Four tsRNAs (tRF-SeC-TCA001, tiRNAGly–CCC–003, tRF-Gly-GCC-002, and tRF-Tyr-GTA-007) were validated via quantitative reverse transcriptionpolymerase chain reaction. Bioinformatic analysis revealed these tsRNAs target genes associated with cell adhesion and various cellular processes mediated by plasma membrane adhesion molecules. KEGG analysis suggested these target genes may contribute to AF pathogenesis through processes such as glycosaminoglycan biosynthesis, AMP-activated protein kinase activity, and the insulin signaling pathway. Our findings unveil altered tsRNA expression profiles in EAT from AF patients, hinting at potential interactions between tsRNAs and mRNA within EAT that could impact AF pathogenesis.

Dynamic whole-transcriptome landscape of acute bilirubin encephalopathy in newborns

Hyperbilirubinemia in newborns may progress to acute bilirubin encephalopathy (ABE), posing short- and long-term health risks. Despite extensive research identifying numerous mRNAs, lncRNAs, circRNAs, and miRNAs associated with brain injury, their roles in neonatal bilirubin-induced brain injury remain elusive. This study employed whole-transcriptome sequencing to ascertain the differentially expressed (DE) RNA profiles in a newborn ABE rat model, followed by bioinformatic analysis. A time-series competing endogenous RNA (ceRNA) regulatory network was established, and the expression trends of 9 arbitrarily chosen RNAs were verified through quantitative real-time polymerase chain reaction(qRT-PCR). In comparison with the control group, we identified 595, 888, and 1448 DE mRNAs; 22, 37, and 37 DE miRNAs; 1945, 1869, and 1997 DE lncRNAs; and 31, 28, and 36 DE circRNAs at 6 h, 12 h, and 24 h, respectively. Predominantly, these DERNAs contribute to biological functions and pathways associated with inflammation, immunity, metabolism, cell death, and neurodevelopmental regulation. Moreover, we constructed ceRNA networks of DE lncRNA/circRNA-DE miRNA-DE mRNA based on time series. The qRT-PCR expression trends for the selected 9 RNAs were generally similar to the RNA-seq outcomes. This investigation uniquely delineated the temporal expression patterns of mRNA and non-coding RNA in ABE, establishing ceRNA networks and identifying potential molecular mechanisms underlying bilirubin-induced hippocampal damage. Nonetheless, further studies are warranted to corroborate these findings in humans.

The Expression Patterns of Non-Coding RNAs in Periodontal Disease.

During the last few decades there has been a growing interest in understanding the involvement of epigenetics in the pathogenesis and treatment of periodontal disease. Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), may serve as epigenetic modifiers affecting the expression of genes involved in the pathogenesis of inflammatory and autoimmune diseases. There is increasing evidence supporting the idea that the function of all three types of ncRNAs seems to be interdependent. LncRNAs can act as miRNA decoys, while circRNAs can act as miRNA sponges, leading to the re-expression of miRNA target genes. The purpose of this review is to evaluate the expression patterns of ncRNAs in periodontal disease. Studies demonstrate a positive correlation between miRNA expression and periodontitis; however, this cannot be claimed for lncRNAs and circRNAs, which appear to be differentially expressed in periodontitis patients. Several studies have also suggested utilizing ncRNAs as diagnostic and prognostic biomarkers in periodontitis, or even as potential therapeutic targets; Nevetheless, the evidence to support this is premature. Future well-designed research remains necessary to establish the functional role of ncRNAs in the evolution and progression of periodontal disease.

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs

ABSTRACT Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Integrative analyses of whole-transcriptome sequencing reveals CeRNA regulatory network in pulmonary hypertension treated with FGF21

Noncoding RNAs have been shown to play essential roles in hypoxic pulmonary hypertension (HPH). Our preliminary data showed that HPH is attenuated by fibroblast growth factor 21 (FGF21) administration. Therefore, we further investigated the whole transcriptome RNA expression patterns and interactions in a mice HPH model treated with FGF21. By whole-transcriptome sequencing, differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs were successfully identified in normoxia (Nx) vs. hypoxia (Hx) and Hx vs. hypoxia + FGF21 (Hx + F21). Differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs regulated by hypoxia and FGF21 were selected through intersection analysis. Based on prediction databases and sequencing data, differentially co-expressed mRNAs, miRNAs, lncRNAs, and circRNAs were further screened, followed by functional enrichment analysis. MAPK signaling pathway and epigenetic modification were enriched and may play fundamental roles in the therapeutic effects of FGF21. The ceRNA regulatory network of lncRNA–miRNA–mRNA and circRNA–miRNA–mRNA was constructed with miR-7a-5p, miR-449c-5p, miR-676-3p and miR-674-3p as the core. In addition, quantitative real-time PCR experiments were employed to verify the whole-transcriptome sequencing data. The results of luciferase reporter assays highlighted the relationship between miR-449c-5p and XR_878320.1, miR-449c-5p and Stab2, miR-449c-5p and circ_mtcp1, which suggesting that miR-449c-5p may be a key regulator of FGF21 in the treatment of PH. Taken together, this study provides potential biomarkers, pathways, and ceRNA regulatory networks in HPH treated with FGF21 and will provide an experimental basis for the clinical application of FGF21 in PH.

Transcriptional and functional profiling identifies inflammation and endothelial-to-mesenchymal transition as potential drivers for phenotypic heterogeneity within a cohort of endothelial colony forming cells

BackgroundEndothelial colony-forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease. ObjectivesTherefore, we aimed to determine phenotypic and gene expression differences between control ECFCs. MethodsA total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n = 15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by quantitative polymerase chain reaction. Phenotypic profiling was performed and migration speed of the ECFCs was measured using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed. ResultsThrough hierarchical clustering of RNA expression profiles, we could distinguish 2 major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1 and inflammation and endothelial-to-mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1, which contained more and longer Weibel–Palade bodies. Migration speed in cluster 1 was also significantly higher. ConclusionWe observed a range of different RNA expression patterns between ECFC clones, mostly associated with inflammation and clear differences in Weibel–Palade body count and structure. We developed a quantitative polymerase chain reaction panel that can be used for the characterization of ECFC clones, which is essential for the correct analysis of pathogenic mechanisms in vascular disorders.

Dysregulated Expression Patterns of Circular RNAs in Cancer: Uncovering Molecular Mechanisms and Biomarker Potential.

Circular RNAs (circRNAs) are stable, enclosed, non-coding RNA molecules with dynamic regulatory propensity. Their biogenesis involves a back-splicing process, forming a highly stable and operational RNA molecule. Dysregulated circRNA expression can drive carcinogenic and tumorigenic transformation through the orchestration of epigenetic modifications via extensive RNA and protein-binding domains. These multi-ranged functional capabilities have unveiled extensive identification of previously unknown molecular and cellular patterns of cancer cells. Reliable circRNA expression patterns can aid in early disease detection and provide criteria for genome-specific personalized medicine. Studies described in this review have revealed the novelty of circRNAs and their biological ss as prognostic and diagnostic biomarkers.

Abstract 874: Expanded detection of BAP1 alterations in cancer and comparison of tumor type-specific gene expression scores

Abstract BAP1 is a tumor suppressor gene that was originally studied in uveal melanoma (UVM), kidney renal cell clear cell carcinoma (KIRC), and malignant mesothelioma (MESO). Early analyses focused on single-nucleotide variants, but other alteration types such as larger indels and gene-level copy number (CN) loss can also lead to loss of BAP1 expression. By incorporating these, we can improve our assessment of cancer type-specific consequences of BAP1 loss and broaden our findings beyond the initial small subset of cancer types which were studied in the context of mutations alone. We performed integrated multi-omic analyses using data from The Cancer Genome Atlas (TCGA), for 33 cancer types and more than 10,000 individuals. By combining and manually reviewing existing variant calls and new calls derived from a de novo local realignment pipeline across multiple independent variant callers including indel callers, we increased detection of high-quality somatic variant calls by 30% from 85 to 111, including 14 indels ≥5bp. Including CN loss alterations, we found a total of 1561 samples from 32 cancer types to be BAP1-altered, with alterations being predominantly CN-driven. We confirmed that expression of BAP1 was lower in altered versus unaltered tumors (p&amp;lt;0.001). Alteration frequencies across cancer types were highly variable, ranging from 0% in thyroid cancer to &amp;gt;30% in KIRC, cholangiocarcinoma (CHOL), UVM, MESO, lung squamous carcinoma (LUSC), and head-neck squamous cell carcinoma (HNSC). Tumor types such as CHOL, UVM, MESO, and liver hepatocellular carcinoma (LIHC) were relatively more mutation-driven whereas other tumor types like KIRC, LUSC, and HNSC were almost entirely CN-driven. To better understand the tissue and context-specific consequences of BAP1 alteration, we performed differential expression analyses across BAP1-altered and -unaltered samples. For each cancer type, we separately computed per-sample BAP1 alteration signature z-scores summarizing expression of the set of differentially expressed genes. In UVM, we observed distinct patterns of RNA expression, consistent with established literature on BAP1 loss and serving as a baseline for comparison for the biological effects of BAP1 loss in other less-studied cancer types. The BAP1 altered scores for 66% of cancer types were positively correlated with the UVM BAP1 altered signature, suggesting shared biological responses and affected pathways including immune responses, epithelial-mesenchymal transition, oxidative phosphorylation, and proliferation – all known roles of BAP1. In LIHC, we noted enrichment of bile duct gene expression in BAP1-altered tumors, suggesting a possible role for BAP1 in maintaining cell identity distinguishing hepatocytes and cholangiocytes. Our findings broadly emphasize the improvements that are gained by using new computational approaches in large cancer-genome studies such as TCGA. Citation Format: Ian R. Sturgill, Jesse R. Raab, Katherine A. Hoadley. Expanded detection of BAP1 alterations in cancer and comparison of tumor type-specific gene expression scores [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 874.

Abstract 5035: Use of Streck nucleic acid BCT with plasma nucleic acid next-generation sequencing workflows

Abstract Introduction: Liquid biopsies, which interrogate body fluids in search of analytes released by cancer cells, are becoming increasingly common in clinical oncology. Foremost among the technologies used are those that employ nucleic acids, such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA). Currently, many NGS-based tests perform near the limit of assay detection, making the identification of critical mutations, fusions, or expression patterns like finding a needle in a haystack. Further hindering successful assay development is the variability in analyte availability and detectability that can occur prior to analysis due to blood collection, transport, and storage. Here, we demonstrate that Nucleic Acid BCT stabilizes draw-time levels of plasma nucleic acids for downstream use with NGS-based assays. Methods: Blood from self-proclaimed healthy donors was drawn into EDTA and Nucleic Acid BCT and stored at ambient temperature for up to 7 days before plasma isolation with a general double-spin protocol and storage at -80 °C. Plasma nucleic acids were isolated using the MAGicBead cfDNA Kit (Zymo, cfDNA) or the QIAamp Circulating Nucleic Acid Kit (QIAGEN, cfRNA). Library prep for cfDNA was carried out with added synthetic mutant spike-in oligonucleotides using the Illumina TruSight Tumor 15 kit (Illumina). cfRNA library prep was done with the NEBNext Ultra II RNA Library Prep Kit for Illumina with upfront rRNA depletion (New England Biolabs). Sequencing was performed using an Illumina NextSeq 500 system, and all data were saved in and analyzed with Illumina BaseSpace applications. Results: When blood samples were stored in EDTA tubes for up to 7 days, spiked mutant alleles (EGFRL858R, EGFRdel19, KRASG12D, and PIK3CAE545K) were no longer detected, likely due to a dilution of the spiked DNA concentration following the release of genomic DNA containing wildtype alleles during white blood cell degradation. In contrast, equivalent blood samples stored in Nucleic Acid BCTs displayed mutant allele frequencies reflective of draw-time levels. Interrogation of plasma RNA expression patterns in samples stored in Nucleic Acid BCT versus EDTA revealed similar results, where differentially expressed plasma transcripts in blood samples stored in EDTA were found at high levels (&amp;gt;1,000 transcripts) versus donor-matched blood samples stored in Nucleic Acid BCT (&amp;lt;50 transcripts). Conclusions: Our data demonstrate that Nucleic Acid BCT maintains mutant allele frequencies and the plasma transcriptome profile of blood samples during ambient temperature storage. This provides precious sample integrity during whole blood storage and transport, offering laboratories and assay developers reduced preanalytical variability for NGS-based analysis of gene mutations, fusions, indels, and the plasma transcriptome. Nucleic Acid BCT is for Research Use Only. Not for use in diagnostic procedures. Citation Format: Nicholas M. George, Lisa Bartron. Use of Streck nucleic acid BCT with plasma nucleic acid next-generation sequencing workflows [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 5035.