Non-protein Coding Genes Research Articles

Utility of genome sequencing in exome-negative pediatric patients with neurodevelopmental phenotypes.

Exome sequencing (ES) has emerged as an essential tool in the evaluation of neurodevelopmental disorders (NDD) of unknown etiology. Genome sequencing (GS) offers advantages over ES due to improved detection of structural, copy number, repeat number and non-coding variants. However, GS is less commonly utilized due to higher cost and more intense analysis. Here, we present nine cases of pediatric NDD that were molecularly diagnosed with GS between 2017 and 2022, following non-diagnostic ES. All individuals presented with global developmental delay or regression. Other features present in our cohort included epilepsy, white matter abnormalities, brain malformation and dysmorphic features. Two cases were diagnosed on GS due to newly described gene-disease relationship or variant reclassification (MAPK8IP3, CHD3). Additional features missed on ES that were later detected on GS were: intermediate-size deletions in three cases who underwent ES that were not validated for CNV detection, pathogenic variants within the non-protein coding genes SNORD118 and RNU7-1, pathogenic variant within the promoter region of GJB1, and a coding pathogenic variant within BCAP31 which was not sufficiently covered on ES. GS following non-diagnostic ES led to the identification of pathogenic variants in this cohort of nine cases, four of which would not have been identified by reanalysis alone.

The long intergenic non-coding RNA LINC01140 modulates gastric cancer phenotypes and cancer cell lines aggressiveness

BackgroundLong-intergenic non-protein coding gene 01140 (LINC01140) a long non-coding RNA is highly expressed in various cancers. However, its biological functions in gastric cancer progression is still unknown. MethodTo elucidate LINC01140 function, 70 GC tumor samples and 30 normal gastric tissues were collected. LINC01140 expression level were determined by qRT-PCR analysis and correlated with different clinico-pathological parameters. Then we tried to see the impact of LINC01140 on gastric cell line aggressiveness by knocking down the target gene and performing cell viability assay, migration assay and invasive capacity of the cell lines along with immunoblotting to check several protein levels. ResultLINC01140 RNA is found to be positively correlated with FGF9 and significantly up regulated in GC tissues. LINC01140 knockdown inhibited the viability, migratory capacity and invasive capacity of AGS cells. LINC01140 targets miR-140–5p, while miR-140–5p targeted FGF9 to form lncRNA–miRNA-mRNA axis. The affect of miR-140–5p inhibition on gastric cancer cell aggressiveness were opposite to those of LINC01140 or FGF9 knockdown. Additionally, inhibition partially reversed the effects of LINC01140 knockdown on FGF9 protein levels, gastric cancer cell phenotypes. ConclusionLINC01140, miR-140–5p and FGF9 form a lncRNA-miRNA-mRNA axis that modulates the gastric cancer phenotypes and in turn affects gastric cancer cell aggressiveness.

The forgotten variable? Does the euthanasia method and sample storage condition influence an organisms transcriptome – a gene expression analysis on multiple tissues in pigs

BackgroundTranscriptomic studies often require collection of fresh tissues post euthanasia. The chosen euthanasia method might have the potential to induce variations in gene expressions that are unlinked with the experimental design. The present study compared the suitability of ‘nitrogen gas in foam’ (ANOXIA) in comparison to a non-barbiturate anaesthetic, T-61® (T61), for euthanizing piglets used in transcriptome research. Further, the effect of common tissue storage conditions, RNAlater™ (RL) and snap freezing in liquid nitrogen (LN2), on gene expression profiles were also analysed.ResultsOn comparison of the 3’mRNA-Seq data generated from pituitary, hypothalamus, liver and lung tissues, no significant differential expression in the protein coding genes were detected between the euthanasia methods. This implies that the nitrogen anoxia method could be a suitable alternative for euthanasia of piglets used in transcriptomic research. However, small nuclear RNAs (snRNAs) that constitute the eukaryotic spliceosomal machinery were found to be significantly higher (log2fold change ≥ 2.0, and adjusted p value ≤ 0.1) in pituitary samples collected using ANOXIA. Non-protein coding genes like snRNAs that play an important role in pre-mRNA splicing can subsequently modify gene expression. Storage in RL was found to be superior in preserving RNA compared to LN2 storage, as evidenced by the significantly higher RIN values in representative samples. However, storage in RL as opposed to LN2, also influenced differential gene expression in multiple tissues, perhaps as a result of its inability to inhibit biological activity during storage. Hence such external sources of variations should be carefully considered before arriving at research conclusions.ConclusionsSource of biological variations like euthanasia method and storage condition can confound research findings. Even if we are unable to prevent the effect of these external factors, it will be useful to identify the impact of these variables on the parameter under observation and thereby prevent misinterpretation of our results.

Human Breast Milk miRNAs: Their Diversity and Potential for Preventive Strategies in Nutritional Therapy.

The endogenous miRNAs of breast milk are the products of more than 1000 nonprotein-coding genes, giving rise to mature small regulatory molecules of 19-25 nucleotides. They are incorporated in macromolecular complexes, loaded on Argonaute proteins, sequestrated in exosomes and lipid complexes, or present in exfoliated cells of epithelial, endothelial, or immune origins. Their expression is dependent on the stage of lactation; however, their detection depends on progress in RNA sequencing and the reappraisal of the definition of small RNAs. Some miRNAs from plants are detected in breast milk, opening the possibility of the stimulation of immune cells from the allergy repertoire. Each miRNA harbors a seeding sequence, which targets mRNAs, gene promoters, or long noncoding RNAs. Their activities depend on their bioavailability. Efficient doses of miRNAs are estimated to be roughly 100 molecules in the cytoplasm of target cells from in vitro and in vivo experiments. Each miRNA is included in networks of stimulation/inhibition/sequestration, driving the expression of cellular phenotypes. Three types of stress applied during lactation to manipulate miRNA supply were explored using rodent offspring: a foster mother, a cafeteria diet, and early weaning. This review presents the main mature miRNAs described from current mothers' cohorts and their bioavailability in experimental models as well as studies assessing the potential of miR-26 or miR-320 miRNA families to alter offspring phenotypes.

Expanding the Natural History of SNORD118-Related Ribosomopathy: Hints from an Early-Diagnosed Patient with Leukoencephalopathy with Calcifications and Cysts and Overview of the Literature.

Leukoencephalopathy with calcifications and cysts (LCC) is a rare autosomal recessive disorder showing a pediatric or adult onset. First described in 1996 by Labrune and colleagues, it was only in 2016 that bi-allelic variants in a non-protein coding gene, SNORD118, were found as the cause for LCC, differentiating this syndrome from coats plus (CP). SNORD118 transcribes for a small nucleolar RNA, which is necessary for correct ribosome biogenesis, hence the classification of LCC among ribosomopathies. The syndrome is characterized by a combination of white matter hyperintensities, calcifications, and cysts on brain MRI with varying neurological signs. Corticosteroids, surgery, and recently bevacizumab, have been tried with unclear results since the natural history of the disease remains elusive. To date, 67 patients with a pediatric onset of disease have been described in the literature, with a clinical-radiological follow-up carried out in only eleven of them. We described the clinical-radiological follow-up from birth to almost five years of age of a late-preterm patient diagnosed with LCC and carried out a thorough overview of pediatric patients described in the literature. It is important to gather serial clinical-radiological data from other patients to depict the natural history of this disease, aiming to deeply depict genotype-phenotype correlations and make the role of new therapeutics clearer.

Temporal expression analysis of microRNAs and their target GRAS genes induced by osmotic stress in two contrasting wheat genotypes.

MicroRNAs (miRNAs) are important nonprotein-coding genes in plants which participate in almost all biological processes during abiotic and biotic stresses. Understanding how plants respond to various environmental conditions requires the identification of stress-related miRNAs. In recent years, there has been an increased interest in studying miRNA genes and gene expression. Drought is one of the common environmental stresses limiting plant growth and development. Stress-specific miRNAs and their GRAS gene targets were validated to understand the role of miRNAs in response to osmotic stress. In this study, expression patterns of the ten stress-responsive miRNAs involved in osmotic stress adaptation were examined in order to undertand the regulation behavior of abiotic stress and miRNAs in two contrasting wheat genotype C-306 (drought tolerant) and WL-711 (drought sensitive). Three miRNAs were discovered to be upregulated under stress, whereas seven miRNAs were showed to be down-regulated as a consequence of the study. In contrast to miRNA, it was also discovered that GRAS genes as their targets were up-regulated during osmotic stress. In addition, the expression level of miR159, miR408 along with their targets, TaGRAS178 and TaGRAS84 increased in response to osmotic stress. Nevertheless, miR408 is highly conserved miRNA that regulates plant growth, development and stress response. As a result, variation in the expression levels of studied miRNAs in the presence of target genes provides a plausible explanation for miRNA-based abiotic stress regulation. A regulatory network of miRNA and their targets revealed that fourteen miRNA interact with 55 GRAS targets from various subfamilies that contribute in the plant growth and development. These findings provide evidence for temporal and variety-specific differential regulation of miRNAs and their targets in wheat in response to osmotic shock, and they may aid in determining the potential.

Leveraging GWAS data derived from a large cooperative group trial to assess the risk of taxane-induced peripheral neuropathy (TIPN) in patients being treated for breast cancer: Part 2-functional implications of a SNP cluster associated with TIPN risk in patients being treated for breast cancer.

Using GWAS data derived from a large collaborative trial (ECOG-5103), we identified a cluster of 267 SNPs which predicted CIPN in treatment-naive patients as reported in Part 1 of this study. To assess the functional and pathological implications of this set, we identified collective gene signatures were and evaluated the informational value of those signatures in defining CIPN's pathogenesis. In Part 1, we analyzed GWAS data derived from ECOG-5103, first identifying those SNPs that were most strongly associated with CIPN using Fisher's ratio. After identifying those SNPs which differentiated CIPN-positive from CIPN-negative phenotypes, we ranked them in order of their discriminatory power to produce a cluster of SNPs which provided the highest predictive accuracy using leave-one-out cross validation (LOOCV). An uncertainty analysis was included. Using the best predictive SNP cluster, we performed gene attribution for each SNP using NCBI Phenotype Genotype Integrator and then assessed functionality by applying GeneAnalytics, Gene Set Enrichment Analysis, and PCViz. Using aggregate data derived from the GWAS, we identified a 267 SNP cluster which was associated with a CIPN+ phenotype with an accuracy of 96.1%. We could attribute 173 genes to the 267 SNP cluster. Six long intergenic non-protein coding genes were excluded. Ultimately, the functional analysis was based on 138 genes. Of the 17 pathways identified by Gene Analytics (GA) software, the irinotecan pharmacokinetic pathway had the highest score. Highly matching gene ontology attributions included flavone metabolic process, flavonoid glucuronidation, xenobiotic glucuronidation, nervous system development, UDP glycosyltransferase activity, retinoic acid binding, protein kinase C binding, and glucoronosyl transferase activity. Gene Set Enrichment Analysis (GSEA) GO terms identified neuron-associated genes as most significant (p = 5.45e-10). Consistent with the GA's output, flavone, and flavonoid associated terms, glucuronidation were noted as were GO terms associated with neurogenesis. The application of functional analyses to phenotype-associated SNP clusters provides an independent validation step in assessing the clinical meaningfulness of GWAS-derived data. Functional analyses following gene attribution of a CIPN-predictive SNP cluster identified pathways, gene ontology terms, and a network which were consistent with a neuropathic phenotype.

A comprehensive overview of miRNA targeting drought stress resistance in plants.

MicroRNAs (miRNAs) are essential nonprotein-coding genes. In a range of organisms, miRNAs has been reported to play an essential role in regulating gene expressions at post-transcriptional level. They participate in most of the stress responsive processes in plants. Drought is an ultimate abiotic stress that affects the crop production. Therefore understanding drought stress responses are essential to improve the production of agricultural crops. Throughout evolution, plants have developed their own defense systems to cope with the adversities of environmental stresses. Among defensive mechanisms include the regulations of gene expression by miRNAs. Drought stress regulates the expression of some of the functionally conserved miRNAs in different plants. The given properties of miRNAs provide an insight to genetic alterations and enhancing drought resistance in cereal crops. The current review gives a summary to regulatory mechanisms in plants as well as miRNAs response to drought stresses in cereal crops. Some possible approaches and guidelines for the exploitation of drought stress miRNA responses to improve cereal crops are also described.

Uterine fibroids: a look at the problem

Despite scientific progress, there is currently no sigle opinion about the cause of the occurrence and recurrence of uterine fibroids, but due to the high level of molecular medicine, progress is being made in the hormonal and molecular genetic mechanisms of initiation, formation and growth of the fibroisds. The issue of pathogenetic treatment and prevention of recurrence of uterine fibroids in reproductive age remains relevant. The aims of the review. The aim of this review is to summarize current data about microRNA in biology of uterine leiomyoma (LM). This information can improve our understanding of the broad molecular interaction of signaling pathways in the formation of LM, and further maintaining epigenetic regulation as an important mechanism in the pathogenesis of uterine leiomyoma. In leiomyomas, the expression of a number of non-proteincoding genes is altered, such as microRNAs (miRNAs), which target genes that code protein. Material and research methods. Original and review articles, book chapters in the PubMed database related to the study of the pathogenesis of uterine fibroids in the period from 2004 to 2022 were found and analyzed. Results and discussions. Based on an analytical review of the literature, it becomes obvious that as evidence should be considered: 1. Abnormal myometrial and fibroid stem cells show an increased response to estrogen and progesterone exposure, stimulating processes such as cell proliferation, inhibition of apoptosis, and extracellular matrix (ECM) formation. 2. A number of tumor suppressor genes are abnormally hypermethylated in the LM when compared to normal myometrium, genes that form and regulate collagen, and a subset of estrogen receptor genes. 3. Multiple studies using microarray analysis or sequencing have demonstrated the existence of dysregulation of a number of protein-coding genes involved in cell proliferation and apoptosis, which are critical for the growth and progression of uterine fibroids. There are no reliable evidence base and do not provide an opportunity for practical application of clinically significant risk factors, the possibility of mathematical prediction of the growth of uterine fibroids in women of reproductive age. Data on the effect of the expression of a number of microRNAs on the growth of uterine fibroids in vivo are rather contradictory. The epigenetic processes of regulation and pathogenesis of the growth of leiofibromyomas in reproductive age have not been fully studied and substantiated. There are practically no data on predicting the growth of uterine fibroids in reproductive age, which will allow us to assess the risk of growth and determine further treatment tactics. Conclusion. Further work on the identification of specific genes, miRNAs, that are involved in the pathogenesis of LM may inspire the creation of new pathogenetic treatments. Such treatment is especially relevant for those groups of patients of reproductive age for whom surgical treatment may be ineffective. Targeted treatment can also prevent the recurrence of uterine fibroids, hence the need for repeat surgery.

P875: ACTIVATION OF LNCRNA NEAT1 LEADS TO SURVIVAL ADVANTAGE OF MULTIPLE MYELOMA CELLS BY SUPPORTING A REGULATORY LOOP WITH DNA REPAIR PROTEINS: RATIONAL BASES FOR NEAT1 THERAPEUTIC TARGETING IN THE DISEASE

Background: Multiple myeloma (MM) is a fatal malignant proliferation of antibody-secreting bone marrow plasma cells characterized by marked genomic instability. Long non-coding RNA (lncRNA) represents the largest class of non-protein coding genes in the human genome. Their crucial role in solid tumor and hematological malignances, including MM, is well documented. NEAT1 is a highly expressed nuclear lncRNA, representing the core structural component of paraspeckle organelles. We previously demonstrated that NEAT1 silencing negatively regulates proliferation and viability of MM cells, both in vitro and in vivo, highlighting its pivotal role in DNA integrity maintenance, by regulating the homologous recombination. Despite the increasing information obtained by loss-of-function approaches, there is still a lack of information regarding possible oncogenic benefits acquired by MM cells upon NEAT1 overexpression. Aims: Taking advantage of the use of the CRISPR/Cas9 SAM genome editing approach, the present study aimed to shed light on the biological and molecular implication of NEAT1 overexpression in MM. Methods: We adopted the CRISPR/Cas9 Synergistic Activation Mediator editing system to transactivate NEAT1 in AMO-1 MM cell line. LNA-gapmeR antisense oligonucleotide technology was used to silence NEAT1 in MM cells by gymnotic delivery. Hypoxia was induced by culturing the cells into a modular incubator chamber flushed with a mixture of 1% O2, 5% CO2 and 94%N2 at 37 °C. NEAT1 expression was assessed by qRT-PCR and RNA-FISH. Cell cycle phases distribution and apoptotic cells percentage were assessed by flow cytometry. Clonogenic potential was evaluated by methylcellulose assay. Protein expression was investigated by WB and IF. Cell morphological analysis was performed after May-Grunwald Giemsa staining. Transcriptomic analysis was performed by RNA sequencing, following the TruSeq Stranded Total RNA protocol. Libraries with optimal quality and quantity were run on NextSeq 500. Statistical significance was determined by Student t test analysis; differences were considered significant when P-value was <0.05. Results: We performed a transcriptomic analysis of AMO-1 cells either transactivated or silenced for NEAT1. Our data revealed a NEAT1 pivotal role in the maintenance of DNA integrity, showing a significant deregulation of almost all the crucial cellular DNA repair mechanisms for both single and double strand breaks. In particular, we found that NEAT1 transactivation affects DNA repair mechanisms through the activation of a molecular axis including two fundamental kinase proteins, i.e. ATM and DNA-PKcs, and the direct target pRPA32. Furthermore, our data strongly confirmed the activation of this NEAT1-orchestrated molecular axis whether MM cells are exposed to nutrient starvation and hypoxia, suggesting its implication in conferring oncogenic and pro-survival properties to MM cells. The disruption of this oncogenic loop leads to MM cell death and the loss of pro survival advantages, thus suggesting that NEAT1 should be considered a crucial factor for MM cells survival. Summary/Conclusion: Overall, we provided novel important insights into the role of NEAT1 in MM pathobiology, demonstrating that its deregulation strongly correlates with adaptation to stress condition, often associated with late stages of the disease. Taken together, our results suggest that NEAT1 could represent Achilles’ heel for MM cells and should be considered a potential therapeutic target for MM treatment.

Abstract 3278: Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer

Abstract Among the hallmarks of non-small cell lung cancer (NSCLC) is cancer immunoediting. Immunoediting results when cancer cells evade immune recognition via positive selection for cells that do not present immunogenic antigens recognized by CD8+ T cells. An epigenetic therapy-based approach has been developed in our lab to enhance expression of tumor antigens using combinatorial DNA methyltransferase inhibition (DNMTi) paired with histone deacetylase inhibition (HDACi). However, durable responses from the application of epigenetic therapy alone or in combination with other therapies have remained elusive in NSCLC. This may be because enhanced proliferation and infiltration of tumor-associated antigen specific CD8+ T cells are necessary to recognize epigenetically induced antigens to halt disease progression. In the current study, we identified epigenetic therapy inducible self-antigens that can potentially be used to activate and guide the differentiation of CD8+ T cells specific to NSCLC tumor antigens. The identification of these candidates was derived from analyses of paired RNAseq data from NSCLC patients treated with combination epigenetic therapy. From these analyses, differentially expressed transcripts selected from total transcriptome data were filtered to remove non-protein coding genes and genes with detectable expression in normal tissues as indicated by GTEx. HLA binding affinity (HLA-A*0201) of selected targets was defined by NetMHC 4.0 artificial neural network-based prediction to identify strong binding peptide candidates. The combined results of these data filtering facilitated the identification of 34 epigenetic therapy induced genes and associated peptides (9mers) with predicted high binding affinity for HLA-A*0201. The predicted peptides of target genes were selected for single peptide pulsing in allogenic dendritic cells to generate antigen specific CD8+ T cells for TCR sequencing (TCRseq). TCRseq libraries derived from peptide stimulation experiments revealed distinct V and J gene frequencies and emergence of antigen specific clonotypes, which suggest clonal expansion. To validate whether epigenetic therapy could induce target genes expression in an independent experimental model, in vitro human NSCLC cell lines were treated with DNMTi and HDACi to mimic the clinical drug paradigm and transcriptional augmentation was assessed by qRT-PCR. In this orthogonal system, we validated eight target genes that were induced by epigenetic therapy in vitro. Altogether, these data suggest that a conserved subset of genes, whose derived proteins are predicted to be presented in class I MHC and act as T cell epitopes, is induced by combination epigenetic treatment. Our studies provide a basis for the development of novel and personalized strategies to improve outcomes and quality of life for more patients with NSCLC. Citation Format: Julia An, Valsamo Anagnostou, Kristen A. Marrone, Victor E. Velculescu, Julie R. Brahmer, Stephen B. Baylin, Michael J. Topper. Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3278.

Effects of a High Glycemic Diet and Soluble Epoxide Hydrolase Inhibitor on Hippocampal Microvascular Function, and Multigenomic Modifications

ObjectivesDiet is a modifiable risk factor for cardiovascular disease (CVD) and dementia, yet relatively little is known about the effect of a high glycemic diet (HGD) on the brain microvasculature. The objective of our study was to determine the multigenomic effects of a HGD on hippocampal microvessels, and to determine if a soluble epoxide hydrolase (sEH) inhibitor (sEHI), known to be vasculoprotective and anti-inflammatory, modulates these effects.MethodsWild type male and female mice were fed a low glycemic diet (LGD, 12% sucrose/weight) or a HGD (34% sucrose/weight) with/without the sEHI, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), for 12 wks. Brain hippocampal microvascular gene expression was assessed by microarray and data analyzed using a multiomic approach for differential expression of protein and non-protein coding genes, gene networks, functional pathways, and transcription factors.ResultsGlobal hippocampal microvascular gene expression was fundamentally different for mice fed the HGD vs the LGD. The HGD modulated expression of genes involved in cell signaling, neurodegeneration, metabolism, and cell adhesion/inflammation/oxidation effects reversible by t-AUCB and hence sEH inhibitor correlated with protection against Alzheimer's dementia. However, in female, HGD had very minor genomic modifications, while sEHI presented significant effect in female mice on LGD.ConclusionsOurs is the first study to demonstrate that high dietary glycemia contributes to brain hippocampal microvascular inflammation, through sEH, effects that were sex-dependant.Funding SourcesThis work was supported by an award from the Richard A. and Nora Eccles Foundation (A20-0111), the Richard A. and Nora Eccles Harrison Endowed Chair in Diabetes Research (J.C.R.), and the Frances Lazda Endowed Chair in Women's Cardiovascular Medicine (A.C.V).

Natural antisense RNA Foxk1-AS promotes myogenic differentiation by inhibiting Foxk1 activity

BackgroundNatural antisense RNAs are RNA molecules that are transcribed from the opposite strand of either protein-coding or non-protein coding genes and have the ability to regulate the expression of their sense gene or several related genes. However, the roles of natural antisense RNAs in the maintenance and myogenesis of muscle stem cells remain largely unexamined.MethodsWe analysed myoblast differentiation and regeneration by overexpression and knockdown of Foxk1-AS using lentivirus and adeno-associated virus infection in C2C12 cells and damaged muscle tissues. Muscle injury was induced by BaCl2 and the regeneration and repair of damaged muscle tissues was assessed by haematoxylin–eosin staining and quantitative real-time PCR. The expression of myogenic differentiation-related genes was verified via quantitative real-time PCR, Western blotting and immunofluorescence staining.ResultsWe identified a novel natural antisense RNA, Foxk1-AS, which is transcribed from the opposite strand of Foxk1 DNA and completely incorporated in the 3′ UTR of Foxk1. Foxk1-AS targets Foxk1 and functions as a regulator of myogenesis. Overexpression of Foxk1-AS strongly inhibited the expression of Foxk1 in C2C12 cells and in tibialis anterior muscle tissue and promoted myoblast differentiation and the regeneration of muscle fibres damaged by BaCl2. Furthermore, overexpression of Foxk1-AS promoted the expression of Mef2c, which is an important transcription factor in the control of muscle gene expression and is negatively regulated by Foxk1.ConclusionThe results indicated that Foxk1-AS represses Foxk1, thereby rescuing Mef2c activity and promoting myogenic differentiation of C2C12 cells and regeneration of damaged muscle fibres.DfdN9uwC-a513-TBDoKHuCVideo

Association of PSORS1C3, CARD14 and TLR4 genotypes and haplotypes with psoriasis susceptibility.

Psoriasis is a common chronic, immune-mediated inflammatory disease of the skin. PSORS1C3 is a non-protein coding gene, of which the RNA transcript is found in psoriatic patients. CARD14 is mainly expressed in epidermal keratinocytes. TLR4 is a transmembrane protein to recognize microbial antigens. Our study aimed to assess the relationship among PSORS1C3, CARD14 and TLR4 polymorphisms, inflammatory expression and psoriasis susceptibility. To the end, 71 patients with psoriasis and 46 healthy individuals with the well-characterized clinical profiles were enrolled. Gene polymorphisms were determined by Sanger DNA sequencing and secretion of cytokines by ELISA. As a result, genetic analysis of PSORS1C3 gene identified nine SNPs and three haplotype blocks. Sequencing of the CARD14 gene determined eight SNPs and one haplotype block. Sequencing of TLR4 gene identified nine SNPs, in which a SNP rs1018673641 was found to exert deleterious effect. The linkage disequilibrium analysis showed that seven variants in PSORS1C3 gene and three SNPs in CARD14 gene were in tightly linked. More importantly, a significant association between IL-6 level and rs1018673641 AT genotype in TLR4 gene was detected in psoriatic patients. In conclusion, the PSORS1C3, CARD14 and TLR4 polymorphisms and haplotypes may be correlated with risk of suffering psoriasis and the IL-6-mediated chronic inflammation in psoriasis could be partially regulated by the TLR4 functional variant.

Comprehensive Mapping of the Cell Response to Borrelia bavariensis in the Brain Microvascular Endothelial Cells in vitro Using RNA-Seq.

Borrelia bavariensis can invade the central nervous system (CNS) by crossing the blood-brain barrier (BBB). It is predicted that B. bavariensis evokes numerous signaling cascades in the human brain microvascular endothelial cells (hBMECs) and exploits them to traverse across the BBB. The complete picture of signaling events in hBMECs induced by B. bavariensis remains uncovered. Using RNA sequencing, we mapped 11,398 genes and identified 295 differentially expressed genes (DEGs, 251 upregulated genes and 44 downregulated genes) in B. bavariensis challenged hBMECs. The results obtained from RNA-seq were validated with qPCR. Gene ontology analysis revealed the participation of DEGs in a number of biological processes like cell communication, organization of the extracellular matrix, vesicle-mediated transport, cell response triggered by pattern recognition receptors, antigen processing via MHC class I, cellular stress, metabolism, signal transduction, etc. The expression of several non-protein coding genes was also evoked. In this manuscript, we discuss in detail the correlation between several signaling cascades elicited and the translocation of BBB by B. bavariensis. The data revealed here may contribute to a better understanding of the mechanisms employed by B. bavariensis to cross the BBB.

Markedly reduced myocardial expression of γ-protocadherins and long non-coding RNAs in patients with heart disease

BackgroundAdverse cardiac remodeling and tissue damage following heart disease is strongly associated with chronic low grade inflammation. The mechanisms underlying persisting inflammatory signals are not fully understood, but may involve defective and/or non-responsive transcriptional and post-transcriptional regulatory mechanisms. In the current study, we aimed to identify novel mediators and pathways involved in processes associated with inflammation in the development and maintenance of cardiac disease. Methods and resultsWe performed RNA sequencing analysis of cardiac tissue from patients undergoing coronary artery bypass grafting (CABG) or aortic valve replacement (AVR) and compared with control tissue from multi-organ donors.Our results confirmed previous findings of a marked upregulated inflammatory state, but more importantly, we found pronounced reduction of non-protein coding genes, particularly long non-coding RNAs (lncRNA), including several lncRNAs known to be associated with inflammation and/or cardiovascular disease. In addition, Gene Set Enrichment Analysis revealed markedly downregulated microRNA pathways, resulting in aberrant expression of other genes, particularly γ-protocadherins. ConclusionsOur data suggest that aberrant expression of non-coding gene regulators comprise crucial keys in the progression of heart disease, and may be pivotal for chronic low grade inflammation associated with cardiac dysfunction. By unmasking atypical γ-protocadherin expression as a prospective genetic biomarker of myocardial dysfunction, our study provides new insight into the complex molecular framework of heart disease. Creating new approaches to modify non-coding gene regulators, such as those identified in the current study, may define novel strategies to shift γ-protocadherin expression, thereby normalizing part of the molecular architecture associated with heart disease.

Transcriptional Landscapes of Long Non-coding RNAs and Alternative Splicing in Pyricularia oryzae Revealed by RNA-Seq

Pyricularia oryzae causes the rice blast, which is one of the most devastating crop diseases worldwide, and is a model fungal pathogen widely used for dissecting the molecular mechanisms underlying fungal virulence/pathogenicity. Although the whole genome sequence of P. oryzae is publicly available, its current transcriptomes remain incomplete, lacking the information on non-protein coding genes and alternative splicing. Here, we performed and analyzed RNA-Seq of conidia and hyphae, resulting in the identification of 3,374 novel genes. Interestingly, the vast majority of these novel genes likely transcribed long non-coding RNAs (lncRNAs), and most of them were localized in the intergenic regions. Notably, their expressions were concomitant with the transcription of neighboring genes thereof in conidia and hyphae. In addition, 2,358 genes were found to undergo alternative splicing events. Furthermore, we exemplified that a lncRNA was important for hyphal growth likely by regulating the neighboring protein-coding gene and that alternative splicing of the transcription factor gene CON7 was required for appressorium formation. In summary, results from this study indicate that lncRNA transcripts and alternative splicing events are two important mechanisms for regulating the expression of genes important for conidiation, hyphal growth, and pathogenesis, and provide new insights into transcriptomes and gene regulation in the rice blast fungus.

MicroRNA-1205 Regulation of FRYL in Prostate Cancer.

High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (CRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). The 8q24 chromosomal locus is a region of very high PCa susceptibility that carries genetic variants associated with high risk of PCa incidence. This region also carries frequent amplifications of the PVT1 gene, a non-protein coding gene that encodes a cluster of microRNAs including, microRNA-1205 (miR-1205), which are largely understudied. Herein, we demonstrate that miR-1205 is underexpressed in PCa cells and tissues and suppresses CRPC tumors in vivo. To characterize the molecular pathway, we identified and validated fry-like (FRYL) as a direct molecular target of miR-1205 and observed its overexpression in PCa cells and tissues. FRYL is predicted to regulate dendritic branching, which led to the investigation of FRYL in neuroendocrine PCa (NEPC). Resistance toward ADT leads to the progression of treatment related NEPC often characterized by PCa neuroendocrine differentiation (NED), however, this mechanism is poorly understood. Underexpression of miR-1205 is observed when NED is induced in vitro and inhibition of miR-1205 leads to increased expression of NED markers. However, while FRYL is overexpressed during NED, FRYL knockdown did not reduce NED, therefore revealing that miR-1205 induces NED independently of FRYL.

Large-Scale Transcriptomics-Driven Approach Revealed Overexpression of CRNDE as a Poor Survival Prognosis Biomarker in Glioblastoma.

Simple SummaryMost glioblastoma patients succumb to the disease within 12 to 18 months, and only 9% are alive 2 years after diagnosis. Even with extensive research, the life expectancy of glioblastoma patients has not changed in decades. We aimed to identify differences in the transcriptomic profiles of glioblastoma patients with long and short survival. With large-scale transcriptomic analysis, we examined information from publicly available datasets (TCGA and CGGA) in combination with FFPE patient tissue samples. We identified one gene, the long noncoding RNA CRNDE, whose overexpression is directly correlated with poor patient survival. Therefore, we suggest its further confirmation as a negative prognostic glioblastoma biomarker. Glioblastoma management still lacks suitable diagnostic, predictive, and prognostic biomarkers for early disease diagnosis, and treatment follow-up. We believe our findings can serve as the basis for identification of new and potential suitable disease biomarkers by looking beyond the classical molecules (DNA, RNA, and proteins) into the noncoding genome.Glioblastoma is the most common and malignant brain malignancy worldwide, with a 10-year survival of only 0.7%. Aggressive multimodal treatment is not enough to increase life expectancy and provide good quality of life for glioblastoma patients. In addition, despite decades of research, there are no established biomarkers for early disease diagnosis and monitoring of patient response to treatment. High throughput sequencing technologies allow for the identification of unique molecules from large clinically annotated datasets. Thus, the aim of our study was to identify significant molecular changes between short- and long-term glioblastoma survivors by transcriptome RNA sequencing profiling, followed by differential pathway-activation-level analysis. We used data from the publicly available repositories The Cancer Genome Atlas (TCGA; number of annotated cases = 135) and Chinese Glioma Genome Atlas (CGGA; number of annotated cases = 218), and experimental clinically annotated glioblastoma tissue samples from the Institute of Pathology, Faculty of Medicine in Ljubljana corresponding to 2–58 months overall survival (n = 16). We found one differential gene for long noncoding RNA CRNDE whose overexpression showed correlation to poor patient OS. Moreover, we identified overlapping sets of congruently regulated differential genes involved in cell growth, division, and migration, structure and dynamics of extracellular matrix, DNA methylation, and regulation through noncoding RNAs. Gene ontology analysis can provide additional information about the function of protein- and nonprotein-coding genes of interest and the processes in which they are involved. In the future, this can shape the design of more targeted therapeutic approaches.

Abstract 2365: Intracellular interaction of downstream molecular mediators of miR-1207-3p in prostate cancer cells

Abstract Prostate cancer (PCa) is the second most common cancer diagnosed among men in the United States. Human chromosome 8q24 is the most important PCa susceptibility locus. This region contains the MYC oncogene, which is involved in early PCa initiation. Downstream to MYC is the PVT1 gene, which is often amplified in PCa. PVT1 is a non-protein coding gene that encodes six annotated microRNAs (miRNAs), including miR-1207-3p. Our laboratory has previously demonstrated miR1207-3p to be a significant modulator in PCa and revealed that miR-1207-3p is significantly underexpressed in PCa cells and histologically confirmed PCa tissues compared to normal prostatic cells and tissues. We have also shown that miR-1207-3p has tumor suppressive activity in vivo. Moreover, we discovered that miR-1207-3p directly targets fibronectin type III domain containing 1 (FNDC1), which was found to be overexpressed in PCa cells and there is a concomitant overexpression of (FN1)/androgen receptor (AR)/c-MYC. In an effort to better understand the role of FNDC1/FN1/AR/c-MYC in PCa progression we examined the interaction between FNDC1/FN1/AR/c-MYC and found direct physical interaction through coimmunoprecipitation. We also examined the spatial localization of the FNDC1/FN1/AR/c-MYC pathway by performing immunofluorescence staining in PCa cells. Single staining analysis revealed that FNDC1 and c-MYC localize to the nucleus and cytoplasm while AR localizes to the nucleus and FN1 localizes to the cytoplasm in PCa cells. However, in normal prostate epithelial cells FNDC1, AR and c-MYC localize to the nucleus whereas FN1 localize to the cytoplasm. These results suggest that the underexpression of miR1207-3p may be affecting the localization of FNDC1 to cytoplasm and thus promoting interaction between FNDC1/FN1/AR/c-MYC. Future studies include examining the effect of miR-1207 on the spatial organization and interactions of the FNDC1/FN1/AR/c-MYC pathway through immunofluorescence and co-immunoprecipitation assays. Thus, understanding this molecular interaction could reveal interesting insight into the biology of microRNA-1207-3p regulation in PCa. Citation Format: Priyanka Ghosh, Oloronseun Ogunwobi. Intracellular interaction of downstream molecular mediators of miR-1207-3p in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2365.