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Functional Network of the Long Non-coding RNA Growth Arrest-Specific Transcript 5 and Its Interacting Proteins in Senescence.

ABSTRACT: Increasing studies show that long non-coding RNAs (lncRNAs) play essential roles in various fundamental biological processes. Long non-coding RNA growth arrest-specific transcript 5 (GAS5) showed differential expressions between young and old mouse brains in our previous RNA-Seq data, suggesting its potential role in senescence and brain aging. Examination using quantitative reverse transcription-polymerase chain reaction revealed that GAS5 had a significantly higher expression level in the old mouse brain hippocampus region than the young one. Cellular fractionation using hippocampus-derived HT22 cell line confirmed its nucleoplasm and cytoplasm subcellular localization. Overexpression or knockdown of GAS5 in HT22 cell line revealed that GAS5 inhibits cell cycle progression and promotes cell apoptosis. RNA-Seq analysis of GAS5-knockdown HT22 cells identified differentially expressed genes related to cell proliferation (e.g., DNA replication and nucleosome assembly biological processes). RNA pull-down assay using mouse brain hippocampus tissues showed that potential GAS5 interacting proteins could be enriched into several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and some of them are involved in senescence-associated diseases such as Parkinson's and Alzheimer's diseases. These results contribute to understand better the underlying functional network of GAS5 and its interacting proteins in senescence at brain tissue and brain-derived cell line levels. Our study may also provide a reference for developing diagnostic and clinic biomarkers of GAS5 in senescence and brain aging.

SUBMITTER: Wang S

PROVIDER: S-EPMC7987947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe diagnosis of severe asthma (SA) is difficult due to a necessary long-term treatment history currently, while there are few studies on biomarkers in the diagnosis of SA. Long non-coding RNA (lncRNA) growth arrest specific-5 (GAS5) has the potential of playing this role because its binding with glucocorticoid receptor (GR). The purpose of this article is to explore the possibility of lncRNA GAS5 acting as a biomarker for early diagnosis of severe asthma (SA).MethodsPeripheral blood was obtained from healthy volunteers, patients with non-severe asthma (nSA) and SA, and peripheral blood mononuclear cells (PBMCs) were separated. Twenty-four female BALB/c mice (aged 6 weeks) were randomly and averagely divided into 3 groups, i.e., control group, asthma group and dexamethasone group. The mice were sensitized and challenged with ovalbumin (OVA) and lipopolysaccharide (LPS) to establish a murine model of steroid-insensitive asthma. Human bronchial epithelial cells (HBECs) were cultured, transfected with miR-9 mimics, JNK1 inhibitor and treated with interleukin (IL)-2 + IL-4 and dexamethasone. Western blot was used to detect glucocorticoid receptor phosphorylation at serine 226 (GRser226), and quantitative real-time PCR was used to detect GAS5 level.ResultsThe level of GAS5 in PBMCs from nSA group elevated 20-fold higher after dexamethasone treatment in vitro, while it reduced 15-fold lower in SA group (P<0.001). The expression of GRser226 in PBMCs from SA group was significantly higher than that from control group and nSA group after dexamethasone treatment (P<0.001). In the lung tissue of mice, the GAS5 level of dexamethasone group was lower than that of asthma group (P<0.001) and control group (P<0.05). Both treatment with IL-2 + IL-4 and transfection of miR-9 mimics could increase the expression of GRser226 in HBECs (P<0.001). The GAS5 level in HBECs after IL-2 + IL-4 + Dexamethasone treatment was lower than that in HBECs only treated with IL-2 + IL-4 (P<0.001). Similarly, dexamethasone treatment also decreased the level of GAS5 in HBECs transfected with miR-9 mimics (P<0.05). Moreover, transfecting with JNK1 inhibitor could reverse the expression of GAS5 in HBECs transfected with miR-9 mimics and treated with dexamethasone. However, the level of GAS5 in HBECs interfered with IL-2 + IL-4 + Dexamethasone was not affected by JNK1 inhibitor.ConclusionsThe expression of GAS5 is different in PBMCs between nSA and SA, and is affected by glucocorticoids treatment, which is due to GRser226 phosphorylation. GAS5 can be used as a potential biomarker for diagnosis of severe asthma by comparing GAS5 level in PBMCs from patients before and after glucocorticoids treatment in vitro.

Project description:Long non-coding RNA (lncRNA) genes encode non-messenger RNAs that lack open reading frames (ORFs) longer than 300 nucleotides, lack evolutionary conservation in their shorter ORFs, and do not belong to any classical non-coding RNA category. LncRNA genes equal, or exceed in number, protein-coding genes in mammalian genomes. Most mammalian genomes harbor ~20,000 protein-coding genes that give rise to conventional messenger RNA (mRNA) transcripts. These coding genes exhibit sweeping evolutionary conservation in their ORFs. LncRNAs function via different mechanisms, including but not limited to: (1) serving as "enhancer" RNAs regulating nearby coding genes in cis; (2) functioning as scaffolds to create ribonucleoprotein (RNP) complexes; (3) serving as sponges for microRNAs; (4) acting as ribo-mimics of consensus transcription factors binding sites in genomic DNA; (5) hybridizing to other nucleic acids (mRNAs and genomic DNA); and, rarely, (6) as templates encoding small open reading frames (smORFs) that may encode short proteins. Any given lncRNA may have more than one of these functions. This review focuses on one fascinating case-the growth-arrest-specific (GAS)-5 gene, encoding a complicated repertoire of alternatively-spliced lncRNA isoforms. GAS5 is also a host gene of numerous small nucleolar (sno) RNAs, which are processed from its introns. Publications about this lncRNA date back over three decades, covering its role in cell proliferation, cell differentiation, and cancer. The GAS5 story has drawn in contributions from prominent molecular geneticists who attempted to define its tumor suppressor function in mechanistic terms. The evidence suggests that rodent Gas5 and human GAS5 functions may be different, despite the conserved multi-exonic architecture featuring intronic snoRNAs, and positional conservation on syntenic chromosomal regions indicating that the rodent Gas5 gene is the true ortholog of the GAS5 gene in man and other apes. There is no single answer to the molecular mechanism of GAS5 action. Our goal here is to summarize competing, not mutually exclusive, mechanistic explanations of GAS5 function that have compelling experimental support.

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Functional Network of the Long Non-coding RNA Growth Arrest-Specific Transcript 5 and Its Interacting Proteins in Senescence.

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