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Functional network of the long non-coding RNA growth arrest specific transcript 5 (GAS5) and its interacting proteins in senescence

ABSTRACT: Increasing studies show that long non-coding RNAs (lncRNAs) play essential roles in various fundamental 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 RT-qPCR revealed that GAS5 had a significantly higher expression level in old mouse brain hippocampus region than the young one. Cellular fractionation using hippocampus-derived HT22 cell line confirmed its nucleoplasm and cytoplasm subcellular localization. We then performed overexpression (OE) or knockdown (KD) of GAS5 in HT22 cell line and found that GAS5 inhibits cell cycle progression and promotes cell apoptosis. RNA-Seq analysis of GAS5-knockdown HT22 cell identified differential expressed genes related to cell proliferation (e.g., DNA replication and nucleosome assembly biological processes). RNA pull-down assay using mouse brain hippocampus tissues revealed that potential GAS5 interacting proteins can be enriched into several KEGG pathways and some of them are involved in senescence associated diseases such as Parkinson and Alzheimer's Disease. These results contribute to better understand 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 reference for developing diagnostic and clinic biomarkers of GAS5 in senescence and brain aging.

ORGANISM(S): Mus musculus

PROVIDER: GSE163237 | GEO | 2020/12/16

REPOSITORIES: GEO

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Project description:Lung cancer is one of the most common and fatal cancer worldwide. There are two major types of lung cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Recently, gefitinib, a small molecule, which target tyrosine kinase, has been regarded as the first-line treatment in NSCLC patients. However, several patients have been observed tumor recurrence and eventually developed progressive outcomes after target therapy. Thus, an effective therapeutic approach need to be explored. Here, we found that ectopically expressed ATP synthase on plasma membrane exhibited gefitinib-resistance properties in lung cancer cell lines. Furthermore, we unraveled that citreoviridin, an ectopic ATP synthase inhibitor, suppressed the abilities of both proliferation and colony formation in lung cancer cell lines. To elucidate the comprehensive mechanism regulated by citreoviridin, we performed microarray analysis. The results indicated that not only mRNAs but also long non-coding RNAs (lncRNAs) are involved in citreoviridin-treated cell death. One of the well-known lncRNAs, growth arrest-specific transcript (GAS5), was robustly upregulated after citreoviridin treatment. In order to investigate the upstream modulator of GAS5, we utilized chromatin immunoprecipitation (ChIP) assay and revealed that E2F transcription factor 1 (E2F1) could bind to the promoter of GAS5. Consistently, both microarray and qPCR data showed the expression level of E2F1 was negatively correlated to GAS5 after citreoviridin treatment. The evidence suggests that E2F1 might be a potential repressor of GAS5. Furthermore, combining microarray and Gene Set Enrichment Analysis (GSEA) analysis as well as qPCR demonstrated that p53 pathway was activated. To further realize the GAS5-p53 regulating network, RNA-protein pull-down assay followed by LC-MS/MS will be utilized to dissect the GAS5-interacting proteomic profiling.From the results, we identified 107 GAS5-interacting proteins in common from A549 and H1975 cell lines. Our proteomics experiments identified topoisomerase 2-alpha (TOP2A) as the key protein involved in the citreoviridin-regulated gefitinib-resistance pathway, therefore, we picked out TOP2A for further study. Additionally, we further validated the interaction between TOP2A and GAS5 by western blot and RNA immunoprecipitation (RIP). Taken together, this study suggests targeting E2F1/GAS5/p53 axis is a potential therapeutic strategy for gefitinib-resistant lung cancer.

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Functional network of the long non-coding RNA growth arrest specific transcript 5 (GAS5) and its interacting proteins in senescence

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