The effect of lncRNA GAS5 on lens epithelial cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE317222 | GEO | 2026/01/26
REPOSITORIES: GEO
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Project description:Long non-coding RNAs (lncRNAs) are recently characterized players that are involved in the regulatory circuitry of self-renewal in human embryonic stem cells (hESCs). However, the specific roles of lncRNAs in this circuitry are poorly understood. Here, we determined that growth-arrest-specific transcript 5 (GAS5), which is a known tumor suppressor and growth arrest gene, is abundantly expressed in the cytoplasm of hESCs and essential for hESC self-renewal. GAS5 depletion in hESCs significantly impaired their pluripotency and self-renewal ability, whereas GAS5 overexpression in hESCs accelerated the cell cycle, enhanced their colony formation ability and increased pluripotency marker expression. By RNA sequencing and bioinformatics analysis, we determined that GAS5 activates NODAL-SMAD2/3 signaling by sustaining the expression of NODAL, which plays a key role in hESC self-renewal but not in somatic cell growth. Further studies indicated that GAS5 functions as a competing endogenous RNA (ceRNA) to protect NODAL mRNA against degradation and that GAS5 transcription is directly controlled by the core pluripotency transcriptional factors (TFs). Taken together, we suggest that the core TFs, GAS5 and NODAL-SMAD2/3 form a feed-forward loop to maintain the hESC self-renewal process. These findings are specific to ESCs and did not occur in the somatic cell lines we tested; therefore, our findings also provide evidence that the functions of lncRNAs vary in different biological contexts. We analyzed long non-coding RNAs in two hESC cell lines (X-01 and H1), and found GAS5 is highly expressed and functional in maintaining hESC self-renewal. We generate stable overexpressed or knockdown hESC cell lines using lentiviral approach. We transfected cells initialy after passage, and lentiviruses are added with daily medium change for three days (at a final concentration of 10^5 IU/ml). Puromycin is added for selection and supplied with daily medium change. Stable cell lines are established after two passages and verified under fluorescence scope. Total RNAs and miRNAs are extracted separately of all three cell lines (LV-NC, LV-GAS5 and LV-shGAS5) and put to sequencing.
2016-03-17 | E-GEOD-66993 | biostudies-arrayexpress
Project description:Long non-coding RNAs (lncRNAs) are recently characterized players that are involved in the regulatory circuitry of self-renewal in human embryonic stem cells (hESCs). However, the specific roles of lncRNAs in this circuitry are poorly understood. Here, we determined that growth-arrest-specific transcript 5 (GAS5), which is a known tumor suppressor and growth arrest gene, is abundantly expressed in the cytoplasm of hESCs and essential for hESC self-renewal. GAS5 depletion in hESCs significantly impaired their pluripotency and self-renewal ability, whereas GAS5 overexpression in hESCs accelerated the cell cycle, enhanced their colony formation ability and increased pluripotency marker expression. By RNA sequencing and bioinformatics analysis, we determined that GAS5 activates NODAL-SMAD2/3 signaling by sustaining the expression of NODAL, which plays a key role in hESC self-renewal but not in somatic cell growth. Further studies indicated that GAS5 functions as a competing endogenous RNA (ceRNA) to protect NODAL mRNA against degradation and that GAS5 transcription is directly controlled by the core pluripotency transcriptional factors (TFs). Taken together, we suggest that the core TFs, GAS5 and NODAL-SMAD2/3 form a feed-forward loop to maintain the hESC self-renewal process. These findings are specific to ESCs and did not occur in the somatic cell lines we tested; therefore, our findings also provide evidence that the functions of lncRNAs vary in different biological contexts.
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Project description: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.
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Project description:In this study monoclonal cell lines carrying mutations in the small nucleolar RNA host-gene GAS5 were obtained. Next, Poly(A) RNA-seq and Small RNA-seq analyses of obtained cell lines were performed on an Illumina NextSeq 500 platform. Poly(A) RNA-seq libraries was constructed on polyA mRNA-enriched fraction. Small RNA-seq libraries was constructed on small RNA fraction (<200 nucleotide length). The reads were aligned via STAR to the human genome (GRCh37). The comparison of the mRNA levels of genes and small RNA levels in RNA-Seq experiments was carried out using CuffDiff tool. Sashimi plots for RNA-Seq analyses of isoform expression were generated by IGV. It was shown that specific mutations in SNORD74 led to the downregulation of all GAS5-encoded SNORDs and GAS5 lncRNA. Obtained results demonstrate the SNORD-dependent manner of the GAS5 maturation.
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