Pvt1 silencing in C2C12 cell cultures
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ABSTRACT:
PROVIDER: PRJNA449371 | ENA |
REPOSITORIES: ENA
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Project description:In order to understand the role of lncRNA Pvt1 in skeletal muscle physiopathology we silenced this transcript in-vitro, using C2C12 cell cultures, and in-vivo, in leg muscles of CD1 wild-type and denervated mice.
2019-01-07 | GSE112881 | GEO
Project description:Long non-coding RNAs (lncRNAs) are emerging as important players in the regulation of several aspects of cellular biology. For a better comprehension of their function it is fundamental to determine their expression in single cells, to identify their subcellular localization and eventually DNA interacting regions. In fact, lncRNAs present a cell-type specific expression and different functions depending on their subcellular localization. C2C12 cells are a model to study muscle pathophysiology and differentiation. We used this model to evaluate Pvt1-DNA interacting regions. We demonstrated that the lncRNA Pvt1, early activated during muscle atrophy, regulate the transcription of DNA and influence mitochondrial respiration and morphology ultimately impinging mito/autophagy and myofiber size in vivo. We evidenced that the long non-coding RNA Pvt1 is highly expressed during skeletal muscle atrophy. Moreover, it is preferentially expressed in fast contracting myofibers. By using high throughput techniques and Fluorescent In Situ Hybridization we evidenced that Pvt1 localize inside the nucleai of myofibers and C2C12 cells. C2C12 cells are widely used as in vitro model to study different aspects of muscle biology such as development, differentiation and metabolism. Because Pvt1 nuclear localization we checked its capacity to interact with DNA by Chromatin isolation by RNA purification (ChIRP).
2020-04-29 | GSE112977 | GEO
Project description:Each gene exists as two copies in the same nucleus of mammalian cells. Although most genes are equivalently controlled on both alleles, Random Monoallelic Expressing (RME) genes stably maintain the expression from only one of two alleles, but the mechanisms and functional consequences of RME remains unclear. Here we performed allele-specific RNA-seq on over 100 neural progenitor cell (NPC) clonal lines to reveal the extent of RME for genes across clonal lines. Interestingly out of 297 autosomal RME genes, Pvt1, a well-studied oncogenic long non-coding RNA, is monoallelically expressed with a skewed genetic bias towards the Castaneous allele in F1-hybrid NPCs. In the absence of genetic differences, allelic tracking by gene editing reveals Pvt1 undergoes balanced RME that is maintained across somatic cell generations. Monoallelic expression is maintained by a poised Pvt1 promoter with bivalent active histone marks, H3K4me3 and H3K27ac, and silencing histone marks, H3K4me3 and H3K27me3, demonstrating its ability to switch transcriptional states depending on the cellular context. Leveraging our large RNA-seq data set and the genetic skew, we use differential gene expression analysis on clonal lines based on their Pvt1 allelic status to reveal a transcription factor, Tfap2a, with differential binding at the Pvt1 promoter, providing a mechanism for the initiation and allelic choice for RME genes. Additionally, we demonstrate monoallelic Pvt1 expression results in an increase in Pvt1 expression leading to a growth advantage relative to Pvt1 biallelic expressing clonal lines These findings provide an example of how genetic differences can skew a stochastic process which results in an epigenetic phenomenon with a phenotypic consequence in early development.
2025-09-26 | GSE287458 | GEO
Project description:lncRNA PVT1 is an emerging lncRNA of significance in cancer due to alterations in both the RNA and genomic locus in multiple cancers and its established relationship to the oncogene MYC. Several recent studies have documented potential important roles for the lncRNA in ovarian cancer. Herein RNA sequencing was performed to determine the impact of PVT1 on global gene expression by performing RNA sequencing in SK-OV3 cells after silencing PVT1 (siPVT1) of cells grown upon transient knockdown of the lncRNA PVT1. SK-OV3 cells were cultured to 50% confluence in 6 well plates. Pooled siRNA’s to human PVT1 or non targeting control siRNA’s from Dharmacon were used to transfect SK-OV3 cells for 48 hrs in full serum media carefully maintaining cell confluence to not exceed approximately 80%. This was followed by RNA extraction and verification of knockdown using primers to PVT1 followed by sequencing. We find that 450 protein coding genes were differentially expressed between control (siControl) and siPVT1 cells with 50 additional found to be non-protein coding. The top 50 differentially expressed genes include 12 that were downregulated by siPVT1 and 32 that were upregulated. Several pathways associated with metabolic and stress processes, ribosome biogenesis and ncRNA processing were altered based on GO pathway analysis. Additional pathways included pathways associated with cell motility and differentiation.
2022-07-06 | GSE185933 | GEO
Project description:Each gene exists as two copies in the same nucleus of mammalian cells. Although most genes are equivalently controlled on both alleles, Random Monoallelic Expressing (RME) genes stably maintain the expression from only one of two alleles, but the mechanisms and functional consequences of RME remains unclear. Here we performed allele-specific RNA-seq on over 100 neural progenitor cell (NPC) clonal lines to reveal the extent of RME for genes across clonal lines. Interestingly out of 297 autosomal RME genes, Pvt1, a well-studied oncogenic long non-coding RNA, is monoallelically expressed with a skewed genetic bias towards the Castaneous allele in F1-hybrid NPCs. In the absence of genetic differences, allelic tracking by gene editing reveals Pvt1 undergoes balanced RME that is maintained across somatic cell generations. Monoallelic expression is maintained by a poised Pvt1 promoter with bivalent active histone marks, H3K4me3 and H3K27ac, and silencing histone marks, H3K4me3 and H3K27me3, demonstrating its ability to switch transcriptional states depending on the cellular context. Leveraging our large RNA-seq data set and the genetic skew, we use differential gene expression analysis on clonal lines based on their Pvt1 allelic status to reveal a transcription factor, Tfap2a, with differential binding at the Pvt1 promoter, providing a mechanism for the initiation and allelic choice for RME genes. Additionally, we demonstrate monoallelic Pvt1 expression results in an increase in Pvt1 expression leading to a growth advantage relative to Pvt1 biallelic expressing clonal lines These findings provide an example of how genetic differences can skew a stochastic process which results in an epigenetic phenomenon with a phenotypic consequence in early development.
2025-09-26 | GSE287455 | GEO
Project description:Each gene exists as two copies in the same nucleus of mammalian cells. Although most genes are equivalently controlled on both alleles, Random Monoallelic Expressing (RME) genes stably maintain the expression from only one of two alleles, but the mechanisms and functional consequences of RME remains unclear. Here we performed allele-specific RNA-seq on over 100 neural progenitor cell (NPC) clonal lines to reveal the extent of RME for genes across clonal lines. Interestingly out of 297 autosomal RME genes, Pvt1, a well-studied oncogenic long non-coding RNA, is monoallelically expressed with a skewed genetic bias towards the Castaneous allele in F1-hybrid NPCs. In the absence of genetic differences, allelic tracking by gene editing reveals Pvt1 undergoes balanced RME that is maintained across somatic cell generations. Monoallelic expression is maintained by a poised Pvt1 promoter with bivalent active histone marks, H3K4me3 and H3K27ac, and silencing histone marks, H3K4me3 and H3K27me3, demonstrating its ability to switch transcriptional states depending on the cellular context. Leveraging our large RNA-seq data set and the genetic skew, we use differential gene expression analysis on clonal lines based on their Pvt1 allelic status to reveal a transcription factor, Tfap2a, with differential binding at the Pvt1 promoter, providing a mechanism for the initiation and allelic choice for RME genes. Additionally, we demonstrate monoallelic Pvt1 expression results in an increase in Pvt1 expression leading to a growth advantage relative to Pvt1 biallelic expressing clonal lines These findings provide an example of how genetic differences can skew a stochastic process which results in an epigenetic phenomenon with a phenotypic consequence in early development.
2025-09-26 | GSE287457 | GEO
Project description:Long intergenic non-coding RNA (lincRNA) PVT1 is an oncogene known to be overexpressed in various types of cancer. PVT1 high expression is associated with increased prostate cancer (PCa) risk while androgen-independent PCa progression is correlated with increased androgen receptor (AR) expression. However, the mechanism of PVT1 and AR involvement in the development of prostate cancer is still unclear. Here, we tested the hypothesis that PVT1 participates along with AR and the methyltransferase EZH2 from the Polycomb repressive complex 2 in the repression of gene expression in LNCaP prostate cancer cells. Native RNA-binding proteins immunoprecipitation followed by quantitative PCR of co-precipitated RNAs (RIP-qPCR) revealed that in LNCaP, PVT1 lincRNA is associated both with AR (10 – 12 % of PVT1 input) and EZH2 (36 – 42 % of input) in the presence or absence of androgen. PVT1 knockdown in LNCaP in the presence of androgen increased the expression of 160 genes whose expression was repressed by androgen, including genes involved in regulation of cell differentiation, in inhibition of cell migration/invasion and in triggering apoptosis. Analysis by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) of the histone marks occupancy at the promoter region of the tumor suppressor gene NOV, one of the genes that had an increased expression upon PVT1 silencing, showed a significant epigenetic remodeling at its promoter and enhancer regions upon PVT1 knockdown. We provide evidence for a genome-wide transcriptional repressive role of PVT1 lincRNA on tumor suppressor genes in prostate cancer cells.
2021-01-19 | GSE133372 | GEO
Project description:Background Cutaneous squamous cell carcinoma (cSCC) is one of the most common and fastest increasing forms of cancer worldwide with metastatic potential. Long non-coding RNAs (lncRNAs) are a group of RNA-molecules with essential regulatory functions for both physiological and pathological processes. Objectives To investigate the function and mode of action of lncRNA plasmacytoma variant translocation 1 (PVT1) in cSCC. Methods The expression level of PVT1 was quantified in healthy skin, benign skin diseases and cSCCs by qRT-PCR and single molecule in situ hybridization. The function of PVT1 in cSCC was investigated both in vivo (tumour xenograft) and in vitro (competitive cell growth assay, EdU-incorporation assay, colony formation assay and tumour spheroid formation assay) by CRISPR-Cas9-mediated PVT1 or PVT1 exon 2 knockout and by locked nucleic acid (LNA) GapmeR-mediated PVT1-knockdown. RNAseq-analysis was conducted to identify genes and processes regulated by PVT1. Results We identified PVT1 as a lncRNA upregulated in cutaneous squamous cell carcinoma in situ (cSCCIS) and cSCC and associated with oncogenic phenotype of cSCC. The increased expression of PVT1 in cSCC was regulated by MYC. Both CRISPR-Cas9-deletion of the entire PVT1 locus and LNA GapmeR-mediated knockdown of PVT1-transcript impaired malignant behaviour of cSCC cells which suggested that PVT1 is an oncogenic transcript in cSCC. Furthermore, knockout of PVT1 exon 2 inhibited cSCC tumour growth both in vivo and in vitro demonstrating that exon 2 is a critical element for the oncogenic role of PVT1. Mechanistically, we show that PVT1 is localized in the cell nucleus and acts as a suppressor of cellular senescence by inhibiting CDKN1A expression and preventing cell cycle arrest. Conclusions Our study reveals a previously unrecognized role for exon 2 of PVT1 in its oncogenic role and that PVT1 suppresses cellular senescence. PVT1 may be a biomarker and therapeutic target in cSCC.
2023-11-30 | GSE235606 | GEO
Project description:Each gene exists as two copies in the same nucleus of mammalian cells. Although most genes are equivalently controlled on both alleles, Random Monoallelic Expressing (RME) genes stably maintain the expression from only one of two alleles, but the mechanisms and functional consequences of RME remains unclear. Here we performed allele-specific RNA-seq on over 100 neural progenitor cell (NPC) clonal lines to reveal the extent of RME for genes across clonal lines. Interestingly out of 297 autosomal RME genes, Pvt1, a well-studied oncogenic long non-coding RNA, is monoallelically expressed with a skewed genetic bias towards the Castaneous allele in F1-hybrid NPCs. In the absence of genetic differences, allelic tracking by gene editing reveals Pvt1 undergoes balanced RME that is maintained across somatic cell generations. Leveraging our large RNA-seq data set and the genetic skew, we use differential gene expression analysis on clonal lines based on their Pvt1 allelic status to reveal a transcription factor, TFAP2a, with differential binding at the Pvt1 promoter, providing a mechanism for the initiation and allelic choice for RME genes. Additionally, we demonstrate monoallelic Pvt1 expression results in an increase in Pvt1 expression leading to a growth advantage relative to Pvt1 biallelic expressing clonal lines These findings provide an example of how genetic differences can skew a stochastic process which results in an epigenetic phenomenon with a phenotypic consequence in early development.
2025-09-26 | GSE287456 | GEO
Project description:Mus musculus strain:C2C12 cell Genome sequencing
| PRJDA72887 | ENA