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Pvt1 silencing in C2C12 cell cultures

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE112881 | GEO | 2019/01/07

REPOSITORIES: GEO

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Pvt1 silencing in C2C12 cell cultures

Project description:Pvt1 silencing in C2C12 cell cultures

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Chromatin isolation by RNA immunoprecipitation (ChIRP) for the long non-coding RNA Pvt1

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).

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Murine fast-twitch muscle: two-week progression of denervation atrophy

Project description:The right legs of 3 months old CD1 mice were denervated by unilaterally cutting the sciatic nerve at the level of trochanter, resulting in a permanent denervation of the lower hindleg. At the indicated times, animals were killed by cervical dislocation and tibialis anterior (TA) muscles were dissected out and frozen in liquid nitrogen. The expression analysis was carried out at five time points, namely just after denervation (time 0) and 1, 3, 7 and 14 days after nerve interruption. The contralateral, left TA muscle from the same individual served as innervated control. We reversed labeling of denervated and contralateral samples in each experiment, thus carrying out two separate hybridizations for each time point. Keywords = mouse Keywords = fast-twitch muscle Keywords = denervation Keywords = atrophy Keywords: time-course

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Long non-coding RNA PVT1 is overexpressed in cutaneous squamous cell carcinoma and exon 2 is critical for its oncogenicity

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.

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N6-methyladenosine Modification of lncRNA Pvt1 Governs Epidermal Stemness

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Next Generation Sequencing Facilitates Quantitative Analysis of PVT1 overexpressed MDA-MB-231 cells.

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Gene expression changes associated with knockdown of lncRNA PVT1 in ovarian cancer cell line SK-OV3

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.

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Next Generation Sequencing Facilitates Quantitative Analysis for Chromatin Immunoprecipitation of Wild Type and PVT1 Knockdown by CRISPRi in MDA-MB-231 human breast cancer cell line

Project description:CRISPRi targeting PVT1 specifically increased H3K9me3 at PVT1

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