Dataset Information

P53-Dependent induction of PVT1 and miR-1204.

ABSTRACT: p53 is a tumor suppressor protein that acts as a transcription factor to regulate (either positively or negatively) a plethora of downstream target genes. Although its ability to induce protein coding genes is well documented, recent studies have implicated p53 in the regulation of non-coding RNAs, including both microRNAs (e.g. miR-34a) and long non-coding RNAs (e.g. lincRNA-p21). We have identified the non-protein coding locus PVT1 as a p53-inducible target gene. PVT1, a very large (>300 kb) locus located downstream of c-myc on chromosome 8q24, produces a wide variety of spliced non-coding RNAs as well as a cluster of six annotated microRNAs: miR-1204, miR-1205, miR-1206, miR-1207-5p, miR-1207-3p, and miR-1208. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and luciferase assays reveal that p53 binds and activates a canonical response element within the vicinity of miR-1204. Consistently, we demonstrate the p53-dependent induction of endogenous PVT1 transcripts and consequent up-regulation of mature miR-1204. Finally, we have shown that ectopic expression of miR-1204 leads to increased p53 levels and causes cell death in a partially p53-dependent manner.

SUBMITTER: Barsotti AM

PROVIDER: S-EPMC3268411 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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p53-Dependent induction of PVT1 and miR-1204.

Barsotti Anthony M AM Beckerman Rachel R Laptenko Oleg O Huppi Konrad K Caplen Natasha J NJ Prives Carol C

The Journal of biological chemistry 20111122 4

p53 is a tumor suppressor protein that acts as a transcription factor to regulate (either positively or negatively) a plethora of downstream target genes. Although its ability to induce protein coding genes is well documented, recent studies have implicated p53 in the regulation of non-coding RNAs, including both microRNAs (e.g. miR-34a) and long non-coding RNAs (e.g. lincRNA-p21). We have identified the non-protein coding locus PVT1 as a p53-inducible target gene. PVT1, a very large (>300 kb) l ...[more]

PMID: 22110125

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Project description:Prostaglandin (PG) A2, one of cyclopentenone PGs, is known to induce activation of apoptosis in various cancer cells. Although PGA2 has been reported to cause activation of apoptosis by altering the expression of apoptosis-related genes, the role of p53, one of the most critical pro-apoptotic genes, on PGA2-induced apoptosis has not been clarified yet. To address this issue, we compared the apoptosis in HCT116 p53 null cells (HCT116 p53-/-) to that in HCT116 cells containing the wild type p53 gene. Cell death induced by PGA2 was associated with phosphorylation of histone H2A variant H2AX (H2AX), activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase 1 in HCT116 cells. Induction of apoptosis in PGA2-treated cells was almost completely prevented by pretreatment with a pan-caspase inhibitor, z-VAD-Fmk, or an inhibitor of protein synthesis, cycloheximide. While PGA2 induced apoptosis in HCT116 cells, phosphorylation of p53 and transcriptional induction of p53-target genes such as p21WAF1, PUMA, BAX, NOXA, and DR5 occurred. Besides, pretreatment of pifithrin-? (PFT-?), a chemical inhibitor of p53's transcriptional activity, interfered with the induction of apoptosis in PGA2-treated HCT116 cells. Pretreatment of NU7441, a small molecule inhibitor of DNA-activated protein kinase (DNA-PK) suppressed PGA2-induced phosphorylation of p53 and apoptosis as well. Moreover, among target genes of p53, knockdown of DR5 expression by RNA interference, suppressed PGA2-induced apoptosis. In the meanwhile, in HCT116 p53-/- cells, PGA2 induced apoptosis in delayed time points and with less potency. Delayed apoptosis by PGA2 in HCT116 p53-/- cells was also associated with phosphorylation of H2AX but was not inhibited by either PFT-? or NU7441. Collectively, these results suggest the following. PGA2 may induce p53-dependent apoptosis in which DNA-PK activates p53, and DR5, a transcriptional target of p53, plays a pivotal role in HCT116 cells. In contrast to apoptosis in HCT116 cells, PGA2 may induce apoptosis in a fashion of less potency, which is independent of p53 and DNA-PK in HCT116 p53-/- cells.

Project description:Introduction: Amplification at chromosome 8q24 is one of the most frequent genomic abnormalities in human cancers and is associated with reduced survival duration in breast and ovarian cancers. The minimal amplified region encodes c-MYC and the non-coding RNA, PVT1 including miR-1204 encoded in exon 1b. Here we analyzed the genomic changes at chromosome 8q24.21 in breast cancer and the functional roles of miR-1204 in breast and ovarian cancer progression. Methods: The genomic changes at chromosome 8q24.21 were detected in 997 breast cancer tumors and 40 breast cancer cell lines. Expression of miR-1204 in breast and ovarian cancer cell lines was investigated by qRT-PCR method. The role of miR-1204 in the tumorigenesis of breast and ovarian cancer was explored using both knockdown and overexpression of miR-1204 in vitro. Candidate miR-1204 target genes from two independent expression microarray datasets and computational predict programs were identified and further validated by qRT-PCR and western blot methods. The role of inhibition of miR-1204 on tamoxifen sensitivity in breast cancer cells was also investigated. Results: MiR-1204 is frequently co-amplified with MYC and expression of miR-1204 is strongly correlated with the expression and amplification of the noncoding PVT1 transcript and less so with MYC in human breast and ovarian cancer cells. Inhibition of miR-1204 decreases cell proliferation and increased apoptosis in breast and ovarian cancer cell lines with 8q24 amplification, but not in lines without amplification and so may be involved in Myc-induced apoptosis. Additionally, overexpression of miR-1204 enhances both breast and ovarian cancer cell growth and Myc-initiated Rat1A cell transformation. Computational and experimental analyses 30 promising candidate miR-1204 target genes. mRNA levels for these genes were assessed after over expression and knockdown of miR-1204 as were protein levels for 10 genes for which antibodies were available. These studies implicated VDR and ESR1 as miR-1204 targets. Inhibition of miR-1204 increased response to tamoxifen in Estrogen Receptor negative breast cancer cell lines. Conclusions: We conclude that amplification of miR-1204 contributes to breast and ovarian pathophysiology at least in part, by increasing proliferation and down regulating apoptosis and by decreasing expression of VDR and ESR1. Seven cell line sample pairs, where samples are LNA transfected with antimiR-1204 or antimiR-1204 control

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P53-Dependent induction of PVT1 and miR-1204.

Publications

p53-Dependent induction of PVT1 and miR-1204.

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