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| Status |
Public on Dec 21, 2019 |
| Title |
RNA-seq analysis of gene regulation by miR-34a and expression changes induced by DNA damage in zebrafish |
| Organism |
Danio rerio |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Li-Fraumeni syndrome (LFS) is a disorder due to inherited mutations in the TP53 gene resulting in an increased risk of developing several types of cancer. MicroRNA miR-34a has been implicated downstream of p53 on the basis of being a direct transcriptional target and, when over-expressed, having pro-apoptotic phenotypes in cell lines. Moreover, miR-34a has been shown to be a modifier gene in the context of LFS, since its epigenetic silencing increases the likelihood of tumour development in patients with mutant TP53. However, the in vivo consequences of miR-34 loss are still unclear. For example, mice lacking all three (a,b,c) miR-34 homologs show no detectable abnormalities in p53 responses. The relative expression of different miR-34 genes in zebrafish was studied using qRT-PCR with specific assays. The miR-34a, miR-34b and miR-34c display unique onset of developmental expression and expression levels, with miR-34a being the most abundant and constant in expression. All of the miR-34 genes also showed clear induction by p53 when DNA-damaging treatments are performed. Using CRISPR-Cas9 technology, we generated a zebrafish miR-34a deletion mutant to further investigate the roles of miR-34a on its own and its association with the p53 pathway. Predictably, a miR-34a deletion mutant demonstrated absence of miR-34a, though without miR-34b and miR-34c compensation beyond baseline expression levels. Mutants survive to adulthood, show no overt phenotypes and have normal apoptotic responses to DNA-damaging irradiation or camptothecin treatments. To further explore the effects of miR-34a, we performed gene expression profiling using RNA-seq of wild-type and miR-34a deletion mutant zebrafish embryos at 28 hpf with or without treatment with a DNA-damaging drug camptothecin. The results of this RNA-seq experiments showed that the loss of miR-34a does not strongly affect induction of genes by DNA-damage. However, the overall pattern of gene expression is significantly different as shown by Principal Component Analysis and there is a group of about 100 genes which are differentially expressed due to loss of miR-34a. The dataset we present in this submission was used to reach these conclusions.
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| Overall design |
Zebrafish embryos at 24 hpf of wild-type and miR-34a-/- genotypes were dechorionated and the control (DMSO) or 1 uM camptothecin treatment for 4 hours was performed followed by RNA extraction from all samples.
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| Contributor(s) |
Prykhozhij SV, Wajnberg G, Crapoulet N, Berman JN |
| Citation missing |
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| Submission date |
Dec 20, 2019 |
| Last update date |
Dec 22, 2019 |
| Contact name |
Sergey Prykhozhij |
| E-mail(s) |
[email protected]
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| Phone |
9028178846
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| Organization name |
CHEO Research Institute/University of Ottawa
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| Lab |
Berman Lab
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| Street address |
Room 318/319 | CAREG, 20 Marie-Curie Private
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| City |
Ottawa |
| State/province |
ON |
| ZIP/Postal code |
K1N 9B4 |
| Country |
Canada |
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| Platforms (1) |
| GPL24059 |
Ion Torrent Proton (Danio rerio) |
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| Samples (12)
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| GSM4227404 |
28 hpf wild-type treated with 1μM CPT rep1 |
| GSM4227405 |
28 hpf wild-type treated with 1μM CPT rep2 |
| GSM4227406 |
28 hpf wild-type treated with 1μM CPT rep3 |
| GSM4227407 |
28 hpf miR-34a-/- DMSO control rep1 |
| GSM4227408 |
28 hpf miR-34a-/- DMSO control rep2 |
| GSM4227409 |
28 hpf miR-34a-/- DMSO control rep3 |
| GSM4227410 |
28 hpf miR-34a-/- treated with 1μM CPT rep1 |
| GSM4227411 |
28 hpf miR-34a-/- treated with 1μM CPT rep2 |
| GSM4227412 |
28 hpf miR-34a-/- treated with 1μM CPT rep3 |
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| Relations |
| BioProject |
PRJNA597022 |
| SRA |
SRP238398 |
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