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| Status |
Public on Dec 01, 2014 |
| Title |
Gene expression profile (GEP) of siRNA-JARID2 CD34+ cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
JARID2 is a chromatin remodeler, member of the Jumonji family of transcription factor genes that belongs to the polycomb repressive complex 2 (PRC2) (Peng JC et al. Cell 2009) and is frequently deleted in leukemic transformation of chronic myeloid malignancies (Puda A et al. Am J Hematol. 2012). In this work, we compared gene expression profile (GEP) of CD34+ cells from Primary Myelofibrosis (PMF) patients with healthy donors and we found JARID2 among downregulated genes. In addition, integrative analysis of gene and miRNA profiles highlighted JARID2 as a shared target of several miRNAs aberrantly expressed in PMF CD34+ cells. Since the role of JARID2 in normal and malignant hematopoiesis has never been investigated, we performed JARID2 silencing experiments on normal Cord Blood (CB) CD34+ cells to evaluate its involvement in proliferation and commitment. Therefore, CD34+ cells were transfected with a mixture of 3 Silencer Select siRNAs targeting JARID2 mRNA and with a non-targeting siRNA as control (NegCTR). The expression level of JARID2 in control samples and JARID2-siRNA cells was assessed by QRT-PCR at 24h (RQ 0,2 ± SEM 0,036, p <.001) and 48h (RQ 0,32 ± SEM 0,026, p<.001) after the last nucleofection.
Flow cytometric analysis of CD41 Megakaryocyte (MK) lineage differentiation marker performed on serum-free multilineage culture at day 8, 10 and 12 pointed out that JARID2 inhibition induces a significant increase in the MK fraction compared to NegCTR sample. In addition, we demonstrated that JARID2 silencing induces a remarkable increase in percentage of CFU-megakaryocyte (CFU-MK) colonies and a strong decrease of non-megakaryocyte colonies (CFU non-MK) in collagen-based clonogenic assay. Moreover, morphological evaluation of May-Grunwald-Giemsa (MGG)–stained cytospins of TPO treated cells at day 8, 10 and 12 after the last nucleofection clearly displayed a considerable enrichment in MK precursors at different stages of maturation in JARID2-siRNA sample compared to NegCTR. Finally, to better characterize changes in gene expression induced by JARID2 gene silencing, we performed mRNA expression profile microarray analysis in NegCTR and JARID2-siRNA CD34+ cells.
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| Overall design |
Gene expression profile (GEP) was performed on total RNA derived from three independent experiments at 24h after the last nucleofection.
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| Contributor(s) |
Pennucci V, Norfo R, Zini R, Salati S, Bianchi E, Prudente Z, Sacchi G, Ruberti S, Tagliafico E, Manfredini R |
| Citation(s) |
25097177 |
| Submission date |
Dec 03, 2013 |
| Last update date |
Mar 21, 2019 |
| Contact name |
Rossella Manfredini |
| E-mail(s) |
manfredini.rossella@unimore.it
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| Phone |
+390592058065
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| Organization name |
Centre for Regenerative Medicine
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| Department |
Life Sciences
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| Street address |
Via Gottardi 100
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| City |
Modena |
| ZIP/Postal code |
41100 |
| Country |
Italy |
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| Platforms (1) |
| GPL13667 |
[HG-U219] Affymetrix Human Genome U219 Array |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA230468 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE52921_RAW.tar |
12.0 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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