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Gene expression profile (GEP) of siRNA-JARID2 CD34+ cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE52921 | GEO | 2014/12/01

SECONDARY ACCESSION(S): PRJNA230468

REPOSITORIES: GEO

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Project description:As recently reported by our group, we performed miRNA and gene expression profiling of CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 42 PMF patient samples compared with 31 healthy controls. Integrative analysis of these profiles by means of Ingenuity Pathway Analysis (IPA) allowed the identification of several aberrantly regulated miRNA-mRNA target pairs organized in interaction networks. In particular, our results highlighted the up-regulation of miR-494-3p in CD34+ cells from PMF patients (Norfo R et al, Blood, 2014). Interestingly, among the most upregulated miRNAs, miR-494-3p emerges as being associated to the highest number of downregulated target mRNAs. In order to understand the biological role of miR-494-3p during the hematopoietic commitment and differentiation, we overexpressed this miRNA in cord blood (CB) derived-CD34+ cells. Cells were electroporated with either miR-494-3p miRNA mimic (mimic miR-494) or a negative control mimic (mimic Neg CTR). qRT-PCR confirmed miR-494-3p overexpression 24h and 4 days after transfection (RQ ± SEM, 512.60 ± 137.37, p<.01, and 20.63 ± 3.03, p<.01, respectively). Immunophenotypic analysis of CD41 and CD42b megakaryocyte (MK) lineage differentiation markers, performed on serum-free megakaryocytic ultilineage culture at day 3, 5, 8, 10 and 12, demonstrated that miR-494-3p overexpression induces a significant increase in the MK fraction compared to control samples. Accordingly, morphological analysis of May-Grünwald-Giemsa stained cytospins revealed an expansion of megakaryocytic lineage in samples overexpressing miR-494-3p. These data were further confirmed by collagen-based clonogenic assays which showed a significant increase in the percentage of megakaryocytic colonies in miR-494-3p overexpressing samples compared with controls. In order to better characterize the molecular mechanisms underlying the effects of miR-494-3p on HSPCs differentiation, we performed gene expression analysis of miR-494-3p overexpressing cells 24 hours after the last nucleofection.

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Gene expression profile (GEP) of siRNA-JARID2 CD34+ cells

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