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Gene expression profile (GEP) of CALR-overexpressingCD34+ cells

ABSTRACT: In this work, we compared gene expression profile (GEP) of CD34+ cells transduced with the retroviral vector LCALRIDN with CD34+ cells transduced with the empty vector control LXIDN and of CD34+ cells treated with CALR siRNA with control cells transfected with a Non-Targeting siRNA In order to explore the role of CALR in the proliferation and differentiation of hematopoietic stem/progenitor cells (HPSCs), we studied the effects of CALR overexpression in Cord Blood (CB) CD34+ cells. Assessment of cell differentiation unveiled that CALR enforced expression is able to skew the haematopoietic commitment towards the MK and erythroid cell lineages. Conversely, CALR silencing induced a marked repression of MK and erythroid differentiation. In order to characterize the effects of CALR mutations on human hematopoietic cells at the molecular level, we analyzed the coding RNA profiles of CB CD34+ overexpressing CALR. This analysis identified a list of differentially expressed genes. Among upregulated genes, we found genes involved in platelet aggregation, inflammation and erythroid differentiation, potentially related to the characteristics of the disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE97807 | GEO | 2020/04/12

REPOSITORIES: GEO

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Project description: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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Gene expression profile (GEP) of CALR-overexpressingCD34+ cells

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