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RNAseq in JARID2 siRNA-treated in vitro differentiated human primary adipocytes

ABSTRACT: RNAseq in JARID2 siRNA-treated in vitro differentiated human primary adipocytes

PROVIDER: PRJNA690568 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13380400.fastq.gz	Fastqsanger.gz
	SRR13380401.fastq.gz	Fastqsanger.gz
	SRR13380402.fastq.gz	Fastqsanger.gz
	SRR13380403.fastq.gz	Fastqsanger.gz
	SRR13380404.fastq.gz	Fastqsanger.gz

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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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RNAseq in JARID2 siRNA-treated in vitro differentiated human primary adipocytes

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