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RHOB regulates megakaryocytes and erythroid differentiation by altering the cell cycle and cytoskeleton.

ABSTRACT: RHOB, a member of the Rho GTPase family, is a target of miR-1915-3p. miR-1915-3p has been proved by us to affect the differentiation of MEPs, promoting the differentiation of megakaryocytes and inhibiting the erythroid lineage, which may lead to an increase in the number of platelets and a decrease in the number of erythrocytes. Although RHOB has been confirmed to be involved in the process of proplatelets production in mice, its role in early hematopoietic differentiation, especially its impact on the megakaryocytic and erythroid differentiation of human HSCs and MEPs, has not yet been elucidated. In this study, we confirmed that the function of the RHOB gene was opposite to miR-1915-3p by knocking down the gene in progenitor cells and inducing them into different cells. Silencing of RHOB significantly decreased the erythroid surface markers, reduced the diameter of the cell and inhibited the expression of erythroid-related genes in human cells. Furthermore, RHOB knockdown in megakaryocytic-induced cells increased the megakaryocytic surface markers, enlarged cell and nuclear volume and promoted cell polyploidy and megakaryocyte-related gene expression. The results of RNA sequencing suggest that the RHOB may play a role in differentiation by influencing the cell cycle and the cytoskeleton. Based on that, we found RHOB could affect the expression of differential CDKs and cyclins to alter the proportions of G0/1 and S phase cells and influent the expression or aggregation of β-ACTIN during erythroid or megakaryocytic differentiation. Taken together, these results suggest that RHOB plays an important role in megakaryocytic and erythroid differentiation by cell cycle and cytoskeleton regulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE282217 | GEO | 2025/04/23

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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RHOB regulates megakaryocytes and erythroid differentiation by altering the cell cycle and cytoskeleton.

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