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

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 ß-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. Overall design: To investigate the function of RHOB in megakaryocyte and erythroid differentiation, we knocked down the expression of RHOB in K562 cells by siRNA transfection. We performed gene expression profiling analysis using data obtained from RNA-seq in K562 cell which cultured in growth medium (Ctl) and megakaryocyte differentiation medium (MK) for 3d. Comparative gene expression profiling analysis of negative control group (NC) and RHOB knockdown group (siRHOB).