Project description:The goal of the study was to compare gene expression of Robo1+/+ and Robo1-/- luminal progenitors. Total RNAs were then extracted from FACS purified luminal progenitor cells, harvested from Robo1+/+ or Robo1-/- mice (n=3 per genotype, two animals per n) using TRIreagent LS (Sigma, T3934). Poly(A)+ RNA sequencing libraries were made from each sample using the TruSeq RNA library preparation kit v.1 (Illumina). Illumina RNA PolyA library preparation guide. A total of 6 libraries were created by PCR amplification with Illumina barcoding primers using kit recommended conditions and quantified using a Bioanalyzer DNA 1000 kit (Agilent).
Project description:Id1 and its closely related family member Id3 are expressed by a diversity of stem and progenitor cells. We show that Id1/3 are required for the self-renewal and proliferation of triple negative breast cancer (TNBC) cells both in vitro and in vivo. Furthermore, we identified that Id1/3 negatively regulates the tumour suppressor gene Robo1. Depletion of Robo1 could rescue the proliferative defect induced by Id1/3 knockdown. To understand the mechanisms by which Robo1 rescues cell proliferation in Id1/3 depleted cells, we performed RNA-Sequencing on 4T1 cells with Dox-inducible Id1/3 KD and/or Robo1 depletion using siRNA. We conclude that following Id1/3 knockdown, Robo1 is induced and exerts anti-proliferative effects via suppression of a Myc transcriptional program.
Project description:We previously identified inactivating mutations in roundabout guidance receptor 1 (ROBO1) in patients with myelodysplastic syndrome (MDS), which was associated with poor prognosis and susceptibility to acute myeloid leukemia (AML) transformation. Nonetheless, the exact role of ROBO1 in hematopoiesis remains poorly delineated. Here we report that the ablation of Robo1 in mice is sufficient to confer MDS-like disease with anemia and multilineage dysplasia. More specifically, Robo1 deficiency impairs the self-renewal and differentiation capacity of hematopoietic stem progenitor cells (HSPCs) and disrupts HSPC pool, especially the reduction of megakaryocyte erythroid progenitors (MEPs), which causes a blockage in the early stages of erythropoiesis in mice. Similarly, Robo1-deficienct HSPCs recapitulated MDS-like disease with anemia and multilineage dysplasia in transplanted mice. Correspondingly, clinical data also reveal that low expression of ROBO1 is associated with shortened survival, severe anemia and a high risk of AML transformation in patients with MDS. Mechanistically, transcriptional profiling indicates that Cdc42, a member of the Rho-GTPase family, serves as a downstream target gene for Robo1 in HSPCs. Overexpression of Cdc42 partially restores the proliferation of erythroid colonies in Robo1 deficient mice. In contrast, Cdc42 inhibitor (CASIN) effectively attenuates the erythroid colony formation of HSPCs. Collectively, our results suggest that Robo1 deficiency may provide a promising therapeutic target against MDS by impairing HSPC self-renewal and erythropoiesis via CDC42, predicting a poor prognosis for severe anemia and a high risk of AML transformation in MDS.
Project description:Seven patient paired primary human HLA-G+ extravillous trophoblasts (EVT) and Villous trophoblasts (VT) obtained from 1st trimester (7-9 weeks) villous tissue were obtained. RNA was isolated directly after isolation and purification using FACS sort for CD45-HLA-G+ (EVT) and CD45-HLA-G-EGFR1+ (VT) fractions. Expression profiles were compared to two samples of the choriocarcinoma cell line JEG-3 and four samples of decidual stromal cells (DSC) at passage 2 after cell culture.