RNAi knock-down of MKL1 in human megakaryocytes derived from cord blood or cytapheresis
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ABSTRACT: Characterize the genes regulated by MKL1/SRF complex in human megakaryocytes (MKs) derived from cord blood or cytapheresis : Using a knock down approach by small interference of MKL1 in MK progenitors, we observed a decrease in the percentage of cells with actin polymerization after adhesion on various substrates, an increase of mean ploidy level and apoptosis. Furthermore, MKL1 inhibition induced a major defect in pro-platelet formation and MKs migration. These results clearly demonstrate that MKL1 is involved in the cytoskeleton organization and maturation of MKs as well as in platelet formation. The goal of gene profiling was to identify new potential MKL1/SRF targets the expression level of which was down regulated after MKL1 inhibition: Human CD34+ cells isolated from cord blood or cytapheresis were transduced by a control lentivirus (designed as SCR; scramble) or by the lentivirus encoding for shRNA of MKL1 (designed as shMKL1) both containing a GFP expressed under the control of PGK promoter. The cells were than grown in serum free medium supplemented by thrombopoietin leading to a generation of megakaryocytes. At day 9 of culture (80% of MKs), the GFP positive cells were sorted, RNAs were extracted and submitted to hybridization as described in annex protocols. Two lists of genes (one for cord blood samples and one for cytapheresis samples) deregulated after the repression of MKL1 were done comparing the intensity of hybridization between SCR and shMKL1 samples. The common list of genes deregulated in MKs from cord blood and cytapheresis after the repression of MKL1 was than generated.
ORGANISM(S): Homo sapiens
SUBMITTER: Philippe DESSEN
PROVIDER: E-TABM-640 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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