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RNA-SEQ Quantitative Analysis of WT and Mpl-del-overexpressing AMKL cell Transcriptomes

ABSTRACT: In the previous studies, we reported that Mpl alternative splicing form Mpl-del overexpressed in acute megakaryoblasttic leukemia (AMKL) patients and mediated thrombopoietin signaling to promote AMKL cells malignant proliferation and chemotherapy resisitance. To investigate the detailed mechanism of Mpl-del mediated AMKL cells proliferation and survival, the AMKL cell Dami stably expressing GFP or Mpl-del-GFP were established by transduction with GFP or Mpl-del-GFP lentiviruses and treated with 20ng/ml TPO for RNA-SEQ analysis to examine the expression of proliferation and survival-related genes. Our results indicated that the levels of the "leading edge" genes that account for survival and prolieration were significantly higher in Mpl--del overexpressed AMKL cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE127762 | GEO | 2019/04/04

REPOSITORIES: GEO

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Project description:Polyamines are aliphatic polycations that have emerged as important determinants of cell growth and viability in rapidly proliferating cells, including in the pathogenic protozoan parasite Leishmania donovani. In L. donovani, the polyamine spermidine is synthesized by the successive conversion of ornithine into putrescine (catalyzed by ornithine decarboxylase or ODC) and putrescine into spermidine (catalyzed by spermidine synthase or SPDSYN). Deletion of either ODC (del-odc) or SPDSYN (del-spdsyn) from the L. donovani genome renders these parasites auxotrophic for polyamines and these mutants are impaired in their ability to survive both in culture and within the mammalian host without the addition of exogenous polyamine supplementation. Significantly, del-odc parasites immediately cease proliferation after putrescine is removed from the culture media and perish within two weeks, while spermidine starved del-spdsyn mutants, which retain intracellular putrescine pools, show a slow-growth phenotype, and persist for several weeks in culture. To elucidate the key differences within the proteome of putrescine-starved del-odc cells and spermidine-starved del-spdsyn parasites, a shotgun quantitative proteomics approach was undertaken using TMT labeling and LC-MS/MS analysis. Briefly, three biological replicates each for mid-log phase del-odc and del-spdsyn promastigotes grown in the presence of exogenous putrescine (for del-odc) or spermidine (for del-spdsyn) supplementation were washed to remove the exogenous polyamine supplementation and incubated in polyamine-free media. At 24 and 48 h, cells from each biological replicate were isolated and prepared for tandem mass tag (TMT) labeling and downstream LC-MS/MS analyses. Peptides were identified using a database generated from the reference genome of L. donovani BPK282A1 strain. Changes in relative protein abundance for the polyamine-starved del-odc and del-spdsyn cell lines at 24 and 48 h were calculated by comparing aggregate total reporter ion intensities for each protein to that of the corresponding polyamine-supplemented 0-h timepoint.

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RNA-SEQ Quantitative Analysis of WT and Mpl-del-overexpressing AMKL cell Transcriptomes

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