Project description:Primary Myelofibrosis (PMF) is a Philadelphia negative chronic myeloproliferative neoplasm characterized by bone marrow fibrosis, enhanced oxidative stress and high levels of serum pro-inflammatory cytokines. To identify genes and miRNAs potentially involved in PMF pathogenesis, we have previously carried out an integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem cells (HSPCs), which allowed us to identify miR-382-5p as up-regulated miRNA (Norfo R. et al, Blood 2014). Overexpression experiments in normal CD34+ cells have already demonstrated its central role in HSPC fate decision toward granulocyte lineage (Zini R. et al, Stem Cell Dev 2016). In this study, to further characterized the role of miR-382-5p in PMF pathogenesis, we performed a gene expression profile analysis in normal CD34+ HSPCs overexpressing miR-382-5p. Among the down-regulated genes upon miR-382-5p upregulation, we selected the anti-oxidant superoxide dismutase 2 (SOD2), depicted as the most favorable predicted target by TatgetScan 7.0. Firstly, luciferase reporter assay confirmed SOD2 as a real target of miR-382-5p. Furthermore, we showed that enforced miR-382-5p expression in CD34+ cells reduced SOD2 expression and activity, induced ROS accumulation, and oxidative DNA damages, as well as enhanced CD34+ cell proliferation. Afterwards, to confirm miR-382-5p as a key player in PMF pathogenesis, we performed inhibition experiments in PMF CD34+ cells revealing that miR-382-5p silencing restored SOD2 expression and activity, induced ROS disposal, decreased DNA oxidation and impaired cell proliferation.
Project description:In order to identify the targets of miR-193a-5p in osteosarcoma U2OS cell line, we used a lentivirus-mediated expression system to overexpressing miR-193a precusor, miR-193a-5p target sequence and non-target sequence, respectively, in osteosarcoma cell line U2OS. A tandem mass tag (TMT)-based quantitative proteomic strategy was employed to identify the global profile of miR-193a-5p-regulated proteins. order to identify the targets of miR-193a-5p, we used a lentivirus-mediated expression system to overexpressing miR-193a precusor, miR-193a-5p target sequence and non-target sequence, respectively, in osteosarcoma cell line U2OS. A tandem mass tag (TMT)-based quantitative proteomic strategy was employed to identify the global profile of miR-193a-5p-regulated proteins.