citations

                    miRNA-mRNA integrative analysis in primary myelofibrosis CD34+ cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis.
                

Authors:

Norfo R1, Zini R1, Pennucci V1, Bianchi E1, Salati S1, Guglielmelli P2, Bogani C2, Fanelli T2, Mannarelli C2, Rosti V3, Pietra D4, Salmoiraghi S5, Bisognin A6, Ruberti S1, Rontauroli S1, Sacchi G1, Prudente Z1, Barosi G3, Cazzola M4, Rambaldi A5, Bortoluzzi S6, Ferrari S7, Tagliafico E7, Vannucchi AM2, Manfredini R1; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Investigators.

In:

Source: Blood
Publication Date: (2014)
Issue: 124 (13): e21-32

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Cells used in publication:

CD34+ cell, human: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

The electroporation of CD34 cells was based on a previously published protocol, which was optimized to be performed on the 4D NucleofectorSystem (Lonza). Briefly, each sample was electroporated 3 times once every 24 hours with a mix of 3 Silencer Select small interfering RNAs (siRNAs) targeting human JARID2 (Life Technologies), starting from the day after CD34 cell purification. For each electroporation, 4 x 105 CD34 cells were resuspended in 100 mL of P3 Primary Cell Solution (Lonza), containing 3 mg of siRNA mix, and pulsed with the program DS112. As described for siRNA transfections, the number of nucleofections and the quantities of miRNA mimics/inhibitors were modified from a previously described protocol to best fit the properties of the second-generation of miRNA mimics/inhibitors (Life Technologies). Briefly, CD34 cells were nucleofected twice, once every 24 hours, with 3 mg of mirVana miR-155-5p mimic or mirVana miRNA mimic Negative Control #1 (Neg-mimic), by using the previously mentioned electroporation protocol DS112. PMF and CBCD341 cells were nucleofected 4 times, once every 24 hours, with 3mg of mirVana miR-155-5p inhibitor or mirVana miRNA inhibitor Negative Control #1 (NegINH), by using the electroporation protocol DS-112. Cells were analyzed 24 and 48 hours after the last nucleofection for both cell viability and JARID2 or miR-155-5p Expression.

Abstract

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34(+) cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34(+) cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41(+) MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF.

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