title: Cardiomyocyte protection by GATA-4 gene engineered mesenchymal stem cells is partially mediated by translocation of miR-221 in microvesicles.
abstract: BACKGROUND
  microRNAs (miRs), a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs) plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC).
  
  
  RESULTS
  Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM) were harvested as primary cultures. MSC were transduced with GATA-4 (MSC(GATA-4)) using the murine stem cell virus (pMSCV) retroviral expression system. Empty vector transfection was used as a control (MSC(Null)). The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH). MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM) from MSC cultures was evaluated by lactate dehydrogenase (LDH) release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSC(GATA-4). MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA). Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System.
  
  
  CONCLUSIONS
  Our results demonstrate that cardioprotection by MSC(GATA-4) may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Bin
authors.initials: B
authors.lastname: Yu
authors.forename: Min
authors.initials: M
authors.lastname: Gong
authors.forename: Yigang
authors.initials: Y
authors.lastname: Wang
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Millard
authors.forename: Zeeshan
authors.initials: Z
authors.lastname: Pasha
authors.forename: Yueting
authors.initials: Y
authors.lastname: Yang
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ashraf
authors.forename: Meifeng
authors.initials: M
authors.lastname: Xu
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia / Q000235:genetics; D018920:Coculture Techniques; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C533952:Bbc3 protein, rat; D050991:GATA4 Transcription Factor; C084365:Gata4 protein, rat; C576526:MIRN221 microRNA, rat; D035683:MicroRNAs; D016159:Tumor Suppressor Protein p53