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Exp Ther Med. 2016 Dec;12(6):3972-3978. doi: 10.3892/etm.2016.3867. Epub 2016 Nov 3.

Effect of miR-146a and miR-155 on cardiac xenotransplantation.

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1
Department of General Surgery, Tianjin Medical University General Hospital, Tianjin 300052, P.R. China.

Abstract

The aim of the present study was to investigate the expression levels of miR-146a and miR-155 in a cardiac xenograft model treated with the immunosuppressant FK506, and to construct lentiviral vectors to further study the roles of miR-146a and miR-155 in cardiac xenotransplantation. Expression levels of miR-146a and miR-155 were examined by quantitative polymerase chain reaction analysis and protein expression of RelA, which is a member of the nuclear factor-κB family, was examined by western blot analysis. Pre-miR-146a and pre-miR-155 fragments were designed and synthesized according to MiRBase and were cloned into the plasmid pCDH1-MCS1-EF1-copGFP. Recombinant plasmids were identified by enzyme digestion and sequencing. Survival time of cardiac grafts in the FK506 treatment group was significantly increased in comparison with the control group (P<0.05). In addition, the histopathological grading results were significantly decreased in the treatment group (P<0.05). A significant decrease in RelA protein expression levels was observed in the treatment group (P<0.05), along with a significant increase in miR-146a expression levels (P<0.05) and a significant decrease in miR-155 expression levels (P<0.05). Digestion and sequencing findings demonstrated that the insertion of miRNA into the plasmid pCDH1-MCS1-EF1-copGFP conformed with the pre-miRNAs, and the lentiviral vectors were concentrated to a titer of 5×107 IFU/ml. These findings demonstrated that FK506 is able to inhibit the rejection effect in a mouse-to-rat cardiac xenotransplantation model. FK506 treatment altered the expression levels of miR-146a and miR-155, indicating that they may have an important role in regulating the immune response to the rejection effect. miR-146a and miR-155 lentiviral vectors were successfully constructed for further experiments both in vitro and in vivo.

KEYWORDS:

FK506; acute vascular rejection; hyperacute rejection; lentiviral vector; miR-146a; miR-155; xenotransplantation

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