MicroRNA Expression and Functions During Allograft Rejection

Abstract

Introduction: MicroRNAs (miRNAs) are single-stranded noncoding RNA molecules of ˜22 nucleotides in length that bind to mRNAs and prevent their translation. They are an important gene regulatory step in many biological processes, and abnormal expression of miRNAs is associated with many disease states. Recent reports have demonstrated altered patterns of miRNAs in recipients of kidney and small bowel allografts. We sought to profile the miRNA expression patterns during unmodified rejection using a well-characterized experimental model of heart transplantation. Methods: Heart grafts from syngeneic (C57BL/6 —> C57BL/6) and allogeneic (BALB/c —>C57BL/6) (n=9) heterotopic heart transplants were collected 7 days after transplantation. Graft function was assessed through abdominal palpation and rejection was confirmed by histopathological analyses using H&E and Masson Trichrome staining. Samples were profiled for miRNAs using TaqMan microfluidic cards and microarray data were analyzed to define miRNAs that are differentially expressed (P< 0.05). Target predictions using both mouse and human homolog miRNA were made using TargetScan, PicTar and pathway predictions were performed using PANTHER. A luciferase reporter assay was used to determine binding of miR-132 to a predicted target USP9X. Results: Eight miRNAs, miR-10a, -20a, -132, -147b, -155, 188-5p, -221, and -424/322, were chosen for further investigation. Quantitative PCR was used to detect the kinetics of expression in heart grafts on days 1, 3, 5, and 7 post-transplant (n=3). Four distinct patterns were observed when the rejecting, allogeneic group was compared to the non-rejecting, syngeneic group. MiRs -10a, -20a and -132 demonstrated significantly elevated (3X), peak expression on day 5 post-transplant with a subsequent drop on day 7. MiRs-147b and -155 were also significantly elevated on day 5 and continued to increase through day 7 post-transplant (5X). MiRs-188-5p and -221 were moderately increased (2X) and reached a plateau by day 5 post-transplant. In contrast, expression of miR-424 was moderately decreased on day 7 post-transplant. Putative targets of each miRNA were predicted and used for pathway analysis. Results indicate that miR-20a is involved in NF-κB pathways. Mir-424 is important in cell cycle control and the Wnt pathways. Mir-221 may target genes involved in calcineurin regulation. Of note, miR-132 is predicted to target USP9X, a gene involved in deubiquination of SMAD4 in TGF-beta signaling. To confirm binding of miR-132 to USP9X, the 3' UTR of USP9X was inserted downstream of the luciferase ORF, specific binding to miR-132 prevented luciferase reporter gene expression, however mutation of the USP9X binding site decreased specific binding to miR-132 and restored luciferase activity indicating that USP9X is a target of miR-132. Thus increased expression of miR-132 post-transplant may be important in decreasing USP9X gene expression. Interestingly, altered expression of USP9X has been reported in ischemic reperfusion injury, cardiomyopathy and during allograft rejection in the PBMC of heart transplant patients. Conclusion: Studying the roles of specific miRNA will give us insights into the regulation of key genes that control immune activation, signaling, and cell growth and suggest new targets for therapeutic intervention.