MicroRNA-182 is a key modulator of FOXO1 expression and induced in response to alloantigens during acute rejection

Abstract

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the post-transcriptional expression of target genes and have been shown to be important regulators of many biologic processes including innate and adaptive immune responses. The roles of specific miRNAs in alloimmune responses remain unknown. We used a mouse model of heart transplantation and microarray analysis to profile miRNAs differentially expressed during allograft rejection with the goal of identifying miRNAs that regulate alloimmune responses and graft outcomes. Syngeneic (C57BL/6 → C57BL/6) and allogeneic (BALB/c → C57BL/6) (n=3-9) heterotopic heart transplants were performed. Heart grafts were collected on days 1-7 and graft infiltrating lymphocytes (GILs) were isolated on day 5 post-transplant. Samples were profiled using TaqMan microfluidic cards that contain 335 mature mouse miRNAs. Microarray data were analyzed to define miRNAs that are differentially expressed (P< 0.05). Differentially expressed miRNAs were validated by Taqman miRNA assays using samples from additional groups (n=3-5) of transplanted mice. Sixteen miRNAs were significantly differentially expressed in both the heart grafts and the GILs of allogeneic recipients, compared to syngeneic recipients, including the miRNA miR-182, a dominantly expressed member of miR-183-96-182 cluster. Forkhead Box (FOX) proteins are a family of important transcription factors and FOXO1 is a predicted target of miR-182. We used a luciferase reporter assay system and confirmed FOXO1 is a target of miR-182 as mutation of the Foxo1 seed sequence decreased specific binding to miR-182. FOXO1 expression in grafts was analyzed by qPCR and western blot on days 1-5 post-transplant and immunohistochemistry was used to detect FOXO1 expression in cardiomyocytes and GILs. We show that as miR-182 increases after transplant, there is a concomitant post-transcriptional decrease in FOXO1 expression in heart allografts. Further FOXO1 expression is localized to both the cardiomyocytes and CD3+ T cells. Importantly, miR-182 was significantly increased in both the PBLs and plasma from recipients of allogeneic grafts as compared to recipients of syngeneic grafts and normal mice. These data indicate that miR-182 expression is specifically expressed in the circulation of graft recipients and suggest that since miRNAs are incredibly stable in blood, they will be important as biomarkers of graft status. Our results suggest that miRNAs may have important regulatory functions post-transplant.