MicroRNA signatures in cardiac biopsies and detection of allograft rejection

BACKGROUND: Identification of heart transplant (HTx) rejection currently relies on immunohistology and immunohistochemistry. We aimed to identify specific sets of microRNAs (miRNAs) to characterize acute cellular rejection (ACR), antibody-mediated rejection (pAMR), and mixed rejection (MR) in monitoring formalin-fixed paraffin-embedded (FFPE) endomyocardial biopsies (EMBs) in HTx patients. METHODS: In this study we selected 33 adult HTx patients. For each, we chose the first positive EMB for study of each type of rejection. The next-generation sequencing (NGS) IonProton technique and reverse transcript quantitative polymerase chain reaction (RT-qPCR) analysis were performed on FFPE EMBs. Using logistic regression analysis we created unique miRNA signatures as predictive models of each rejection. In situ PCR was carried out on the same EMBs. RESULTS: We obtained >2,257 mature miRNAs from all the EMBs. The 3 types of rejection showed a different miRNA profile for each group. The logistic regression model formed by miRNAs 208a, 126-5p, and 135a-5p identified MR; that formed by miRNAs 27b-3p, 29b-3p, and 199a-3p identified ACR; and that formed by miRNAs 208a, 29b-3p, 135a-5p, and 144-3p identified pAMR. The expression of miRNAs on tissue, through in situ PCR, showed different expressions of the same miRNA in different rejections. miRNA 126-5p was expressed in endothelial cells in ACR but in cardiomyocytes in pAMR. In ACR, miRNA 29b-3p was significantly overexpressed and detected in fibroblasts, whereas in pAMR it was underexpressed and detected only in cardiomyocytes. CONCLUSIONS: miRNA profiling on FFPE EMBs differentiates the 3 types of rejection. Localization of expression of miRNAs on tissue showed different expression of the same miRNA for different cells, suggesting different roles of the same miRNA in different rejections.