Specific miRNAʼs and their Downstream Targets Are Associated with Acute Rejection in Pediatric Renal Allografts

Abstract

Background: miRNA's are small RNA molecules that are involved in post-transcription mRNA regulation through degradation or translational inhibition, via natural cellular processes. A single miRNA or families of miRNA's may target many different mRNA species resulting in the regulation of sections or even whole functional pathways. To explore the function of miRNA signaling and impact of their targets in acute rejection, we investigated the expression of various miRNA's using microfluidic QPCR and matched mRNA microarrays. Methods: The kidney transplant tissue biopsy samples used in this study were from pediatric renal transplant patient samples (n=88) that have been extracted for RNA (Trizol, Invitrogen) and banked from various NIH funded studies. Samples were chosen to represent the distinct pathology of acute rejection (n=28) or stable (Banff=0 for all grades, n=60). 25ng of total-RNA was used for target specific reverse transcription, followed by target specific pre-amplification using Taqman MegaPlex pool A (ABI). 5ng of pre-amp sample was run on 96.96 dynamic microfluidic arrays (Fluidigm), using TaqMan miRNA assays and universal master mix (ABI). The relative change for each of the miRNA species were calculated using the standard Detla-Delta Ct method, using the expression of miR191 as an internal reference and universal human total RNA (Stratagene, Ambion) as an external reference. Standard T-test was used for significance analysis and Spearman's rank analysis for correlations. Various bioinformatics databases were used to define targets and gene annotation, with Affymetrix Hu133+2.0 microarrays (mRNA) of demographically matched samples were used for target expression (n=97). Results: We observed quite high expression of a number of miRNA species and associated family members. Using the microfluidic QPCR we identified 7 miRNA's that were differentially expressed in acute rejection (AR) compared to normal allograft samples. 6 were significantly down-regulated and one up-regulated. We also observed that specific miRNA's and not whole families were differentially altered, indicating sequence specific and not family wide signaling mechanisms playing a role in AR. The expression of a number of miRNA's correlated with allograft pathology of infiltrate (r=-0.3, P=0.005), tubulitis (r=-0.25, P=0.01) and tubular atrophy (r=0.25, P=0.01). Experimentally verified mRNA targets of these significant miRNAs were identified using bioinformatics databases and were investigated for altered expression on microarrays of matched samples. Of the 46 targets of these 7 significant miRNA, 11 were significantly expressed in the corresponding inverse direction, thus indicating target specific degradation and not a translational inhibition mechanism. Gene annotation of altered targets indicates a strong presence of immune signaling, EMT and various other repair mechanisms. Protein validation is underway. Conclusion: We have identified a number of important miRNA species that are strongly associated with acute rejection on biopsy. These miRNA specific target a number mRNA molecules that appear to play a role in either acute rejection or injury repair mechanisms. These findings have identified a potent source of signally and control during the acute rejection in the renal allograft.