Transcriptional Perturbations in Graft Rejection

Abstract

Background: Understanding the regulatory interplay of relevant microRNAs (miRNAs) and messenger RNAs (mRNAs) in the rejecting allograft will result in a better understanding of the molecular pathophysiology of alloimmune injury.

Methods: One hundred sixty-seven allograft biopsies, with (n = 47) and without (n = 120) central histology for Banff scored acute rejection (AR), were transcriptionally profiled for mRNA and miRNA by whole genome microarrays and multiplexed microfluidic quantitative polymerase chain reaction, respectively. A customized database was curated (GO-Elite) and used to identify AR-specific dysregulated mRNAs and the role of perturbations of their relevant miRNAs targets during AR.

Results: The AR-specific changes in 1035 specific mRNAs were mirrored by AR-specific perturbations in 9 relevant miRNAs as predicted by Go-Elite and were regulated specifically by p53 and forkhead box P3. Infiltrating lymphocytes and the renal tubules drove the miRNA tissue pertubations in rejection, involving message degradation and transcriptional/translational activation. The expression of many of these miRNAs significantly associated with the intensity of the Banff-scored interstitial inflammation and tubulitis.

Conclusions: There is a highly regulated interplay between specific mRNA/miRNAs in allograft rejection that drive both immune-mediated injury and tissue repair during AR.

Figures

Figure 1

Project design and analysis flow

Figure 2

Differentially regulated miRNAs in transplant tissues biopsies, with miR142-3p (A), miR342-3p (B) and miR25 (C) all up-regulated in those biopsies with histological evidence of AR compared to normal biopsies. In addition, miR181a (D), miR192 (E), miR204 (F), miR215 (G), miR10b* (H), miR615-3p (I) were all down-regulated in AR compared to normal biopsies.

Figure 3

Expression of experimentally verified mRNA targets (via miRTarBase) of the altered miRNA, measured by Affymetrix Hu133+2.0 arrays in transplant biopsies. (A) miR25 up-regulated targets TP53 and PRMT5 in AR. (B) miR181a down-regulated targets CDX2, ATM and HIPK2 in AR. (C) miR192 up-regulated targets DTL, HRH1, LMNB2 and MAD2L1 in AR. (D) miR204 up-regulated targets TGFβR1, TGFβR2, SPDEF and SNAI1 in AR. (E) Up-regulated targets NCOR2 and ACVR2B of miR10B* and miR215, respectively, along with down-regulated target RAC1 of miR142-3p in AR. (F) mRNA target interactions of the altered miRNA leads to various mechanism of cell cycle and DNA repair through p53 signaling.

Figure 4

miR25 expression significantly trends with Banff pathology scores for tubular atrophy (A), along with several other miRNAs for Banff scores of tubulitis (B) and infiltrate (C). Significant positive trends were observed between miR25, miR342-3p and miR142-3p and Banff t and i scores, indicating a likely lymphocyte origin for these miRNAs. Significant negative trends were observed for miR192, miR204 andmiR10b* and Banff t and i scores, with miR215 significantly tending (negative) with Banff t only. This suggests an origin of the tubular cells in response to injury during AR. A significant positive trend was only observed between miR25 and Banff ct score, suggesting a role for this miRNA in tubular injury also.

Figure 5

Transcription factors associated with altered miRNA. (A) Network node displaying the association of various transcription factors (blue) that are predicted to regulate the mRNA targets of the altered miRNA (green). There is a clear cluster of FOXP3 as a consistent transcription factor that regulates a number of the mRNA targets of all altered miRNA except for miR142-3p. (B) FOXP3 is down-regulated in AR (AR), as per mRNA measured by microarrays, which is consistent with its targets also being down-regulated in AR.