Role of Circulating MicroRNAs in the Immunopathogenesis of Rejection After Pediatric Lung Transplantation

Abstract

Background: Acute rejection (AR) and development of chronic rejection, bronchiolitis obliterans syndrome (BOS) remain major limiting factors for lung transplantation (LTx). This retrospective study is to identify differentially expressed circulating microRNAs (miRNAs) that associate with development of AR and BOS in pediatric lung transplant recipients (LTxR).

Methods: We determined the circulating levels of 7 selected candidate miRNAs in 14 LTxR with AR, 7 with BOS, and compared them against 13 stable pediatric LTxR at 1, 6, and 12 months after LTx. In addition, 6 AR, 7 BOS, and 8 stable pediatric LTxR, 16 AR, 17 BOS, and 16 stable adult LTxR were included for validation.

Results: MiR-10a, -195, -133b were significantly lower in AR and miR-144, -142-5p, -155 were higher in AR compared to stable (P < 0.05). In addition, circulating levels of miR-134, -10a, -195, -133b were significantly lower and miR-144, -142-5p, -155 were higher (P < 0.05) with development of BOS. The receiver-operating characteristic demonstrated that miR-142-5p, miR-155, and miR-195 strongly discriminated patients with AR from stable LTxR (P < 0.001 for all comparisons): miR-142-5p (area under the curve [AUC], 0.854), miR-155 (AUC, 0.876), and miR-195 (AUC, 0.872). Further, miR-10a, miR-142-5p, miR-144, and miR-155 strongly discriminated BOS from stable LTxR (P < 0.001 for all comparisons).

Conclusions: We demonstrated that differential expression of circulating miRNAs occurs in LTxR with AR and BOS, suggesting that they can provide not only important clues to pathogenesis but also may serve as potential noninvasive biomarkers for AR and BOS after pediatric LTx.

Figure 1

Experiment design and work flow. (A) Longitudinal study design in the pilot study of pediatric LTx patients. For the discovery pilot study, 14 AR, 7 BOS, and 13 stable patients were selected, and serum was collected at 1, 6, and 12 months after LTx. Within the AR group, all 14 patients developed AR in 1 month after LTx, and after treatment, there was no AR found after 6 and 12 months after LTx. There was no clinical diagnosis of BOS at 1 and 6 months in the 7 patients with BOS, while all these patients developed BOS at 12 months after LTx. All 13 stable patients maintained normal graft function at 1 year posttransplant. (B) Statistical methods used in data analysis.

Figure 2

Longitudinal study for differential expression of miRNAs in serum. Quantitative expression of miR-144, -134, -10a, -195, -142-5p, -133b, and -155 was assessed by TaqMan RT-PCR on serum RNA samples from 13 stable LTxRs, 14 LTxRs with AR and 7 BOS LTxRs. Relative expression of these miRNAs was calculated and observed statistically differentially expressed compared to the stable group in 1 month. * p<0.05, ** p<0.01.

Figure 3

Validation of differential miRNAs using independent pediatric patients. Quantitative expression of miR-144, -134, -10a, -195, -142-5p, -133b, and -155 was assessed by TaqMan RT-PCR on serum RNA samples from 8 stable LTxRs, 6 LTxRs with AR and 7 BOS LTxRs. Relative expression of these miRNAs was calculated and observed statistically differentially expressed compared to the stable group. Bonferroni corrections were performed for adjustment to control false positive rate at 0.05, with consideration the number of tested variants. * p<0.05, ** p<0.01.

Figure 4

Validation of differential miRNAs using independent adult patients. Quantitative expression of miR-144, -134, -10a, -195, -142-5p, -133b, and -155 was assessed by TaqMan RT-PCR on serum RNA samples from 16 stable LTxRs, 16 LTxRs with AR and 17 BOS LTxRs. Relative expression of these miRNAs was calculated and observed statistically differentially expressed compared to the stable group. Bonferroni corrections were performed for adjustment to control false positive rate at 0.05, with consideration the number of tested variants. * p<0.05, ** p<0.01.

Figure 5

ROC curves for miRNAs that are significantly different in AR patients as compared to stable patients. ROC curve was performed with AUC for miRNA-142-5p, -155, -195 and combination of miR-155 and miR-195.

Figure 6

ROC curves for miRNAs that are significantly different in BOS patients as compared to stable patients. ROC curve was performed with area under the curve (AUC) for miRNA-10a, -142-5p, -144, -155 and combination of miR-142-5p and miR-144.