miRNA and mRNA Interaction in Circulating Exosomes from Patients with Primary Graft Dysfunction After Lung Transplantation

Purpose Recipient responses to primary graft dysfunction (PGD) after lung transplantation (LTx) have critical implications for the fate of the allograft. Exosome composition is dependent on the parent cell, activation state, and carries biological components including mRNA and miRNA. We hypothesize that there are differences in the miRNA expression and their mRNA targets between subjects with and without PGD. Methods Plasma-derived exosome RNA expression was measured before and 72h after LTx in 10 consented subjects (PGD n=5; non-PGD n=5). RNA libraries were prepared with QIAseq Stranded Total RNA library Kit. miRNA was extracted from exosomes using the exoRNeasy kit. Libraries were sequenced using the NextSeq 500. mRNA-seq and miRNA were normalized with the TMM algorithm, and edgeR was used for differential expression. miRNA targets were assessed for all significant (p<0.05) differentially expressed miRNA using the miRNA Data Integration Portal (mirDIP) and targets scoring in the top 1% were recorded. Comparative analysis between the targets of differentially expressed miRNA and significant differentially expressed mRNA was then performed to find targets for further study. PGD was defined as PGD3 at 48-72 hours after reperfusion. Results In the PGD cohort, comparative analysis between the targets of the overexpressed miRNA and underexpressed mRNA showered that 10 genes were present (TXK, LARS2, MPZL1, BTLA, TRIM63, RAD1, ATLS, CPS2, WDR48, SCRN3) when comparing underexpressed miRNA and overexpressed mRNA, 5 genes were present (CEPT1, FRZB, GPM6A, SLITRK3, UBR3). In the non-PGD cohort when comparing underexpressed miRNA and overexpressed mRNA, 26 genes were present ( CARM1, ELN, PCSK6, PLTP, SLC25A22, SLC6A9, SLC7A2, B4GAT1, TSPAN12, TYW5, ZCCHC2, ZNF615, LMAN2L, SLC16A13, TRPC5, ZNF286B, OLFML2A, SERF1A, ANAPC10, CCNJ, CDH8, IMPG2, LMO3, MIPOL1, TET1, WIPF3). No genes were identified when comparing targets of the overexpressed miRNA and underexpressed mRNA Conclusion We identified a set of miRNAs and their predicted mRNA targets that are consistently differentially expressed between the patients with and without PGD. Future exosome characterization may open the window to noninvasive diagnosis and can lead to new insights into the pathophysiology of PGD. The findings, however, need to be confirmed by other methods in a larger cohort. Recipient responses to primary graft dysfunction (PGD) after lung transplantation (LTx) have critical implications for the fate of the allograft. Exosome composition is dependent on the parent cell, activation state, and carries biological components including mRNA and miRNA. We hypothesize that there are differences in the miRNA expression and their mRNA targets between subjects with and without PGD. Plasma-derived exosome RNA expression was measured before and 72h after LTx in 10 consented subjects (PGD n=5; non-PGD n=5). RNA libraries were prepared with QIAseq Stranded Total RNA library Kit. miRNA was extracted from exosomes using the exoRNeasy kit. Libraries were sequenced using the NextSeq 500. mRNA-seq and miRNA were normalized with the TMM algorithm, and edgeR was used for differential expression. miRNA targets were assessed for all significant (p<0.05) differentially expressed miRNA using the miRNA Data Integration Portal (mirDIP) and targets scoring in the top 1% were recorded. Comparative analysis between the targets of differentially expressed miRNA and significant differentially expressed mRNA was then performed to find targets for further study. PGD was defined as PGD3 at 48-72 hours after reperfusion. In the PGD cohort, comparative analysis between the targets of the overexpressed miRNA and underexpressed mRNA showered that 10 genes were present (TXK, LARS2, MPZL1, BTLA, TRIM63, RAD1, ATLS, CPS2, WDR48, SCRN3) when comparing underexpressed miRNA and overexpressed mRNA, 5 genes were present (CEPT1, FRZB, GPM6A, SLITRK3, UBR3). In the non-PGD cohort when comparing underexpressed miRNA and overexpressed mRNA, 26 genes were present ( CARM1, ELN, PCSK6, PLTP, SLC25A22, SLC6A9, SLC7A2, B4GAT1, TSPAN12, TYW5, ZCCHC2, ZNF615, LMAN2L, SLC16A13, TRPC5, ZNF286B, OLFML2A, SERF1A, ANAPC10, CCNJ, CDH8, IMPG2, LMO3, MIPOL1, TET1, WIPF3). No genes were identified when comparing targets of the overexpressed miRNA and underexpressed mRNA We identified a set of miRNAs and their predicted mRNA targets that are consistently differentially expressed between the patients with and without PGD. Future exosome characterization may open the window to noninvasive diagnosis and can lead to new insights into the pathophysiology of PGD. The findings, however, need to be confirmed by other methods in a larger cohort.