Abstract
Purpose Endomyocardial biopsy (EMB) is a gold standard to detect acute cellar cardiac rejection (ACR), but due to its invasiveness, a new non-invasive approach is warranted. Our final goal is to identify new biomarkers corresponding to ACR severity. Using RNA-seq analysis, we found specific gene expression profiles in EMB specimens corresponding to the grades of ISHLT criteria. We also established a transcriptome analysis method utilizing an FFPE (formalin fixed paraffin embedded) EMB section remained after pathological examination. Methods 1. Gene expression profiling in fresh frozen EMB section: 7 cardiac recipients who had pathological ACR were enrolled. They were divided into two groups: patients with before and at rejection ISHLT grade 1A (G-going; N=3) and with at and after treating rejection grade 1A (G-resolving; N=4). Transcriptome analysis was performed using a fresh frozen section (0.5 - 3 mm wide, 7 µm thickness). 2. Establishment of a 3’mRNA-seq method utilizing thin FFPE EMB section: 28 myocardial specimens (grade 0 in 7, 1A in 12, 2 in 2, 3A in 4 and quilty in 3) were analyzed. 3’mRNA-seq libraries were prepared using total RNA isolated from an FFPE section (1-2 mm wide, 5 µm) remained after pathological examination. 3. GSEA (Gene Set Enrichment Analysis) and DGE analysis: After RNA-sequencing, gene expression profiling was analyzed by GSEA and clustered by k-means. Results In G-going, we identified up-regulation of the genes mainly related to ACR, interferon gamma, inflammation, and ILG-JAK-STAT3 signaling, while in G-resolving, we identified down-regulation of these genes. Inflammatory responses were also observed in 3’mRNA-seq analysis using FFPE section. We also separated 270 up-regulating genes in grade3A and 229 ACR related genes associated with ISHLT criteria. In our analysis, most of these identified genes were not listed in AlloMap ®. Conclusion Although these analyses were preliminary because of the 1st analysis stage, these technique for analyzing gene expressions using a tiny myocardial sample may have a role to find new biomarkers associated with ACR.
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