OR53 In vitro and in silico approach to identify HAS-MIR-374A targets associated with antibody mediated rejection

Abstract

Micro-RNAs are short non-coding RNAs that plays important role in regulating mRNA expression. The role played by micro-RNAs in antibodies mediated rejection (ABMR) is understudied. We utilized a combination of Next Generation Sequencing (NGS) to identify micro-RNAs and mRNAs that are involved in the endothelial cells signaling following Class I and Class II treatment. For these in vitro studies, mRNAs extracted from endothelial cells treated with HLA antibodies were subjected to gene expression analysis using Next Generation Sequencing. NGS was performed using 1 microgram of RNA with RINs ⩾8 used to prepare microRNA-Seq libraries using the small RNA Sample Prep Kit following the protocol as described by the manufacturer (Illumina, San Diego, CA). Libraries were clustered to ensure at least 15 million reads per sample on the cBot as described by the manufacturer (Illumina, San Diego, CA). Clustered RNA-seq libraries were paired-end sequenced using version 4 with 1 × 25 cycles on an Illumina HiSeq2500. De-multiplexing and adapter trimming was performed utilizing Illumina’s BaseSpace. The trimmed files were then analyzed utilizing Illumina’s BaseSpace Small RNA application using multiple aligners to align the sequences to multiple small RNA references. DeSeq2 Bioconductor package was used for annotation and differential expression analysis. A list of mRNAs with significant expression increase or decrease (two folds) was compared to either 5 or 3 prime predicted targets of mir-374a obtained from miRBase the database. The Reactome Pathway browser was used to predict most the significantly affected pathways. hsa-mir-374a were found to be up-regulated and down-regulated following treatment of endothelial cells with HLA Class I and Class II. In order to further investigate the role played by hsa-mir-374a in ABMR, we combined the mRNA expression data with mir-374a known published targets and targets identified in silico. The resultant combination was then used to identify a subset of genes known to be down-stream targets of this micro-RNA and are known to be playing a role in the activation of endothelial cells. A total of 30 mRNAs (i.e. 24 were up-regulated and 6 were down-regulated) were found to be associated with mir-374a in treated endothelial cell, compared with the untreated control. The combinatorial effect of the up- and down-regulated mRNAs is predicted to most significantly affect pathways associated with cell death, differentiation and growth, and several signaling pathways that contribute to ABMR.