B66-10 Endostatin-induced Mir-155 Drives Dysregulation of Tgf-beta Signaling Through Ski and Smurf2 Modulation

Abstract Rationale Endostatin, an angiostatic peptide, is elevated in pulmonary arterial hypertension (PAH) and connective tissue diseases, in particular systemic sclerosis. The proinflammatory microRNA, miR-155, is implicated in autoimmune and cardiovascular diseases. Previously, we showed that recombinant endostatin (rES) represses genes involved with TGF-β signaling at the mRNA level in human lung microvascular endothelial cells (HMVEC-L). Building on these findings, we used miRNA arrays to identify regulatory microRNAs (miRs) and found that hsa-miR-155 was a leading rES-induced candidate. To further explore their regulatory connection, we performed RNAseq using rES and miR-155 mimetics. Methods HMVEC-Ls were challenged with rES, mimetic miR-155, or their appropriate controls for 24 hours and analyzed by bulk RNA-sequencing or miR TaqMan OpenArray. Differential gene expression (DGE) was performed on the filtered and normalized counts using edgeR. Genes were considered differentially expressed if the absolute fold change was 1.2 and FDR was 0.05. Gene set testing using the TargetScan annotation for miR-155 target genes was used to test enrichment of rES DEGs using camera, fry, and romer functions from edgeR. To compare the overlaps of differentially expressed genes after rES or miR-155 treatment, we used UpSet plots. Over-representation analysis (ORA) was then performed on up- and down-regulated genes using the MSigDB Hallmark, DisGeNET, and TargetScan databases to identify common signatures using clusterProfiler. Transcription factor and pathway activity were assessed using decoupleR (dcR). Results DGE analysis revealed 1036 and 3564 genes upregulated at 24 hours by rES and miR-155, respectively, with 1447 and 3103 downregulated. miR-155-treated HMVEC-Ls showed a primarily inflammatory signature by ORA and dcR, indicated by the positive enrichment NF-κB and JAK-STAT pathways. Gene set testing of rES induced DEGs using miR-155 regulated target genes revealed significant enrichment of downregulated genes. UpSet plots revealed 309 downregulated genes shared between rES and miR-155, which were enriched for TGF-β signaling. Overlap of down regulated miR-155 genes and downregulated rES-regulated miR-155 targets resulted in the identification of 22 genes. Functional enrichment of these 22 genes also showed TGF-β signaling as the top affected pathway and pointed to 2 genes (SKI, SMURF2) that are involved in the repression of TGF-β signaling. Conclusions These results identify SKI and SMURF2 as potential downstream mediators of endostatin and miR-155 and offers a mechanistic explanation for how endostatin may disrupt the TGF-β pathway. Further, these results provide a new connection between angiostasis and inflammation, which may be relevant to the pathogenesis of SSc-PAH. This abstract is funded by: NIH HL132153/R01(RLD/PMH)