MicroRNA-23a regulates the tissue factor alternative splicing and thrombogenicity

T. Tabaraie, M. Witkowski, J. Friebel, A. Doerner, U. Landmesser, U. Rauch (Berlin, Germany)

Vascular Wall
Date: 17.02.2017,
Time: 08:00 - 09:15

Objective: Alternative splicing (AS) provides a crucial means to ensure the plasticity of the proteome within the vasculature. Vascular pro-inflammatory cytokines, such as Tumor necrosis factor(TNF)α, induce the transcription of Tissue Factor (TF), the primary initiator of the extrinsic clotting cascade. Constitutive splicing yields the thrombogenic full length (fl)TF, while AS leads to expression of the less procoagulant alternatively spliced (as)TF. Among other factors, the splicing event is governed by serine/arginine-rich proteins (SRPs), which are regulated by splice kinases, such as topoisomerase(Top)I. The role of miR-driven regulation of alternative splicing remains largely unknown. Here, we sought to elucidate the role of miRs in AS of TF.

Methods: In silico analysis were performed to identify potential miRs involved in AS. Next, human microvascular ECs (HMEC-1) were transfected with candidate miRs and then stimulated with TNFα to induce expression of both TF isoforms. Those miRs with an effect on the differential TF isoform expression and thrombogenicity were chosen for further investigation. The expression of splice factors and the phosphorylation pattern of SR proteins were assessed after transfection of ECs with candidate miRs. Finally, the miR-depending effect on the luciferase activity of a Top1-3’UTR reporter plasmid was analyzed to confirm a specific miR target as the underlying effect on AS.

Results: Among the candidate miRs, miR-23a led to enhanced expression of asTF in HMEC-1. In contrast, miR-23a-transfection caused reduced expression of flTF and TF-depending FXa generation. Mechanistically, miR-23a reduced the mRNA and protein level of the splice kinase Top1 and treatment of HMEC with a Top1 inhibitor mimicked the miR-23a effect on TF AS. Western blot analysis revealed distinct changes in the phosphorylation pattern of SRPs involved in TF AS. Finally, we confirmed the Top1 transcript as a direct target for miR-23a.

Conclusion: Vascular miR-23a alters TF alternative splicing via regulation of Top1 and, thereby, reduces TF-mediated procoagulability in human ECs. miR-23a may provide a valuable tool to modulate isoform expression of factors involved in hemostasis and vascular homeostasis.