Large‐scale identification of functional microRNA targeting reveals cooperative regulation of the hemostatic system

Abstract

Essentials MicroRNAs (miRNAs) regulate the molecular networks controlling biological functions such as hemostasis. We utilized novel methods to analyze miRNA-mediated regulation of the hemostatic system. 52 specific miRNA interactions with 11 key hemostatic associated genes were identified. Functionality and drugability of miRNA-19b-3p against antithrombin were demonstrated in vivo. SUMMARY: Background microRNAs (miRNAs) confer robustness to complex molecular networks regulating biological functions. However, despite the involvement of miRNAs in almost all biological processes, and the importance of the hemostatic system for a multitude of actions in and beyond blood coagulation, the role of miRNAs in hemostasis is poorly defined. Objectives Here we comprehensively illuminate miRNA-mediated regulation of the hemostatic system in an unbiased manner. Methods In contrast to widely applied association studies, we used an integrative screening approach that combines functional aspects of miRNA silencing with biophysical miRNA interaction based on RNA pull-downs (miTRAP) coupled to next-generation sequencing. Results Examination of a panel of 27 hemostasis-associated gene 3'UTRs revealed the majority to possess substantial Dicer-dependent silencing capability, suggesting functional miRNA targeting. miTRAP revealed 150 specific miRNA interactions with 14 3'UTRs, of which 52, involving 40 miRNAs, were functionally confirmed. This includes cooperative miRNA regulation of key hemostatic genes comprising procoagulant (F7, F8, F11, FGA, FGG and KLKB1) and anticoagulant (SERPINA10, PROZ, SERPIND1 and SERPINC1) as well as fibrinolytic (PLG) components. Bioinformatic analysis of miRNA functionality reveals established and potential novel links between the hemostatic system and other pathologies, such as cancer, bone metabolism and renal function. Conclusions Our findings provide, along with an in-vivo proof of concept, deep insights into the network of miRNAs regulating the hemostatic system and present a foundation for biomarker discovery and novel targeted therapeutics for correction of de-regulated hemostasis and associated processes in the future. A repository of the miRNA targetome covering 14 hemostatic components is provided.

PCF11 links alternative polyadenylation to formation and spontaneous regression of neuroblastoma

“…This in turn can influence important cellular functions e.g. by modulating the protein output of the affected target RNAs [36][37][38] . Altogether, PCF11 thus represents a global key driver of TREND by directing APA in the BE(2)-C neuroblastoma model system in a direct and most efficient manner ( Fig.…”

A novel rationale for targeting FXI: Insights from the hemostatic miRNA targetome for emerging anticoagulant strategies

“…Several approaches may assist in preventing off-target effects while maintaining the benefits of miRNA target diversity. The functional synergy of several miRNA mimics targeting a single gene, with each individually at sub-effective doses, allows for an increase in effect size and a reduction in side effects (Nourse, Braun, Lackner, Hüttelmaier, & Danckwardt, 2018). For miRNA inhibition a more precise alternative to inhibit miRNA function involves the use of an ASO designed to specifically bind an individual miRNA site within a target mRNA (termed target site blockers, or masks) preventing miRNAs from gaining access to the site (Louloupi & Orom, 2018; Z.…”

“…One of the biggest challenges in the maturation of miRNA-based therapeutics is the identification of key miRNA candidates and targets. There is currently a relatively small number of experimentally validated miRNA:mRNA interactions (Lee, Kim, Muth, & Witwer, 2015) and here knowledge of the miRNA targetome in the hemostatic system is potentially a major trove for future targeted therapeutics (Nourse, et al, 2018), not only for the development of safe anticoagulants but also for developing treatments for coagulopathies.…”

“…This include key genes of the hemostatic system, including the fibrinogens FGA (Z. Chen, et al, 2010;Nourse, et al, 2018), FGB (Fort, et al, 2010) and FGG-alpha (Nourse, et al, 2018); the coagulation factors VII (Nourse, et al, 2018), VIII (Jankowska, Chattopadhyay, Sauna, & Atreya, 2020;Jankowska, McGill, et al, 2020;Nourse, et al, 2018;Sarachana, et al, 2015)], XI (Nourse, et al, 2018); TF (S. Sahu, et al, 2017;Teruel, et al, 2011;Witkowski, et al, 2016;; prekallikrein (Nourse, et al, 2018); TFPI (Ali, et al, 2016;Arroyo, et al, 2017); antithrombin (SERPINC1) (Nourse, et al, 2018); protein c (Nourse, et al, 2018); protein z (Nourse, et al, 2018); protein z-dependent protease inhibitor (SERPINA10) (Nourse, et al, 2018); heparin cofactor 2 (SERPIND1) (Nourse, et al, 2018); VWF (L. ; ADAMTS13 (L. Zhao, Hua, Li, Sun, & Wu, 2015); PAI-1 (SERPINE1) (Luo, et al, 2016;Marchand, Proust, Morange, Lompre, & Tregouet, 2012;Patel, Tahara, Malik, & Kalra, 2011); plasminogen (Nourse, et al, 2018) and the plasminogen activator PLAT (S. . Additionally, miRNAs can impact hemostatic factors indirectly, for example fibrinogen via interleukin-6-mediated signaling (Brock, et al, 2011), factor IX by repressing nonsensemediated mRNA decay (G. Wang, Chai, & Yang, 2016), PAI-1 via SMAD2 signaling (Liao, et al, 2014) and CXCL12 to reduce inflammatory response and thrombosis altering the expression of multiple factors including TF, PAI-1 and VWF (S. Sun, Chai, Zhang, & Lu, 2020).…”

“…In murine models of venous thrombosis, over-expression of miRNAs contributes to thrombus resolution (W. , reduces thrombogenesis (Sahu, et al, 2017), enhances endothelial progenitor cell migration and tubulogenic activity (Meng, et al, 2015) and significantly enhanced angiogenesis and thrombosis recanalization (L. L. Sun, et al, 2019). Antagomir inhibition has been shown to block miR-19b-3p-mediated silencing of SERPINC1 (antithrombin), resulting in an up-regulation of expression and increase in antithrombin activity, illustrating the in-principal druggability of the hemostatic system in a miRNA directed manner (Nourse, et al, 2018). Altogether multiple lines of evidence suggest a critical functional role of miRNA in the hemostatic system.…”