Posttranscriptional Regulation of 14q32 microRNAs by RNA Binding Proteins CIRBP and HADHB during Vascular Regeneration after Ischemia:Posttranscriptional regulation of 14q32 microRNAs

After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the microRNA genes’ order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32, and we aimed to identify these RBPs. To identify proteins responsible for this posttranscriptional regulation, we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of cold-inducible RBP (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) to the precursors of late-upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hindlimb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329 but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB−/− 3T3 cells, which display reduced expression of miR-329 and miR-495 but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs. Keywords: microRNA, 14q32, microRNA cluster, miR-329, miR-495, HADHB, CIRBP, RNA binding proteins, ischemia, hindlimb ischemia mode

Myostatin Inhibits Vascular Smooth Muscle Cell Proliferation and Local 14q32 microRNA Expression, But Not Systemic Inflammation or Restenosis

Myostatin is a negative regulator of muscle cell growth and proliferation. Furthermore, myostatin directly affects the expression of 14q32 microRNAs by binding the 14q32 locus. Direct inhibition of 14q32 microRNA miR-495-3p decreased postinterventional restenosis via inhibition of both vascular smooth muscle cell (VSMC) proliferation and local inflammation. Here, we aimed to investigate the effects of myostatin in a mouse model for postinterventional restenosis. In VSMCs in vitro, myostatin led to the dose-specific downregulation of 14q32 microRNAs miR-433-3p, miR-494-3p, and miR-495-3p. VSMC proliferation was inhibited, where cell migration and viability remained unaffected. In a murine postinterventional restenosis model, myostatin infusion did not decrease restenosis, neointimal area, or lumen stenosis. Myostatin inhibited expression of both proliferation marker PCNA and of 14q32 microRNAs miR-433-3p, miR-494-3p, and miR-495-3p dose-specifically in cuffed femoral arteries. However, 14q32 microRNA expression remained unaffected in macrophages and macrophage activation as well as macrophage influx into lesions were not decreased. In conclusion, myostatin did not affect postinterventional restenosis. Although myostatin inhibits 14q32 microRNA expression and proliferation in VSMCs, myostatin had no effect on macrophage activation and infiltration. Our findings underline that restenosis is driven by both VSMC proliferation and local inflammation. Targeting only one of these components is insufficient to prevent restenosis