MicroRNA-126a Directs Lymphangiogenesis Through Interacting With Chemokine and Flt4 Signaling in Zebrafish

Abstract

Objective— MicroRNA-126 (miR-126) is an endothelium-enriched miRNA and functions in vascular integrity and angiogenesis. The application of miRNA as potential biomarker and therapy target has been widely investigated in various pathological processes. However, its role in lymphatic diseases had not been widely explored. We aimed to reveal the role of miR-126 in lymphangiogenesis and the regulatory signaling pathways for potential targets of therapy. Approach and Results— Loss-of-function studies using morpholino oligonucleotides and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system showed that silencing of miR-126a severely affected the formation of parachordal lymphangioblasts and thoracic duct in zebrafish embryos, although their development in miR-126b knockdown embryos was normal. Expression analyses by in situ hybridization and immunofluorescence indicated that miR-126a was expressed in lymphatic vessels, as well as in blood vessels. Time-lapse confocal imaging assay further revealed that knockdown of miR-126a blocked both lymphangiogenic sprouts budding from the posterior cardinal vein and lymphangioblasts extension along horizontal myoseptum. Bioinformatics analysis and in vivo report assay identified that miR-126a upregulated Cxcl12a by targeting its 5′ untranslated region. Moreover, loss- and gain-of-function studies revealed that Cxcl12a signaling acted downstream of miR-126a during parachordal lymphangioblast extension, whereby Flt4 signaling acts as a cooperator of miR-126a, allowing it to modulate lymphangiogenic sprout formation. Conclusions— These findings demonstrate that miR-126a directs lymphatic endothelial cell sprouting and extension by interacting with Cxcl12a-mediated chemokine signaling and Vegfc-Flt4 signal axis. Our results suggest that these key regulators of lymphangiogenesis may be involved in lymphatic pathogenesis of cardiovascular diseases.

CRISPR/Cas9 editing reveals novel mechanisms of clustered microRNA regulation and function

“…Initial reports demonstrated the feasibility of using the CRISPR/Cas9 platform in targeting miRNA expression albeit inducing a varying degree of inhibition 17 , 18 . Further studies improved the efficiency 19 – 21 . In vivo , the CRISPR/Cas9 platform was used to delete a 53-kb fragment and generate knockout mice to elucidate the function of the largest miRNAs cluster in mice 22 , while the first miRNA double knockout mice using the CRISPR/Cas9 system have also been reported 23 .…”

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“…Confocal imaging was performed as previously described (Chen et al, 2016; Zou et al, 2011). The embryos were anesthetized in 0.04% Tricaine (Sigma-Aldrich, MO) medium, then were mounted in low melting point Agarose (Sigma-Aldrich, MO).…”

Recent Advances in the Genetics of Atherothrombotic Disease and Its Determinants

“…36 Rajput et al 37 demonstrated that suppression by miR-150 of angiopoietin-2 was important for repair of vascular injury. In a related vein, Chen et al 38 observed in zebrafish that miR-126a direct lymphangiogenesis through interactions with chemokine and Flt4 signaling. MiR-126 has also been explored in angiogenesis therapeutics by Cao et al 39 Their study revealed proangiogenic effects of miR-126-3p when coupled with the ultrasound-targeted microbubble destruction delivery method in rodents with chronic hind-limb ischemia.…”