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MiR-342-5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor β Signaling.

ABSTRACT: Endothelial cells (ECs) form blood vessels through angiogenesis that is regulated by coordination of vascular endothelial growth factor (VEGF), Notch, transforming growth factor β, and other signals, but the detailed molecular mechanisms remain unclear.Small RNA sequencing initially identified miR-342-5p as a novel downstream molecule of Notch signaling in ECs. Reporter assay, quantitative reverse transcription polymerase chain reaction and Western blot analysis indicated that miR-342-5p targeted endoglin and modulated transforming growth factor β signaling by repressing SMAD1/5 phosphorylation in ECs. Transfection of miR-342-5p inhibited EC proliferation and lumen formation and reduced angiogenesis in vitro and in vivo, as assayed by using a fibrin beads-based sprouting assay, mouse aortic ring culture, and intravitreal injection of miR-342-5p agomir in P3 pups. Moreover, miR-342-5p promoted the migration of ECs, accompanied by reduced endothelial markers and increased mesenchymal markers, indicative of increased endothelial-mesenchymal transition. Transfection of endoglin at least partially reversed endothelial-mesenchymal transition induced by miR-342-5p. The expression of miR-342-5p was upregulated by transforming growth factor β, and inhibition of miR-342-5p attenuated the inhibitory effects of transforming growth factor β on lumen formation and sprouting by ECs. In addition, VEGF repressed miR-342-5p expression, and transfection of miR-342-5p repressed VEGFR2 and VEGFR3 expression and VEGF-triggered Akt phosphorylation in ECs. miR-342-5p repressed angiogenesis in a laser-induced choroidal neovascularization model in mice, highlighting its clinical potential.miR-342-5p acts as a multifunctional angiogenic repressor mediating the effects and interaction among angiogenic pathways.

SUBMITTER: Yan XC

PROVIDER: S-EPMC4802463 | biostudies-other | 2016 Feb

REPOSITORIES: biostudies-other

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Project description:ObjectiveTo explore the effect of miR-1307-5p which specifically inhibits transforming growth factor beta-induced gene (TGFBI) on the biologic behavior of osteoarthritis (OA) chondrocytes.MethodsWe detected miR-1307-5p and TGFBI expression in the cartilage tissue specimens of OA patients and mice, respectively. RNA22 was applied to predict the target gene of miR-1307-5p, and we further verified the relationship by performing a dual luciferase reporter experiment. Enzyme-linked immunosorbent assay was used to measure the expression of matrix metalloproteinase inhibitor-1 (TIMP-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the culture medium of mouse chondrocytes. Quantitative reverse transcription-polymerase chain reaction and western blot were used to measure the expression of Bax and Bcl-2. MTT method was applied to detect the proliferation activity of chondrocytes, while flow cytometry was implemented to detect the apoptosis of chondrocytes.ResultsThe expression of miR-1307-5p in cartilage tissue specimens of OA patients was up-regulated, while TGFBI expression was down-regulated. Compared with normal mice cartilage tissue specimens, the expression of miR-1307-5p in cartilage tissue specimens of OA mouse was increased, while TGFBI expression was decreased (both P<0.05). The results of the dual luciferase reporter experiment showed that TGFBI was a target gene of miR-1307-5p. In cell experiments, compared with the normal group, TIMP-1 and Bcl-2 expression, and cell proliferation activities in all model groups were decreased. IL-1β, IL-6, TNF-α, Bax expression, and cell apoptosis rates were increased (all P<0.05). Compared with the blank group, TIMP-1 and Bcl-2 expression, and cell proliferation activities in the miR-1307-5p inhibitor group and the TGFBI group were increased, while IL-1β, IL-6, TNF-α, and Bax expression, and cell apoptosis rates were decreased (all P<0.05). The changes in all indicators in the miR-1307-5p mimic group were opposite to those of the miR-1307-5p inhibitor group (all P<0.05). There were no significant differences concerning all indicators between the blank group and the NC group, and between the blank group and the miR-1307-5p mimic + TGFBI group (all P>0.05).ConclusionThe suppression of miR-1307-5p expression can increase TGFBI expression, promoting the proliferation of chondrocytes in OA mice, while inhibiting their apoptosis.

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MiR-342-5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor β Signaling.

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