The Regulation Of Angiogenin On MiR-141Expression And Its Role In Angiogenesis

Angiogenesis involves in tumor growth and metastasis, and thus, anti-angiogenesis has been considered as an effective strategy for tumor therapy. Angiogenin (RNase-5, ANG), one of the key angiogenic factors, plays critical roles in angiogenesis and tumorigenesis and is considered as a potential target for anti-angiogenic therapy. However, the underlying mechanism of action of ANG during angiogenesis is poorly understood. MicroRNAs (MiRNAs) are a class of highly conserved non-coding small RNA molecules that repress target gene expression post-transcriptionally, and play important roles in various of bioprocesses, including angiogenesis and tumorigenesis. This drives us to hypothesize that miRNAs may be involved in ANG regulated angiogenesis. In this study, we have investigated the molecular mechanism of ANG induced angiogenesis by identifying ANG responsive miRNAs, and further exploring the role of the miRNAs in angiogenesis.We firstly applied miRNA array screening assay and analyzed the differentially expressed miRNAs in human umbilical vein endothelial cells (HUVECs) after ANG and bFGF stimulation, and screened out six common miRNAs in response to both ANG and bFGF stimulation, including five down-regulated miRNAs (miR-141, miR-299-3p, miR-329, miR-541and miR-589*), and one up-regulated miRNA (miR-381). MiR-141and miR-381were identified as angiogenesis-related miRNAs by an in vitro angiogenesis model. Furthermore, over-expression of ANG down-regulated miRNA expression level while knocking-down of ANG up-regulated miRNA expression level. Functional analysis showed that miR-141could inhibit HUVECs proliferation, migration and tube formation in vitro, and angiogenesis in vivo. Rescue assay showed that ANG enhanced tube formation in vitro in miR-141over-expressed HUVECs, suggesting that ANG could induce angiogenesis through down-regulation of miR-141.To explore the molecular mechanism of miR-141in inhibition of angiogenesis, six target genes of miR-141, which related to angiogenesis, were screened out by in silico analyses, functional annotation and literature retrieval. Interestingly, two target genes TGFB2and CXCL12β had been reported as miR-141target genes when we were conducting this study. The results by RT-qPCR, western blot and dual-luciferase report assays showed that three new target genes of miR-141(GAB1, GATA6and NRP1) were identified. Furthermore, we found that ANG regulated expression of these target genes by down-regulation of miR-141. Further functional assays showed TGFB2, CXCL12β and GAB1could partially rescue the anti-angiogenic effect of miR-141.To elucidate the molecular mechanism of ANG in down-regulation of miR-141, we firstly analyzed the pri-miR-141and pre-miR-141expression levels to test whether ANG regulates miR-141transcriptionally or post-transcriptionally. RT-qPCR results showed that the levels of pri-miR-141as well as pre-miR-141were not changed after ANG treatment, indicating that ANG regulates miR-141post-transcriptionally. Importantly, ANG with mutant in ribonuclease domain did not affect miR-141expression level, suggesting that the ANG’s ribonuclease activity is critical in down-regulating miR-141. In vitro ribonuclease degradation assay showed that ANG degraded mature miR-141through its endonuclease activity.Taken together, ANG could induce angiogenesis through degradation of mature miR-141, which results in up-regulaton of its downstream target genes TGFB2, CXCL12β, GAB1, GATA6and NRP1. Our study for the first time reports that the ANG regulates miRNAs turnover as an endonuclease, and shed light on a novel mechanism of ANG induced angiogenesis.