Study On The Mechanism Of Hydrogen Sulfide Regulation Of Angiogenesis By MicroRNAs

Hydrogen sulfide (H2S) is commonly recognized as a colorless gas with an offensive odor of rotten eggs. To date, it’s becoming increasingly clear that mammalian cells also produce H2S. The plasma levels detected in rat and normal human are25-65and25-80lmol/L. Endogenous H2S is synthesized from L-cysteine mainly by two enzymes, cystathionine (3-synthase (CBS) and cystathionine y-lyase (CSE), both of which are widely expressed in mammalian tissues and cells. CBS mainly exists in brain and nervous system, whereas CSE in the cardiovascular system. Endogenous H2S is involved in physiological regulation of nervous system, cardiovascular system, digestive system and inspiratory system. Since these descriptions, H2S is now regarded along with carbon monoxide (CO) and nitric oxide (NO) as a third gasotransmitter.Angiogenesis, the formation of new capillaries from preexisting blood vessels, is dependent on endothelial cell proliferation, migration, and apoptosis processes. Angiogenesis occurs in many physiological and pathological processes, such as embryonic development, wound healing and tumor growth.MicroRNAs (miRNAs) are a class of endogenous, conserved, non-coding RNAs that can pair with target sites in3’untranslated regions (3’UTR) in mRNAs of protein-coding genes to repress their expression. In mammals, miRNAs are predicted to control the activity of approximately30%of all protein-coding genes, and they have been shown to participate in the regulation of almost every cellular process examined, including angiogenesis.Our previous studies have found that H2S promotes angiogenesis in vitro by promoting endothelial cells (EC) growth, migration and tube-like structure formation. Neovascularization has also been reported to be promoted by H2S in an in vivo mouse model of a Matrigel plug assay, as well as chicken chorioallantoic membranes.In a rat hindlimb ischaemia model,H2S promotes significant angiogenesis and improves the blood supply of ischemic region. However, the mechanisms underlying H2S-induced angiogenesis remain to be investigated further. At present, there was no report about the connections between microRNAs and H2S biological function. Our subject focused on microRNAs, to observe wether exogenous administration of H2S could induce the changes of miRNAs expression, and wether these miRNAs play part roles on proangiogenic effect of H2S.Firstly, the angiogenic effects of H2S on Human Umbilical Vein Endothelial Cells (HUVECs) were explored. In the past, the proangiogenic effect of H2S was found by using RF/6A cell line, which is different from primary endothelial cells. In our experiments, primary culcured HUVECs were treated with NaHS as a H2S donor. BrdU incorporation assay, scrach wound healing assay and tube-like structure formation assay were used to evaluate cell proliferation, migration and tube formation. The results showed that NaHS (10μmol/L) stimulated endothelial cell growth, and NaHS (30-200μmol/L) promoted wound healing ability and tube formation. Our finding further confirmed the the proangiogenic effect of H2S in endothelial cells.Secondly, using microRNAs microarray analysis,6microRNAs (miR-923, miR-154*, miR-483-5p, miR-195*, miR-640and miR-451) and6microRNAs (miR-421, miR-604, miR-192, miR-29b-1*, miR-455-3p and miR-136*) were found to be down-or upregulated by H2S in HUVECs. Combined the references and predicted microRNA targets,3microRNAs (miR-640, miR-455-3p and miR-192) were further validated by real-time PCR.Thirdly, functions of the3microRNAs (miR-640, miR-455-3p and miR-192) were examined by transfecting specific Anti-MiRNA Inhibitor molecules and Pre-MiRNA Inhibitor molecules. Our results suggested that miR-640acts as a repressor but miR-455-3p and miR-192as promoters in cell migration and formation of capillary-like networks in HUVECs.Finally, bioinformatics predictions by online searching programs and western blotting were performed to analysis possible targets of the3microRNAs. Results were as follows:1、miR-6403binding sites were predicted between miR-640mRNA and hypoxia inducible factor-la mRNA3’UTR. Increased level of miR-640decreased HIF-la expression, and decreased level of miR-640enhanced HIF-la expression investigated by western blotting. In addition, NaHS (50μmol/L) induced HIF-la expression and its downstream VEGF expression. These results suggest that miR-640may play a role in H2S-induced angiogenesis through decreased HIF-la expression. Luciferase reporter experiment needs to be operated for further verification. 2、miR-192According to the references, TGF-β increases miR-192expression, which reduces PTEN expression so that increases the Akt phosphorylation. In our previous study, it was found that H2S elevated levels of TGF-β expression in HUVECs, suggestting the elevated levels of miR-192may be a direct effect of H2S or indirectly mediated by TGF-β. Ectopic re-expression of miR-192decreases PTEN expression, thus increases the Akt phosphorylation; and vice versa. The results suggest that miR-192may promote angiogenesis by targeting the PTEN/Akt pathway. However, NaHS (50μmol/L) didn’t change the level of PTEN expression and Akt phosphorylation. H2S induced endothelial cell migration and tube formation may be not mediated by miR-192/PTEN/Akt pathway. The role of miR-192needs further experiments to explore.3、miR-455-3pBioinformatic predictions showed2binding sites between miR-455-3p and FIH (factor inhibiting HIF1) mRNA3’UTR, that needs further verification.To conclude, our findings indicate that H2S promotes angiogenesis in HUVECs, and also modulates microRNAs expressions. miR-640functions as an repressor of angiogenesis; meanwhile, miR-192and miR-455-3p function as promoters of angiogenesis in vitro. H2S decreases miR-640expression, which is proposed to negatively modulate HIF-la expression, and may mediate the pro-angiogenic activity of H2S.The present study constitutes further understanding towards the proangiogenic effect of H2S, and even provides a new idea that microRNAs involves in the biological effects of the gas molecules.3microRNAs were observed to regulate angiogenesis, which may provide new targets for clinical treatment of illness.