OBJECTIVETo explore the role of microRNA on the myocardial microvascular endothelial cells (CMECs) of ischemic heart rats in the process of angiogenesis and related regulation mechanism.

METHODSMyocardial ischemic rats model was established by coronary ligation.Seven days after operation, the ischemic CMECs were cultured by the method of planting myocardium tissue and identified by immunocytochemistry to observe the biological characteristics of ischemic CMECs angiogenesis, to determine the window period of migration, proliferation, tube formation in the process of its angiogenesis. Dynamic expression changes of microRNA in the process of ischemic CMECs angiogenesis was detected using microRNA chip and further verified by real-time PCR, the core microRNA of the ischemic CMECs was defined and the predicted target genes of core microRNA were determined by bioinformatics methods and real-time PCR. At the same time, the protein expression of target gene and angiogenesis related genes of p38MAPK, PI3K,Akt,VEGF were measured by Western blot.

RESULTSThe CMECs of rats presented typical characteristics of microvascular endothelial cells, and factor VIII, CD31 related antigens were all positively stained by immunocytochemical analysis. The migration window period was on the first day, and the tube formation window period was on the second day of both control and ischemic groups, while the proliferation window period was on the third day for the normal group, and the sixth day for ischemic group. According to the expressional difference and their relationship with angiogenesis, miRNA-223-3p was ultimately determined as the core microRNA in the process of ischemic CMECs angiogenesis, real-time PCR verified this hypothesis. Bioinformatics methods predicted that Rps6kb1 is the target genes of miRNA-223-3p, the pathway analysis showed that Rps6kb1 could regulate angiogenesis via HIF-1α signal pathway. Moreover, the mRNA and protein expression of VEGF, p38MAPK, PI3K,Akt, which were the downstream molecules of Rps6kb1/HIF-1α signal pathway, were also significantly downregulated in ischemic CMECs from migration and proliferation stage.

CONCLUSIONOur results show that the miRNA-223-3p is the core microRNA of ischemic CMECs angiogenesis. MiRNA-223-3p could regulate Rps6kb1/HIF-1α signal pathway, inhibit the process of migration and proliferation of ischemic CMECs angiogenesis. MiRNA-223-3p is thus likely to be a core target for enhancing angiogenesis of ischemic heart disease.

Animals ; Blotting, Western ; Endothelial Cells ; drug effects ; physiology ; Endothelium, Vascular ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; MicroRNAs ; pharmacology ; Myocardial Ischemia ; Myocardium ; Myocytes, Cardiac ; Neovascularization, Pathologic ; Phosphatidylinositol 3-Kinases ; Platelet Endothelial Cell Adhesion Molecule-1 ; RNA, Messenger ; Rats ; Ribosomal Protein S6 Kinases, 70-kDa ; biosynthesis ; Signal Transduction