It has been confirmed that the damage of vascular endothelial function and structure is a key step in the occurrence and development of cardiovascular diseases[24]. Therefore, it is necessary to further explore the potential targets leading to endothelial dysfunction, so as to provide new strategies for the prevention and treatment of various cardiovascular diseases. In recent years, the role of microRNA in cardiovascular diseases has become a research hotspot.
As an important mechanical miRNA closely related to cardiovascular diseases[25, 26], miR-92a plays an important role in regulating the dynamic equilibrium of vascular endothelial cells.In recent years, a large number of studies have shown that miR-92a is associated with cardiovascular diseases. Clinical trials showed that miR-92a increased significantly in the serum of patients with cardiovascular diseases[27–31]. Further ROC analysis confirmed that miR-92a alone showed 85% sensitivity and 82.50% specificity in predicting cardiovascular diseases[32]. The highly expressed miR-92a participated in endothelial cell injury through apoptosis, inflammation, endothelial mesenchymal transformation and other pathways,which leads to the occurrence and development of cardiovascular diseases.Wu et al. found that miR-92a expression was increased in aortic arch EC of mice and pigs exposed to disturbed blood flow, which increased EC inflammation and permeability by inhibiting the expression of Phosphatidic Acid Phosphatase Type 2B (PPAP2B)[25].Wiese et al. transfected miR-92a into human aortic endothelial cells (HCAEC) and found that overexpressed miR-92a can directly regulate FAM220A to increase STAT3 phosphorylation, thereby promoting inflammatory responses to cholesterol and lipid accumulation in HCAEC[23].Wang et al. found that highly expressed miR-92a promoted EC apoptosis and later vascular inflammation by inhibiting the expression of endothelial protective molecules kruppel-like factor 2 (KLF2) and kruppel-like factor 4 (KLF4)[33]. However, there are few reports on the regulation of miR-92a in endothelial cell autophagy.
Therefore, in order to investigate whether miR-92a is related to endothelial cell autophagy, EA.hy926 endothelial cells were treated with the autophagy inducer rapamycin (1000nmol/L) for 6 hours to establish the autophagy model of EA.hy926 endothelial cells.The results showed that the Beclin1 protein expression and LC3II/I ratio of EA.hy926 endothelial cells were increased in the rapamycin group, while the level of p62 was decreased;And the immunofluorescence intensity of LC3B was significantly enhanced; Transmission electron microscope observed that there were more autophagosomes formed in the rapamycin group, suggesting that the autophagy model of EA. hy926 endothelial cells was successfully constructed.Which was consistent with the previous report that endothelial cells could be incubated obvious autophagy after incubation with rapamycin at a concentration of 1000nmol/L for 6 hours[34].RT-PCR results showed that miR-92a was significantly down regulated in EA.hy926 endothelial cell autophagy model, suggesting that miR-92a may be involved in the regulation of endothelial cell autophagy.Therefore, in order to further observe the effect of miR-92a overexpression and low expression on the autophagy activity of EA.hy926 cells, we transfected EA.hy926 endothelial cells with miR-92a mimics or miR-92a inhibitors, and the results showed that the miR-92a overexpression could inhibit the autophagy activity of EA.hy926 cells,and the low expression of miR-92a promoted the autophagy activity of EA.hy926 cells.These experimental results suggest that inhibition of endothelial autophagy may be another potential mechanism of miR-92a leading to endothelial dysfunction. However, the mechanism of miR-92a inhibiting the autophagy activity of endothelial cells has been rarely reported.
Studies have shown that microRNAs bind to the 3'UTR region of downstream target genes to regulate cell biological function[35]. Recent studies have also reported that microRNAs play a new role in the regulation of endothelial autophagy.For example, miR-103 protects coronary endothelial cells from oxidative stress damage induced by H2O2 through BNIP3 mediated end-stage autophagy[36];MiR-130a targets the expression of Runx3 to maintain the normal autophagy levels of endothelial progenitor cells (EPC) and ultimately promote the survival of EPC[37];In addition, Lu et al. proposed that propofol treatment could induce the expression of mir-30b, thereby alleviating HUVEC damage caused by hypoxia / reoxygenation (H/R), and its mechanism is that the up-regulated mir-30b regulates HUVEC autophagy activity by targeting the expression of Beclin1[38].
Therefore, in order to further analyze the mechanism of miR-92a inhibiting endothelial autophagy, we used bioinformatics software to screen and predict the target genes of miR-92a.It was found that FOXO3 was a potential downstream target gene of miR-92a, which was also confirmed by subsequent double luciferase experiment.Further western blot and RT-PCR results showed that miR-92a mimics inhibited FOXO3 mRNA and protein expression, while miR-92a inhibitors promoted FOXO3 mRNA and protein expression.Therefore, the above results confirm that FOXO3 is the target gene of miR-92a, and miR-92a inhibits FOXO3 protein and mRNA expression by binding to FOXO3 3 '-UTR region.FOXO3 is a transcriptional regulator factor involved in a variety of cell functions. Previous studies have shown that FOXO3 is a tumor suppressor that regulates the expression of genes related to apoptosis, cell cycle arrest and oxidative stress resistance[39, 40].Recent studies have shown that FOXO3 is associated with longevity and autophagy by directly activating the expression of autophagy related proteins, thereby complementing the core components of autophagy[41–44].In addition, numerous studies have reported that FOXO3 accepts the negative regulation of microRNA and participates in the occurrence of autophagy.Zhang et al. found that the expression of miR-221 was increased and the level of FOXO3 was decreased after myocardial fibrosis. Inhibition of miR-221 expression could inhibit the autophagy activity of cardiomyocytes, and luciferase reporter gene assay confirmed that FOXO3 was its downstream target gene[45];Song et al. proposed that miR-34a inhibits autophagy of alveolar type II epithelial cells in acute lung injury by inhibiting FOXO3 expression, FOXO3 is considered to be a downstream target gene of miR-34a[46];Zhong et al. showed that miR-223-3p regulates autophagy by acting on its target gene FOXO3 and thus participates in osteogenic differentiation of bone marrow mesenchymal stem cells[47].In conclusion, FOXO3 can play an important role in the process of autophagy as a target gene of microRNA.This study has confirmed that the overexpression of miR-92a can inhibit the autophagy function of EA.hy926 cells, and the target gene of miR-92a is FOXO3. Therefore, we speculated that miR-92a may regulate the autophagy function of EA.hy926 cells by regulating the expression of FOXO3.In order to further verify this hypothesis, this study first studied the effect of FOXO3 on autophagy of EA.hy926 cells. FOXO3 siRNA was transfected into EA. hy926 cells to silence FOXO3 gene, and the results showed that FOXO3 siRNA treatment significantly inhibited the autophagy activity of EA.hy926 cells.We also cotransfected miR-92a and siFOXO3 into EA.hy926 cells to observe whether FOXO3 silencing could reverse the regulation of miR-92a on autophagy of EA.hy926 cells. The results showed that FOXO3 low expression could reverse the promoting effect of miR-92a inhibitor on autophagy activity of EA.hy926 cells.This further confirmed that miR-92a regulates the autophagy activity of EA.hy926 cells through targeted inhibition of FOXO3 expression.
Previous studies have confirmed that miR-92a is associated with autophagy. Rogg et al. showed that miR-92a targeted inhibition of ATG4a (autophagy related 4-cysteine peptidase) expression promotes autophagy of human umbilical vein endothelial cells and myocardial cell metabolism[48].In addition, Xia et al. found that the increased expression of LncRNA-MALAT1 in the exosomes of bone marrow mesenchymal stem cells (MSCs) pretreated by hypoxia could inhibit miR-92a and lead to the activation of ATG4a, thereby improving the mitochondrial metabolism of myocardial cells and synergistically promoting the rejuvenation of myocardial cells. Thus showing a strong protective effect in Dox induced cardiac injury[49].Xu et al. observed that the industrial solvent N, N-dimethylformamide (DMF) can induce hepatocyte death. The mechanism is that DMF can reduce the level of mitochondrial miR-92a in HepG2 cells in a dose-dependent manner. Low level miR-92a regulates DMF-induced mitochondrial dysfunction and mitochondrial autophagy in HepG2 cells by mediating BNIP3L.While miR-92a overexpression can inhibit BNIP3L mediated mitochondrial autophagy, protect mitochondrial function, and reduce DMF induced liver injury in vitro and in vivo. Therefore, this study proposed that miR-92a can be used as a potential therapeutic agent for DMF-induced hepatotoxicity according to the characteristics of miR-92a inhibiting autophagy activity[50].However, the autophagy regulation of miR-92a and its targets in EA.hy926 endothelial cells has rarely been reported.This study proved for the first time that miR-92a inhibits the autophagy activity of EA.hy926 cells by inhibiting FOXO3 expression. Therefore, we believe that miR-92a not only induces EC apoptosis and inflammation, but also inhibits the autophagy activity of EC by inhibiting FOXO3 expression, resulting in the injury of EC.Due to the important role of impaired EC autophagy activity in the pathological process of cardiovascular disease, miR-92a inhibitors may be a EC protective factor and may serve as a new therapeutic approach for cardiovascular disease.However, one microRNA can regulate multiple target genes, and the signaling pathways of microRNA regulating autophagy are extremely complex. Therefore, whether miR-92a also participates in the regulation of EC autophagy by regulating the expression of other target genes or signaling pathways needs further study.In addition, EA.hy926 cell line was used as the research object in vitro experiment. But in vivo experiments such as animals, whether miR-92a can affect autophagy activity through the same mechanism needs further research and confirmation.It is believed that with the in-depth study of miR-92a in cardiovascular diseases, the relationship between cardiovascular diseases and miR-92a will become clearer and clearer, which is bound to provide new ideas for the clinical diagnosis and treatment of various cardiovascular diseases.