The Role Of MicroRNA-126 In Palmitate-induced HUVECs And Its Possible Mechanism

BackgroundThe incidence and mortality of cardiovascular diseases is front row of various kinds of diseases in China. Atherosclerosis (AS) is a chronic,progressive pathological processes characterized by multiple factors involvement constitutes the most important contributor to cardiovascular diseases. Endothelial cells line the inner surface of blood vessels, is a critical interface between blood and all tissues. Endothelial cells is responsible for different functions including secrete various cytokines, antithrombosis and prothrombosis, regulation of vascular inflammation, cholesterol homeostasis. Endothelial dysfunction is the initial step in the pathogenesis of AS. In recent years lots of studies indicated that aberrant expression and function of miRNAs play a significant role in the pathogenesis of AS. Many studies found alteration of microRNA profiles in endothelium dysfuction by microarray. MicroRNA profiles in the circulation may serve as biomarkers for cardiovascular diseases,MicroRNAs (miRNAs) are a class of 21-25 nucleotide long non-coding RNA molecules. They present in the genome of a variety of organisms, ranging from viruses to plants or metazoans. They negatively regulate gene expression by degradation or translational repression of a target mRNA at the posttranscriptional level. MiRNAs are involved in most biological processes including development, differentiation, growth, migration, and apoptosis. Aberrant expression of miRNA has been closely related to AS. MiR-126 is an endothelial cell (EC)-specific miRNA, which modulates vascular development and angiogenesis. Previous studies depicted that miR-126 inhibits endothelial vascular cell adhesion molecule-1 expression and leukocyte adhesion by activated ECs and thus plays an atheroprotective role. Clinical study indicated that miR-126 may act as diagnostic marker in AS. However, the role of miR-126 in endothelium dysfunction is still unclear.Reactive oxygen species (ROS) play a important role in the progression of atheroclerosis. Increased ROS production can injury endothelial cells via increased apoptosis and endothelial vascular cell adhesion molecules. Sirtuin-3(SirT3), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases. Previous researches showed that overexpression of SIRT3 ameliorated ROS accumulation and inflammation stimulated by palmitate in cultured proximal tubular cells. In addition to SIRT3 can regulate mitochondrial ROS production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox),are important enzymes that also regulate ROS generation in the vasculature.The inhibitor of NOX attenutated excessive ROS production induced by palmitate in animal models.Tumor necrosis factor (TNF) exerts a variety of biological effects, including production of inflammatory cytokines, upregulation of adhesion molecules, proliferation, differentiation and cell death.TNF have been shown to increase ROS production in endothelial cells. Tumor necrosis factor receptor-associated factors (TRAFs) are a family of adapter proteins able to couple TNF receptor family proteins.TRAFs ainvolved in most biological processes including adaptive and innateimmunity, embryonic development, stress reaction. There are seven TRAP family members (TRAF1-7), TRAF7 is a recently identified TRAF family member, the function of TRAF7 is still not completely understood. TRAF7 binds to c-Myb and stimulates its sumoylation, thereby inhibiting its trans-activation activity. TRAF7 also interacts with MEKK3 and potentiates MEKK3-mediated signaling. Finally, TRAF7 is involved in the signal transduction pathway that links Toll-like receptor 2 stimulation to activation of NF-?B transcription factor. TRAF2,-5, and -6 are activators of the NF-?B pathway, leading to the degradation of IB, nuclear translocation of the predominant p50/p65 transcription complex, and activation of genes with pleiotropic roles in cell survival and immune and inflammatory responses.Despite this, it is unclear whether TRAF7 involve in palmitate-induced endothelium apoptosis.Previous researches showed that Nox acitivity was also influenced by myosin light chain kinase (MLCK), over expression of MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47phox at the cell periphery and enhanced ROS production. MLCK is able to phosphorylate myosin light chain (MLC), which results in activation of intracellular cytoskeletal contraction-relaxation cycles. Phosphorylation of MLC by activated MLCK plays a critical role in the development of AS.Our purpose for this study was to investigate the role of miR-126 in endothelium injury in response to palmitate. MiR-126 reduces apoptosis,ROS generation,TNF-a levels in palmitate stimulated HUVECs. MiR-126 regulates NOX and SIRT3 expression in palmitate-induced endothelium. MiR-126 ameliorate the effect of palmitate induced motility in HUVECs. MiR-126 regulates MLCK, pMYPT1, pMLC expression in palmitate stimulated HUVECs. expression in palmitate-induced endothelium apoptosis. We found TRAF7 is a target of miR-126 via dual-luciferase reporter assay, Western Blot and Real-time PCR. The effects of miR-126 in palmitate stimulated HUVECs may regulated by MAPK pathway.ObjectivesTo explore the possible mechanism, we evaluated the effects of miR-126 in endothelium injury in response to palmitate.MethodsFirst of all, HUVECs were incubated with 0.1 mM palmitate or oleate, we detected the morphological characteristics of cell by using inverted phase contrast microscope, MiR-126 expression was determined in HUVECs in response to palmitate using the miRNA plate assay.HUVECs were transfected with miR-126 mimic or antagomir,miR-126 expression was determined in HUVECs using the miRNA plate assay. To determine the role of miR-126 in apoptosis, HUVECs were transfected with miR-126 antagomir, mimic or a scrambled oligonucleotide. Twenty-four hours later, HUVECs were incubated with palmitate, oleate, Apoptosis was then assessed by caspase-3 activity, FCAS and DNA fragmentation. To examine whether miR-126 contributes to ROS production and TNF-a expression, we transfected HUVECs with miR-126 antagomir, mimic or a scrambled oligonucleotide. Twenty-four hours after transfection, HUVECs were incubated with palmitate or oleate for another 24 h and ROS production and TNF-? protein level were measured in HUVECs.HUVECs were transfected with miR-126 antagomir, mimic or a scrambled oligonucleotide. Twenty-four hours later, HUVECs were incubated with palmitate, oleate, p67phox, Rac1, SirT3 protein levels were examined by Western Blot.Wound healing assay was applied to test the effects of miR-126 of motility in palmitate stimulated HUVECs. We detected the expression of the protein MLCK, pMYPT1/MYPT1, pMLC/MLC using Western Blot.We transfected HUVECs with miR126 mimic or antagomir, and examined TRAF7 mRNA and protein levels with quantitative real-time PCR and Western Blot, respectively. We investigated TRAF7 expression in HUVECs in response to palmitate.The functional interaction of miR-126 with the 3'UTR sequences of TRAF7 was investigated by dual-luciferase reporter assay. We identified putative binding sites located in the 3'UTR of TRAF7 using the TargetScan5 software. To clarify whether TRAF7 is adirect target of miR-126, we used a reporter vector containing a luciferase gene followed by the 3'UTR of human TRAF7 mRNA (wt-Luc-TRAF7), we mutated the miR-126 putative binding sites on the 3'UTR of TRAF7 of wt-Luc-TRAF7 by substitution.Western Blot were applied to detect the expression levels of the protein ERK, pERKResults1.Palmitate inhibited miR-126 expression in HUVECs. The miRNA plate assay showed miR-126 was significantly decreased in palmitate-compared with oleate-stimulated HUVECs.2.MiR-126 reduces apoptosis in palmitate stimulated HUVECs. Palmitate treatment induced caspase-3 activation and DNA fragmentation in HUVECs compared with oleate. However, these effects of palmitate on apoptosis were significantly attenuated by miR-126 mimic. The miR-126 antagomir further enhanced caspase-3 activity and DNA fragmentation.3.MiR-126 reduces ROS production in palmitate stimulated HUVECs. MiR-126 mimic reduced, whereas miR-126 antagomir enhanced ROS production in palmitate-stimulated HUVECs4.MiR-126 reduces TNF-a expression in palmitate stimulated HUVECs. MiR-126 mimic reduced, whereas miR-126 antagomir enhanced TNF-a in palmitate-stimulated HUVECs5.MiR-126 reduces p67phox,Racl expression whereas increase SirT3 expression in palmitate stimulated HUVECs.6.MiR-126 reduces the effect of palmitate induced motility in HUVECs. MiR-126 reduces MLCK, pMYPT1, pMLC expression in palmitate stimulated HUVECs.7.MiR-126 modulated TRAF-7 expression in HUVECs. MiR-126 mimic and antagomir did not have any effect on TRAF7 mRNA expression. However, miR-126 mimic significantly reduced TRAF7 protein in HUVECs, miR-126 antagomir enhanced TRAF7 protein expression in HUVECs.8.TRAF7 is a target of miR-126. Overexpression of miR-126 by its mimic inhibited the luciferase activity in wtLuc-TRAF7-transfected HUVECs. The mutation abrogated the inhibitory effect of miR-126 on the luciferase activity in HUVECs. On the other hand, inhibition of miR-126 by its antagomir enhanced the luciferase activity in wt-Luc-TRAF7-transfected HUVECs;the mutation of wt-Luc-TRAF7 abrogated the enhanced effect of miR-126 on the luciferase activity in HUVECs.9.The effects of miR-126 in palmitate stimulated HUVECs may regulated by MAPK pathway.Conclusion1.Palmitate inhibited miR-126 expression in HUVECs.2.MiR-126 reduces apoptosis in palmitate stimulated HUVECs.3.MiR-126 reduces ROS production and TNF-? expression in palmitate stimulated HUVECs.4.MiR-126 modulated p67phox, Rac1, SirT3 expression in palmitate stimulated HUVECs.6.MiR-126 ameliorated the motility in palmitate stimulated HUVECs, reduced MLCK expression and reduced phosphorylation of MLC, MYPT1in HUVECs,7.MiR-126 modulated TRAF-7 expression in HUVECs, TRAF7 is a target of miR-1268.The effects of miR-126 in palmitate stimulated HUVECs may regulated by MAPK pathway.