The Mechanisms Of Soluble Epoxide Hydrolase Inhibitor On Regulating MiRNA-1

Objective:Our previous studies suggest that the soluble epoxide hydrolase inhibitor, t-AUCB (trans-4-[4-(3-adamantan-1-yl-ureido) cyclohexyloxy]-benzoic acid) have anti-ischemic arrhythmic effects. And we have demonstrated that t-AUCB can attenuate ischemic arrhythmias by modulate the expression of microRNA-1(miR-1) and its target gene KCNJ2, GJA1mRNA and its encoded protein Kir2.1,Cx43in ischemic myocardium. Some researches found that the expression of miR-1was regulated by serum response factor (SRF), myocyte enhancer factor-2(MEF-2) and other cardiac transcription factor and signal transduction pathways, and there is interaction between transcription factors and signaling pathways. This study aimed to investigated the mechanism of t-AUCB on regulating miR-1by transcription factor SRF, which contribute to provide the new evidence that the role of t-AUCB on ischemic cardiac arrhythmias.Methods:The Kungming male mice of8weeks old were administered with t-AUCB of different-concentraions (0.2,1,5mg/L) or drinking water for7days. After then, they were operated the myocardial infartion or sham surgery.24hours later, the mice were put to death and their hearts were taken out for next experiments. In the transfection experiment, the Kunming male mice were administered with5mg/L t-AUCB for7days, and after the surgery, they were tale-injected with the transfective agent micrONTM mmu-miR-la-3p agomir or micrONTM mmu-miR-la-3p agomir-negative control for control. The pathology of hearts were detected with HE (hematoxylin-eosin) staining. miR-1, SRF mRNA expression were determined by Real-time PCR. SRF protein expression were quantified by Western-blotting.Use three different sequences of siSRF transfection HL-1cells to make SRF silence. Extraction of cellular proteins. SRF protein expression were quantified by Western-blotting. And choose the best sequence for silence SRF protein expression.Results:1. In mice,24hours after the myocardial infarction, the SRFmRNA levels in the ischemic region of mice hearts was increased in the drinking water+MI group than the drinking+sham group, whereas SRF protein expression were significantly decreased(all P<0.05);2. t-AUCB can dose-dependently decreased the SRF mRNA levels in ischemic myocardium, while SRF protein increased (all P<0.05);3. micrONTM mmu-miR-la-3p agomir could increased the expression of miRNA-1,5mg/L t-AUCB could antagonize these effects(all P<0.05); 4. micrONTM mmu-miR-la-3p agomir could increased the expression of SRFmRNA, while SRF protein expression was significantly reduced(all P<0.05);5. In mice,24hours after the myocardial infarction, there is no statistical differences btween5mg/Lt-AUC+agomir+MI group and agomir+MI group on the SRF mRNA levels in the ischemic region of mice hearts(P>0.05). While SRF protein expression was reduced in the5mg/Lt-AUCB+agomir+MI group than in the agomir+MI group (P <0.05)6. In HL-1cells, siSRF1is the best choice for silence SRF protein expression.Conclusions:The expression of miRNA-1is controlled by SRF and some other factors, expression of SRF is repressed by miRNA-1. Thus, these findings identify a complex feedback regulation mechanism in which SRF participate to control miRNA-1expression. t-AUCB can change the expression of SRF and miRNA-1and inhibited ischemic arrhythmias.