Expression Of MiR-146a And TLR4/NF-κB Pathway Protein In HL-1 Cells Cultured In High Glucose,and The Verification Of MiR-146a-5p Target Genes Of Ion Channels

Background: Atrial fibrillation(AF)is one of the most common arrhythmias in clinical sitting.It can cause a series of complications and bring a huge burden to patients and society.Diabetes mellitus is a common metabolic disease and one of the independent risk factor for AF.But the mechanism of diabetes-induced AF is still unclear.Nuclear factor kappa B(NF-κB)plays important roles in apoptosis,inflammation and cell growth.The activated NF-κB in cardiomyocytes in high glucose links to oxidative stress,intracellular inflammatory and fibrosis.But the upstream pathway of NF-κB is still unclear.Toll-like receptor 4(TLR4),Interleukin-1 receptor-associated kinases 1(IRAK1)and tumor necrosis factor receptor associated factor 6(TRAF6)are key molecules in the intracellular signaling pathway and play important roles in the inflammation process.Multiple studies had shown that the TLR4 / IRAK1 / TRAF6 pathway can activate NF-κB,and activated NF-κB can increase its expression by acting on the promoter of miR-146 a.MiR-146 is a one of micro RNA that widely expresses in mammalian cells and participates in various pathophysiological processes.It can act on the m RNA of IRAK1 and TRAF6 to reduce its protein expression.However,the changes of miR-146 a and TLR4 / IRAK1 / TRAF6 in HL-1 cells under high glucose are unclear.Roles of miR-146 a in atrial electrical remodeling need further investigation.Objective: To evaluate expressions of miR-146 a and TLR4/IRAK1/TRAF6/NF-κB proteins in HL-1 cells cultured in high glucose;explore the target relationship between miR-146a-5p,SCN3B and KCNK10 genes.,Reveal the roles that miR-146a-5p may play in atrial electrical remodeling and elucidate the mechanism of atrial remodeling induced by diabetes Method: The study was divided into three parts.Firstly,HL-1 cells were cultured and randomly divided into a control group(5.5mmol/L glucose),a high-glucose group(25mmol/L glucose).Cells in control group was cultured for 24 hours,while the high-glucose group was cultured for 1 hour,6 hours,12 hours and 24 hours.Proteins were extracted from the cells.The levels of IRAK1/TRAF6/NF-κB in the cells were detected by Western Blot.Secondly,HL-1 cells were cultured and randomly divided into a control group,a high-glucose group and a hypertonic mannitol control group(5.5mmol/L glucose +19.5mmol/L mannitol).Cells in the three groups were cultured for 24 hours.RNA and protein were extracted from the cells.The levels of miR-146 a and TLR4/IRAK1/TRAF6/NF-κB in the cells were detected by real-time quantitative polymerase reaction and Western Blot.Thirdly,we constructed recombinant vectors,then co-transfect HEK-293 T cells with miR-146a-5p mimics,miR-146a-5p mimic negative control and 4 recombinant vectors.We compared the ratio of firefly luciferase and Renilla luciferase to verify the target relationship between miR-146a-5p and its predicted target genes.Result: Firstly,compared with the control group,the expression levels of IRAK1,TRAF6 and NF-κB were obviously increased at 12 hours and 24 hours.Secondly,after cultured 24 hours,miR-146 a in HL-1 cells cultured in the high-glucose group was significantly decreased(P<0.05),while the expression levels of TLR4,IRAK1,TRAF6 and NF-κB were significantly increased(P<0.05).There were no statistically significant differences in the levels of TLR4,IRAK1,TRAF6 and NF-κB in the hypertonic group(P>0.05)compared with control group.Thirdly,the dual luciferase reporter assay found that miR-146a-5p could obviously inhibit the luciferase expression of pmir GLO-SCN3B(21.93±0.11 vs.29.75±0.51,P<0.01).And miR-146a-5p had no effect on the luciferase expression of pmir GLO-KCNK10(24.10±0.39 vs.23.45±1.21,P=0.423),pmir GLO-KCNK10-mut(22.90±1.71 vs.22.94±1.10,P=0.977)and pmir GLO-SCN3B-mut(26.00±1.24 vs.26.59±1.18,P=0.580).Conclusion: Firstly,the increaseing tendency of protein expression of IRAK1/TRAF6/NF-κB was obviously at 12 h and 24 h.Secondly,miR-146 a decreased significantly in HL-1 cells while the levels of inflammatory factors TLR4/IRAK1/TRAF6/NF-κB increased significantly in the hyperglycemia state.Thirdly,SCN3B is the target gene of miR-146a-5p,but miR-146a-5p can’t act on the 3’UTR of KCNK10 by binding to the predicted sequence.