Screening Of MicroRNAs Related To Biological Pacing And Its Relationship With Targeting Gene HCN4

Objective: Screening the microRNAs associated with biological pacing,and to explore the possible relationship between microRNAs and the key genes HCN4.Methods: MicroRNA microarray was used to analyze the differential expression of microRNAs between sinoatrial node,atrial and ventricular tissue.MicroRNAs significantly down regulated in the sinoatrial node accepted bioinformatics prediction,according to the order of reduction degree combined with bioinformatics prediction results,6 microRNAs were selected as possible targeting candidate microRNAs,using qRT-PCR method to verify the expression of the 6 candidate microRNAs in sinoatrial node,atrial and ventricle tissue was consistent with microarray results,then constructing 6 microRNAs mimcs respectively.Selecting recognized HCN4 gene as the target gene,and then constructing the target gene plasmid: pMIR-REPORT-HCN4-3` UTR,the firefly luciferase experiment was applied to verify the interaction between the 6 microRNAs and HCN4.The microRNAs had inhibitory effect on target gene in the single luciferase experiment were selected,the sea renal luciferase as a reference,dual luciferase reporter assay system was used to analyzing the regulatory effect of microRNAs on target gene HCN4.Results: 1.MicroRNA microarray analysis showed 39 microRNAs differentially expressed between sinoatrial node,atrial and ventricular tissue(t test,flod change ? 2 or ?0.5,12 up regulated,27 down regulated);2.Miranda was used to predict the target genes of 39 differentially expressed microRNAs: 23 microRNAs associated with sinus node function related target genes,19 microRNAs associated with HCN4 target gene,2 microRNAs(miR370,miR1892)had predicted binding sites with HCN4;3.Combining the degree of down-regulation(>11 times)and bioinformatics prediction results 6 candidates microRNAs: miR370,miR1892,miR654,miR3090,miR380,miR19 b were selected.It was confirmed by qRT-PCR that the differential expression of 6 candidate microRNAs in the sinoatrial node,artrial and the ventricular tissue was consistent with the microarray results.4.Single luciferase experiment proved: mmu-mir-370-3p could inhibit the expression of wild type HCN4 series luciferase activity,but had no significant difference compared with control group(P>0.05);mmu-miR-380-5P had no inhibitory effect,and no significant difference compared with control group(P>0.05);miR654,miR1892,miR3090 and miR19 b had significantly inhibited effect(P<0.01).5.Dual luciferase reporter assay showed that mmu-miR-654 mimics could inhibit the expression of wild type HCN4 series luciferase activity,the inhibition rate was 25%(P<0.001),but which could not inhibit the expression of mutant HCN4 series luciferase activity,indicating there was a direct inhibitory relationship between them.As the other 3 microRNAs,double luciferase reporter assays showed no significant difference between the wild type and mutant target genes,indicating that there was no direct inhibitory relationship between them.Conclusion: The mir-654 had direct targeted inhibition on HCN4 gene.The mir-1892,mir-3090,and mir-19 b had inhibition and no targeted inhibition on HCN4 genes.The mir-370,mir-380 had no inhibition on HCN4 genes.