The Role And Mechanism Of MicroRNAs In The Age-dependent Degeneration Of SAN Function

Objective:The number of elderly patients with sinus node dysfunction increases every year, it becomes a great burden to society, but the current diagnostic tests for this condition obsolete. We can not achieve early diagnosis,and bring a great inconvenience for the treatment of this disease.In recent years, the rapid development of research on disease biomarkers, especially Yang Baofeng research on the role of microRNAs in heart failure and cardiac hypertrophy.We propose not only inspired by the power of older sinus node dysfunction research of biological markers in the diagnosis of disease, and to our research provides a method reference.1.MicroRNAsmicroRNAs, a length of 20-25 nt single-stranded non-coding RNAs, play abiological function by two main ways:microRNAs reducemRNAs stability by binding mRNAs, so that the expression of the target gene has been down-regulated,or block the protein translation process by binding to the mRNA, so that reduce target gene expression.In recent years, studies have shown that microRNAs play an important role in the pathophysiology ofdifferentiation and thedevelopment of disease.In addition, the resule of clinical application made us known that microRNAs can be used as biological markers in disease diagnosis.lt has been widespread attention and applicationin myocardial damage, cancer, and liver function monitoring.2.The age-dependent degeneration of SAN functionSinus nodelocated theCrista, is the pacemaker center of the heart.Increase with age, the sinus node and surrounding tissue gradually fibrosis, sinus node cells reduced by apoptosis, orlon channel protein gene expressionof the sinus node pacemaker cells and the pacing current spectrum changed.Under the joint action of these two mechanisms, sinus node pacemaker function gradually diminish, present to the specific performance of the intrinsic heart rate decrease and sinus node conduction time prolonged.Due to the severity of sinus node dysfunction and mortality was significantly associated with cardiovascular and increasing people’s attention. 3.Pacing related geneThe study found that pacing related genes include the ion channel protein gene in the sinus node cell depolarization, and the effects the signaling connexin gene in the sinus node,like the main hyperpolarization activated cyclic nucleotide-gated cation channel gene, L-type calcium channel gene, potassium ion channel gene connexin gene.Theexpression remodeledwith age in the sinus node, resulting in intrinsic heart rate drops.It has been proved by animal experiments.There is a close relationship between, microRNAs and apoptosis, inflammation, ion channel remodeling.In order tofind older sinus node dysfunction biological markers of disease diagnosis and to clarify the pathogenesis,we will explore the presence of microRNAs in elderly patients’blood,proved by cDNA microarray and quantitative PCR validation spectrum. We pick out three significant differences microRNAs, microRNA-103, microRNA-107, microRNA-1976.Then we will verify that these small molecules whether affect heart function by changing target gene expression,for providing a theoretical basis for clinical diagnosis and treatment.Methods:1.Elderly patients with sinus node dysfunction, serum microRNAs expression profile differences and target gene predictionBlood collected in elderly patients and healthy elderly people.We selectedthe differentially expressed significant microRNAs after the expression by profiling chip. Predicted target genes By Miranda, mirbase, targetscan.2.Luciferase reporter system confirms elderly patients with sinus node dysfunction abnormally elevated microRNAs targeting the sinus node pacemaker-related genesConstruct of microRNA expression plasmid, Luc-UTR reporter gene plasmids.Make it into 293T cells. It is determined whether the microRNA target gene 3’UTR have a role in whether a clear role in the sinus node pacemaker microRNA target genes by detecting luciferase activity.3.Abnormally elevated microRNAs in human sinoatrial node cells derived ips pacing ion channel proteins associated CACNA1C, CACNA1D, CACNA1I, KCNQ1 and KCNH2 expression levels of influenceBy marking HCN4 immunofluorescence experiments, analysis the percentage ofsinoatrial nodecells.Microscopic imaging techniques leave plasmid instant turn around photos and videos.Statistical analysis the change of the beating, verify microRNAs affect sinus node function.Determination ips sinus-derived cells, before and after the instant turn, calcium and potassium channels in each channel mRNA and protein expression levels,to prove microRNAs regulated the pacing-related genes by determinating ips sinus-derived cells, before and after the instant turn, calcium and potassium channels in each channel mRNA and protein expression levels.Results:1. Elderly patients with sinus node dysfunction and blood expression microarray profiling target gene predictionmicroRNAs was significantly differentially expressed,including microRNA-103, microRNA-107, microRNA-1976. Through target gene prediction software Miranda, mirbase, targetscan,wepredicted thattarget genes of microRNA-103 and microRNA-107 was KCNQ1 and KCNH2, the target gene of microRNA-1976 was CACNA1C, CACNA1D and CACNA1I.2. The targets of abnormal increase microRNAs inelderly sinus node dysfunction patientsDeterminethe targets of abnormal increase microRNAs (generally normalized <0.85) after the significance tests (general P<0.05) and the results. Firefly and Renilla Results Results for the ratio:1976+CACNA1I-U1 is 0.836885,1976+CACNA1D-U1 is 0.903125,1976-CACNA1C-U1 is 0.740264,103a+KCNQ1-U1 is 0.693293,103a+ KCNH2-U1 is 0.746934,107+KCNQ1-U1 is 0.820935,107+KCNH2-U1 is 06538193.The percentage of the sinus node cells byImmunofluorescenceImmunofluorescence showed the proportion of sinus node cell is about 15%.4.The statistical analysis by culturing 72h of microRNAs group and control group after transient transfected plasmid about cellbeat frequencyAfter instant turn for 72h of microRNA-103 group and the control plasmid group for statistical analysis,we can clearly see that there is a significant difference between the two groups, microRNA-103 group decreased.72h culture of microRNA-107 group and the control plasmid group for statistical analysis, we can clearly see that there are significant differences between the two, it was reduced.72h culture of microRNA-1976 group and the control plasmid group for statistical analysis, we can clearly see that there are significant differences between the two, it was slowed down.5.Calcium channel and potassium channel expression after inpoting microRNAsThe expression levels of microRNAs target gene mRNA changes, microRNA-103, the target gene mRNA (KCNH2, KCNQ1) microRNA-107 expression were elevated levels.The microRNA-1976 that the target gene CACNA1C, reduce the expression level of mRNA of CACNA1D,but CACNA1I is not too obvious.Western blot results showed that, microRNA-103, microRNA-107 make KCNH2, upregulation of expression of KCNQ1,microRNA-1976 make CACNA1C, and CACNA1I encoded protein CACNA1D down-regulated.The results are consistent with the RTPCR.Conclusion:1.We found that microRNAs differentially expressed in elderly patients blood, like microRNA-103, microRNA-107, microRNA-1976, suggesting that it may be used in the diagnosis of disease.2.We verified that pacing-related targets genes has been targeted by microRNAs differentially expressed.3. We demonstrated microRNA-103, microRNA-107 made KCNH2, KCNQ1 upregulated in human sinoatrial node cells, and microRNA-1976 made calcium channels ganes downregulatedto regulate the beating of sinus rhythm.