The Study About The Regulation Of MicroRNA On The Expression Of HERG Gene

Bachground and Objective: Malignant ventricular arrhythmia is the main cause of suddencardiac death (SCD). Because of the pathogenesis is still unknow and the incentive is very complex,it is difficult to get effective prevention and treatment for the malignant ventricular arrhythmia. Thepoint swinging pattern of Torsade de points (TdP) by the acquired long QT syndrome (aLQTS) is aimportant cause of SCD. A number of studies have demonstrated that the rapidly activatingcomponent of delayed rectifier potassium current (IKr), coded by the KCNH2(hERG) gene, canreduce the myocardial repolarization to produce TdP easily. In recent years, some studies haveshown that the abnormal expression of the microRNA (miRNA) can cause the disorder expressionof ionophorous protein and induce the arrhythmia. Six miRNA we originally predicted may berelated to the hERG gene. The aim of this study is to further verify the correlation between the fourmiRNAs and the hERG gene, meanwhile we will investigate the effect of miRNA on the functionof Herg gene to provide a new way for study the mechanism of aLQTS.Methods: First, the WT-hERG was transfectde into U2OS cells, and the monoclonal cell,stable expressing the WT-hERG gene, was screened by G418. Second, the hERG-U2OS cellmodle were intervented by miRNAs and the AMOs. Then, the related mRNA was detected by realtime PCR(RT-PCR), the related protein expression of hERG was detected by western blot, and therelated protein locatization of the hERG encoded was detected by confocal.Results: Two distinct protein bands (135KD and155KD) showed that the U2OS cells stableexpressing the hERG gene was successfully constructed from the result of western blot. Themolecular biology experimental results are as follow:1. RT-PCR shows that four miRNAs (miR-134、143、103、3619) can reduce theexpression of mRNA, which related to the hERG gene, and antagonizing the miRNAs cancovery the expression of the mRNA; miR-147and miR-185can not change theexpression of the mRNA.2. Western blot shows that four miRNAs (miR-134、143、103、3619) can reduce theexpression of protein, which related to the hERG gene, and antagonizing the miRNAs cancovery the expression of the protein; miR-147and miR-185can not change the expressionof the protein. 3. Confocal results show that four miRNAs (miR-134、143、103、3619) can reduce theexpression of the immature protein in the cytoplasm and the expression of the matureprotein on the cell membrane of hERG-U2OS cells. AMOs sclencing can inhibit thenegative effect of them. But miR-147and miR-185can not change the expression of theprotein either in the cytoplasm or on the cell membrane.Conclusion: The experiment verified that hERG gene was a target gene of miR-134,143,103,3619, confirmed that the four miRNAs could downregulation the expression of mRNA andprotein, which were coded by WT-hERG. While silencing the miRNAs above, could inhibit theeffect..