The Effect Of Mir-19b On KCNA5Gene Expression In H9c2Cells

Objective:To investigate the effects of miR-19b on expression of KCNA5gene in H9c2cells and itsviability.Methods:1. MicroRNAs (miRNAs) microarray was used to analyze the difference of miRNAsexpressed in patients with AF and SR.2. Bioinformatics methods were used to predict the miRNAs, which may effect the genesexpression related with AF, qRT-PCR was used to detect the miRNAs expression level.3. Western blot was used to detect the expression levels of KCNA5protein in patients withAF and SR.4. The GFP-labeled plasmid vectors of miR-19b, anti-miR-19b and NC were constructedand transfected into H9c2cells by lentivirus infection respectively.The expression levelsof GFP fluorescence (fluorescence intensity) were observed with an invertedfluorescence microscope. Then, qRT-PCR was used to detect the expression level ofmiR-19b.5. Western blot was used to detect the expression levels of KCNA5protein in H9c2cells,and qRT-PCR was used to detect the expression levels of KCNA5mRNA.6. H9c2cells were selected to establish the H2O2-induced injury model, CCK-8assay wasused to detect the impaction of miR-19b on the viability of H9c2cells; flow cytometrytechnology was used to detect the impaction of miR-19b on apoptosis of H9c2cell.Results:1. MicroRNAs (miRNAs) microarray analysis showed that there were26miRNAsdifferentially expressed in patients with AF compared with SR. 2. The results of prediction by Bioinformatics methods showed that there may be targetedrelationship between the AF-related gene KCNA5and miR-19b; and qRT-PCR assayshowed that miR-19b expression in patients with AF decreased, compared with SR,which was consistent with the results of MicroRNAs microarray analysis. Therefore，miR-19b was selected as the miRNA to be studied, KCNA5was selected as the gene tobe studied.3. Western blot analysis showed that the expression level of KCNA5protein increasedsignificantly in patients AF compared with SR.4. It was showed that the efficiency of infection into H9c2cells with lentivirus was highby an inverted fluorescence microscope; and qRT-PCR assay showed that, comparedwith the control (con) groups, the expression levels of miR-19b in miR-19boverexpressed (miR-19b) groups increased significantly; miR-19b interfered(anti-miR-19b) groups decreased significantly; while miR-19b negative control (NC)groups did not change significantly, indicating that it was successful of infection intoH9c2cells by lentivirus.5. Western blot analysis showed that, compared with con groups，the expression levels ofKCNA5protein significantly reduced in miR-19b groups; anti-miR-19b groupssignificantly increased; NC group did not change significantly. qRT-PCR assay showedthe expression levels of KCNA5mRNA in all experimental groups did no changesignificantly.6. In H2O2-induced H9c2cell injury models, CCK-8assay showed that miR-19b inhibitedthe viability of H9c2cells and promote its apoptosis.Conclusions:1. MiR-19b inhibits the expression of KCNA5protein in H9c2cells.2. MiR-19b inhibits the viability of H9c2cells.