Effects Of Mir-874 Inhibitors On Cardiomyocyte Apoptosis And Ventricular Remodeling-related Genes

ObjectiveMore and more studies have shown that microRNA(miRNA)plays a key role in the treatment of heart failure.Studies have shown that microRNA-874(miR-874)inhibitors can reduce H2O2-induced cardiomyocyte necrosis and may have a therapeutic effect on heart failure in terms of cardiomyocyte necrosis.Previous reports indicate that miR-874 inhibitors are likely to have therapeutic effects on cardiovascular disease,but many new drugs have rushed into clinical and production due to lack of in-depth study of their possible side effects,resulting in a huge waste of human and material resources and life.With the significant loss of property and property,the new drug was forced to withdraw and stop production after it was put on the market.More drugs at the phase III clinical trial stage had to stop clinical trials due to insufficient initial basic experiments.Heart failure is the result of the progressive loss of cardiomyocytes.Cardiomyocytes are terminally differentiated cells,cardiomyocytes no longer divide and proliferate after the heart matures,so it is difficult to replenish cardiomyocytes once they are lost,causing pathological heart damage and heart failure.Myocardial fibroblasts are also one of the cells that make up the heart.They can secrete too much extracellular matrix,excessive deposition of extracellular matrix to form scar tissue,causing myocardial fibrosis and leading to heart failure.This study tested the activity of caspase-8 in neonatal cardiomyocytes,the number of neonatal cardiac fibroblasts and the activity of caspase-3/7,mRNA levels and activity of MMP2 and MMP9 in H9C2 cells,and the level of SERCA2a mRNA in H9C2 cells,and the calcium concentration in neonatal cardiomyocytes.In order to fully investigate the role of miR-874 inhibitors and explore their potential for the treatment of heart failure.MethodsHeart failure is mainly characterized by the loss of myocardial cells and myocardial fibrosis.Therefore,the main research object is myocardial cells and myofibroblasts that cause fibrosis.In this study,the cells were transfected with 100 nM miR-874 antagomiR or negative control according to manufacturer's instructions of the transfection reagent RNAiMax.The expressions of miR-874 and SERCA2a mRNA,MMP2 mRNA,MMP9 mRNA were detected by real-time fluorescence quantitative PCR in H9C2 cells.Detection of MMP2 and MMP9 activity in H9C2 cells using gelatin zymography.Cell proliferation was measured by CellTiter-FluorTM Cell Viability Assay and Caspase-Glo(?)Assay was used to detect cell apoptosis(including Caspase-Glo(?)8 Assay detection of neonatal cardiac fibroblast apoptosis and Caspase-Glo(?)3/7 Assay detection of neonatal cardiomyocyte apoptosis).The concentration of Ca2+ was measured using Fluo-4 NW Calcium Assay Kits in neonatal cardiomyocytes.ResultsThe miR-874 expression in the miR-874 antagomiR transfected cells was significantly lower than that in the scrambled miRNA control transfected cells(P<0.01).miR-874 level was successfully knocked down by the transfection experiment in miR-874 antagomiR group H9C2 cells.The following experiments were carried out based on the successuful transfection of miR-874 antagomiR into the cells.miR-874 antagomiR had no effect on the proliferation of neonatal cardiac fibroblasts(P>0.05).Compared with the control group,the activity of Caspase-8 in neonatal cardiac fibroblasts of miR-874 antagomiR group was significantly decreased(P<0.01).The activity of Caspase-3/7 in neonatal cardiomyocytes of miR-874 antagomiR group was significantly increased(P<0.01).There was no significant difference in the level of SERCA2a mRNA between the miR-874 antagomiR group and the control group in H9C2 cells(P>0.05).Meanwhile the concentration of Ca2+ had no significant difference between the miR-874 antagomiR group and the control group in neonatal cardiomyocytes(P>0.05).Compared with the control group,the mRNA level of MMP9 in H9C2 cells of miR-874 antagomiR group was significantly decreased(P<0.05),while the mRNA level of MMP2 did not change significantly(P>0.05).At the same time,the results of the gelatin zymogram showed that the changes in the activity of MMP9 and MMP2 were consistent with the changes in their mRNA levels.Conclusions1.Although miR-874 antagomiR has no effect on the proliferation of neonatal cardiac fibroblasts,miR-874 antagomiR can reduce the activity of Caspase-8 in neonatal cardiac fibroblasts,and may inhibit the apoptosis of neonatal cardiac fibroblasts.The miR-874 antagomiR may promote myocardial fibrosis,affect ventricular remodeling and lead to heart failure.2.The miR-874 antagomiR can increase the activity of Caspase-3/7 in neonatal cardiomyocytes,may promote neonatal cardiomyocyte apoptosis,and may accelerate the progression of heart failure.3.The miR-874 antagomiR can down-regulate mRNA and activity of MMP9 in H9C2 cells,but has no effect on MMP2.The miR-874 antagomiR were able to relieve myocardial fibrosis and play a therapeutic role in heart failure.4.The miR-874 antagomiR had no effect on SERCA2a mRNA level in H9C2 cells and had no effect on Ca2+ concentration in neonatal cardiomyocytes.In this regard,miR-874 antagomiR may have no effect on the treatment of heart failure.5.Taken together,miR-874 inhibitors have beneficial and disadvantageous effects on the treatment of heart failure.Therefore,we need to conduct in-depth studies on the relationship between miR-874 inhibitors and heart failure.The results of this study will provide the development of drugs for the treatment of heart failure in the future.in accordance with.In recent years,a combination of several miRNAs has been found to have a good effect in treating myocardial infarction and inducing the conversion of cardiac fibroblasts into cardiomyocytes.Given the active role of miR-874 inhibitors in the treatment of other aspects of myocardial infarction,we can consider the use of miRs.The miR-874 inhibitor is combined with other miRNAs that promote cardiomyocyte proliferation and is used for the treatment of cardiovascular disease.