microRNA-346 Regulates H2O2-mediated Cardiomyocyte Damage by Targeting KLF4
YANG Xuezhen
SONG Lixin
ZHANG Ya'nan
LIU Sishuo
LI Huixian
Abstract：Objective:To investigate the effect of miR-346 on H9c2 cardiomyocyte damage induced by H2O2 and the molecular mechanism.Methods:H9c2 cells were induced with different doses(0,25,50,100,200,and 400 μmol/L)of H2O2 for 6 h.Cell viability was detected by Methyl Thiazolyl Tetrazolium(MTT)assay,and the expression of miR-346 and Krüppel-like factor 4(KLF4)mRNA was detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR).H9c2 cells cultured in vitro were divided into control group,mimic-NC group,miR-346 mimic group,inhibitor-NC group,miR-346 inhibitor group,si-NC group,and si-KLF4 group.The control group did not undergo any special treatment,the other groups were transfected.The transfection efficiency was verified by qRT-PCR.The targeting relationship between miR-346 and KLF4 was verified by TargetScan website prediction and dual-luciferase reporter experiments.The expression of the KLF4 protein in each transfection group was tested by Western Blot.In addition,H2O2 model group,miR-346 inhibitor group,miR-346 inhibitor+si-KLF4 group cells were induced by H2O2for 6 h,cell viability was measured by MTT assay,cell apoptosis rate was measured by TUNEL immunofluorescence,and the content of reactive oxygen species(ROS),superoxide dismutase(SOD),and malondialdehyde(MDA)was measured by kit.The expression levels of proliferating cell nuclear antigen(PCNA),Cyclin D1,B-cell lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax),cleaved poly ADP-ribose polymerase(Cleaved PARP),and the phosphorylation levels of protein kinase B(AKT)/glycogen synthase kinase-3β(GSK-3β)pathway protein were determined by Western Blot.Results:H2O2(concentration not less than 25 μmol/L)showed obvious cytotoxicity to H9c2 cells,and the expression of miR-346 increased,while the expression of KLF4 down-regulated(P＜0.05 or P＜0.01).All groups were successfully transfected,miR-346 targeted and negatively regulated the expression of KLF4.The apoptosis rate,ROS,and MDA production of H9c2 cells in the miR-346 inhibitor group were lower than those in the H2O2 group,while cell viability and SOD levels were higher than those in the H2O2 group(P＜0.05).Western Blot showed that compared with the H2O2 group,the expression of PCNA,Cyclin D1,Bcl-2/Bax,p-AKT,and p-GSK-3β proteins in the miR-346 inhibitor group up-regulated,while the expression of cleaved PARP protein down-regulated(P＜0.05).Compared with the miR-346 inhibitor group,the apoptosis rate,ROS,and MDA production of H9c2 cells in the miR-346 inhibitor+si-KLF4 group increased,cell viability and SOD levels decreased,PCNA,Cyclin D1,Bcl-2/Bax,p-AKT,and p-GSK-3β protein expression down-regulated,and cleaved PARP protein expression up-regulated(P＜0.05).Conclusion:Down-regulating the expression of miR-346 can inhibit H2O2-induced H9c2 cardiomyocyte apoptosis and oxidative stress by targeting KLF4.The mechanism may be related to the AKT/GSK-3β signaling pathway.
Keywords：cardiomyocyte injurymiR-346Krüppel-like factor 4apoptosisreactive oxygen speciesprotein kinase B/glycogen synthase kinase-3β pathwayexperimental study
Publication Date：2025-06-25
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：9( 1820-1828 )