Effect Of LncRNA Malat-1/miR-211-5p/PPARGC1A Pathway In Alleviating Pirarubicin-induced Cardiotoxicity By Rutin

Cancer is the second leading cause of death in humans.In clinical practice,anthracycline drugs such as pirarubicin(THP)are widely used in the treatment of various cancers.However,as the most severe side effect,anthracycline-induced cardiotoxicity jeopardizes patients’health and limits the clinical application of these drugs.Rutin(RUT),a representative flavonoid compound,is one of the main active ingredients in traditional Chinese medicine flos sophorae.It is widely found in plants such as ruta graveolens leave,buckwheat flowers,and jujubes.RUT shows various pharmacological effects,including scavenging of reactive oxygen species,anti-lipid peroxidation,anti-inflammatory,platelet activation inhibition,anti-tumor and cardioprotection.Non-coding RNAs(ncRNAs)play important roles in numerous biological processes in organisms.Long non-coding RNAs(lncRNAs)and microRNAs(miRNAs)have become research hotspots in the field of life sciences.Studies have shown that IncRNAs can competitively bind to miRNAs and regulate the expression of target genes through a competitive endogenous RNA(ceRNA)mechanism.Therefore,is the alleviation of THP-induced cardiotoxicity by RUT regulated by lncRNAs?What biological role do lncRNAs play in THP-induced cardiotoxicity?Does lncRNA act through the regulation of miR-21 1-5p and its target gene PPARGC1A?This study aims to screen for lncRNAs that interact with miR-211-5p,identify the pathway,and investigate the effects and mechanisms of RUT in alleviating THP-induced cardiotoxicity through in vivo and in vitro experiments.It is divided into three parts:(1)Bioinformatics analysis of RUT’s alleviation of THP-induced cardiotoxicity,construction and validation of the lncRNA Malat-1/miR-211-5p/PPARGC1 A pathway.In this part,the expression profiles of miRNAs obtained from previous research on RUT-mediated alleviation of THP-induced cardiotoxicity were analyzed.Differentially expressed miRNAs between groups that also showed conservation across species were selected.The TargetScan and miRDB databases were used to screen for target miRNAs of protective and pathogenic lncRNAs involved in antitumor drug-induced cardiotoxicity.The lncRNA Malat-1/miR-211-5p pathway was selected for further screening of target genes.The KEGG pathway enrichment analysis and TargetScan database were used to identify PPARGC1A as the target gene.Subsequently,a dual-luciferase reporter gene experiments confirmed the presence of one binding site between lncRNA Malat-1 and miR-211-5p,as well as one binding site between miR-211-5p and PPARGC1A.Thus,the lncRNA Malat-1/miR-211-5p/PPARGC1 A pathway was identified,and it serves as the entry point for exploring the mechanisms of RUT in improving THP-induced cardiotoxicity.(2)The role of the lncRNA Malat-1/miR-211-5p/PPARGC1A pathway in RUT-mediated alleviation of THP-induced cardiomyocyte injury.Using THP-induced HL-1 mouse cardiomyocyte injury,it was found that THP upregulated the expression of miR-211-5p in HL-1 cells and downregulated the expression of lncRNA Malat-1 and PPARGC1A mRNA.Cellular deformation and floating death were observed under the microscope.RUT was able to reverse these changes.Experimental methods such as CCK-8 assay,immunofluorescence,and Western blot revealed that THP-induced HL-1 cells showed a significant increase in oxidative stress levels,a significant decrease in cell viability,mitochondrial bioactivity,and membrane potential.After RUT intervention,all these conditions were reversed.To further validate whether RUT inhibits THP-induced cell injury through lncRNA Malat-1,a lentivirus-mediated sh-Malat-1 was constructed and transfected into HL-1 cells to obtain stable transfected cell lines.The results showed that sh-Malat-1 partially blocked the antioxidant stress and mitochondrial protective effects of RUT,while affecting the expression of antioxidant-related proteins,mitochondrial-related proteins,and apoptosis-related proteins.This indicates that lncRNA Malat-1 is involved in the inhibitory effect of RUT on THP-induced HL-1 cell injury.Further rescue experiments found that transfection of sh-Malat-1 upregulated the expression of miR-211-5p and downregulated the expression of PPARGC1A mRNA and its encoded protein PGC-lα.This led to increased ROS levels,decreased mitochondrial bioactivity and membrane potential in HL-1 cells,induction of oxidative stress and mitochondrial damage,and changes in the expression of antioxidant-related proteins,mitochondrial-related proteins,and apoptosis-related proteins.Transfection of a miR-211-5p inhibitor reversed the above regulatory effects of sh-Malat-1,suggesting that lncNA Malat-1 acts as a sponge for miR-211-5p to inhibit THP-induced HL-1 cell damage.Transfection of si-PPARGC1A inhibited the regulatory effects of the miR-211-5p inhibitor,indicating that miR-211-5p targets PPARGC1A to promote THP-induced HL-1 cell damage.These results demonstrate that the role of RUT in improving THP-induced cardiomyocyte injury is mediated through the lncRNA Malat-1/miR-211-5p/PPARGC1 A pathway.(3)The role of l ncRNA Malat-1 in RUT-ediated alleviation of THP-induced cardiotoxicity.THP-induced cardiotoxicity was observed in BALB/c mice,characterized by elevated serum MDA levels,decreased SOD levels,and decreased cardiac enzyme markers.Cardiac hemodynamic monitoring revealed significant abnormalities,and histological examination showed irregular arrangement of myocardial fibers,increased interstitial space,hypertrophic cardiomyocytes,disappearance of nucleoli,cytoplasmic vacuolation,and interstitial edema.After oral administration of RUT to the mice,all these indicators showed improvement.Following tail vein injection of lentivirus-mediated lncRNA Malat-1 silencing in BALB/c mice,decreased serum MDA levels,increased SOD levels,elevated cardiac enzyme markers,and significant changes in cardiac hemodynamic monitoring and histological morphology were observed.RT-qPCR,immunofluorescence,and Western blot experiments were performed to detect the expression of lncRNA Malat-1,miR-211-5p,PPARGC1A mRNA,and various related proteins(PGC-lα,NRF-2,HO-1,Cyt C,caspase-9,and caspase-3)in mouse cardiac tissues.The results showed that THP increased the expression of miR-211-5p in cardiac tissues,while decreasing the expression of lncRNA Malat-1 and PPARGC1A mRNA.It also downregulated the expression of proteins such as PGC-la,NRF-2,HO-1,mitochondrial Cyt C,and upregulated the expression of cytoplasmic Cyt C,cleaved-caspase-9,and cleaved-caspase-3,leading to increased cell apoptosis.After RUT intervention,these changes were reversed.However,when BALB/c mice were injected with sh-Malat-1 via the tail vein,the cardioprotective effect of RUT was reversed.These experimental results indicate that the improvement of THP-induced cardiotoxicity by RUT is mediated through lncRNA Malat-1.In summary,this study has drawn the following conclusions through in vitro and in vivo experiments:(1)Bioinformatics analysis of the gene chip data obtained from the previous studies of the research group,along with dual-luciferase reporter gene experiments,confirmed the lncRNA Malat-1/miR-211-5p/PPARGC1A pathway,which is involved in RUT-mediated improvement of THP-induced cardiotoxicity.(2)In vitro studies demonstrated that RUT improves THP-induced HL-1 mouse cardiomyocyte injury through the lncRNA Malat-1/miR-211-5p/PPARGC1 A pathway.(3)In vivo studies confirmed that the cardioprotective effect of RUT in mice is achieved through lncRNA Malat-1,indicating that lcRNA Malat-1 is an important molecular target of RUT in improving THP-induced cardiotoxicity.