| Objective: Doxorubicin(DOX)is a highly effective anti-tumor drug.However,it induced cardiotoxicity limits its clinical application.Various stimuluses can trigger the endoplasmic reticulum(ER)stress,which is an adaptive protective effect to maintain cell hemostasis.When the adaptive regulation cannot be maintained,it will activate the apoptosis signals.DOX can induce endoplasmic reticulum stress and apoptosis in cardiomyocytes,however the specific mechanism has not been fully elucidated.PAK2(P21 activated kinase 2),located near the endoplasmic reticulum membrane of cardiomyocytes,is a stress response kinase,which can promote protective ER stress response.Micro RNAs(miRNAs)exerts powerful biological effects,and its abnormal expressions is related with the development of various cardiovascular diseases.In recent years,many studies have reported that miRNAs are involved in the regulation of DOX-induced cardiotoxicity.Among them,miR-194-5p,which has been reported significantly up-regulated in patients with myocardial infarction,is a potential heart failure biomarker for this population.However,the pathophysiological effects of miR-194-5p in myocardial diseases are still unclear.It is predicted with the bioinformatic tool that miR-194-5p can directly bind to PAK2.Therefore,this study aims to explore the regulatory role of miR-194-5p in DOX-induced endoplasmic reticulum stress and cardiomyocyte apoptosis by targeting PAK2.Method: Rat cardiomyocyte cell line – H9c2 was used and treated with 2μM DOX to induce apoptosis,which is to stimulate the model of DOX-induced cardiotoxicity.The expression level of miR-194-5p was detected by real-time quantitative PCR;the interaction between miR-194-5p and PAK2 was tested by dual luciferase reporter assay;synthesized miR-194-5p mimic or inhibitor was transfected into H9c2 to enhannce or inhibit miR-194-5p expression;constructed PAK2/ XBP1s(splicing X-box binding protein 1)overexpression plasmid or synthesized corresponding si RNA to enhance or inhibit the endogenous gene expression;TUNEL(terminal deoxinucleotidyl transferase-mediated d UTP-fluorescein nick-end Labeling)assay and apoptosis-related proteins – caspase 3/7 activity were used to assess apoptosis;trypan blue staining was applied to measure cell death;western blotting was perfmored to detect protein expressions;and endoplasmic reticulum-related factors XBP1 s were tested to verify the activation of endoplasmic reticulum stress.Result: Mi R-194-5p was up-regulated under DOX treatment in H9c2 cell in a time-dependent manner,while the protein level of its target--PAK2 decreased.PAK2 was predicted as the target of miR-194-5p,hence,dual luciferase reporter assay indicated that miR-194-5p directly interacted with PAK2 and inhibited its expression.TUNEL assay,caspase-3/7 activity test and typan blue stain results showed that either inhibition of miR-194-5p or overexpression of PAK2 reduced DOX-induced cardiomyocyte apoptosis.In addition,DOX could induce endoplasmic reticulum stress in H9c2,and XBP1 was activated.The expression level of XBP1 s under DOX treatment increased first then dreased.The overexpression of XBP1 s suppressed DOX-induced caspase-3/7 activity elevation,as well as the expression of cleaved caspase-12,which protected cardiomyocyte from apoptosis.Addionally,the activation of XBP1 s was regulated by miR-194-5p and PAK2.Conclusion This study explored the mechanism of miR-194-5p involved in the DOX-induced endoplasmic reticulum stress and apoptosis of H9c2.PAK2,as the downstream target of miR-194-5p,has a protective effect on DOX-induced cardiotoxicity.This protective effect can be achieved by activating XBP1 in the endoplasmic reticulum stress-related pathways.Therefore,this study revealed the novel miR-194-5p/PAK2 axis participated in the regulation of DOX-induced cardiotoxicity via endoplasmic reticulum stress,providing potential prevention/treatment targets for cancer patients receiving DOX treatment. |
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