| Objective Doxorubicin(DOX)is a first-line anti-tumor drug,which has the advantages of strong efficacy and wide anti-tumor spectrum,but its cardiotoxicity is so serious that it can eventually lead to heart failure.A large body of molecular mechanism by which DOX induced cardiotoxicity have been revealed,apoptosis is considered to be the main factors in the progression of DOX induced cardiomyopathy.However,the underlying mechanism remains largely elusive.DDX3X(DEAD-box RNA helicase 3 X-linked)is an ATP-dependent RNA helicase,which is highly genetically conserved and commonly expressed in eukaryotes.Besides involved in the regulation of various RNA metabolism,DDX3X also engage in the regulation of cell cycle,innate immune response,apoptosis,carcinogenesis,cellular stress response and other important cellular physiological processes.However,the role of DDX3X in DOX-induced myocardial injury remains unclear.The Wnt/β-catenin signaling pathway plays an important role in maintaining organ growth and development and tissue homeostasis,and also plays a cardioprotective role in cardiovascular diseases such as myocardial infarction and ischemia-reperfusion injury.The purpose of this study was to investigate whether DDX3X participates in pathologic mechanism of DOX-induced myocardial injury and alleviates DOX-induced cardiotoxicity by regulating Wnt/β-catenin signaling pathway.Method 1.To construct an in vitro model of DOX-induced cardiotoxicity and determine the DDX3X expression in H9c2 cardiomyocytes treated with DOX Western blot was used to detect the protein level of Casp-3 and cleaved Casp-3 after H9c2 cardiomyocytes treated with DOX 2μM for 24 h;Flow cytometer was used to detect the apoptosis level of H9c2 cells with DOX treatment for 24 h;RT-qPCR was used to detect the mRNA expression of DDX3X in H9c2 cells with DOX treatment;Western blot was used to detect the DDX3X expression level after H9c2 cells treated with DOX.2.The effects of DDX3X knockdown on myocardial injury induced by DOX H9c2 cardiomyocytes were transfected with DDX3X siRNA,and RT-qPCR was uesd to test the transfection efficiency;Western blot was used to detect the protein level of DDX3X,Casp-3 and cleaved Casp-3 in H9c2 cardiomyocytes after DOX treatment for 24 h following DDX3X siRNA transfection;Flow cytometer was uesd to detect the apoptosis level of H9c2 cells;Mito-Tracker Red CMXRos and Hoechst 33342 staining were used to detect the level of H9c2 cardiomyocytes mitochondrial damage and apoptosis;The cell viability of H9c2 cardiomyocytes was detected using by CCK-8 assay.3.The influences of the DDX3X ATPase activity inhibition on DOX-induced cardiotoxicity Western blot was used to test the protein level of DDX3X,Casp-3 and cleaved Casp-3 in H9c2 cardiomyocytes after DOX treatment for 24 h following RK-33(7.5μM)pretreatment for 24 h;Mito-Tracker Red CMXRos and Hoechst 33342 staining were used to detect the level of H9c2 cardiomyocytes mitochondrial damage and apoptosis.4.The influences of DDX3X overexpression on DOX-induced cardiotoxicity H9c2 cardiomyocytes was transfected with Ddx3x pcDNA3.1-3XFlag-c plasmids to make DDX3X overexpression.Western blot was used to detect the protein level of DDX3X,Casp-3 and cleaved Casp-3 in H9c2 cardiomyocytes after DOX treatment for 24 h following Ddx3x plasmids transfection;Flow cytometer was uesd to detect the apoptosis level of H9c2 cardiomyocytes;Mito-Tracker Red CMXRos and Hoechst 33342 staining were used to detect the level of H9c2 cardiomyocytes mitochondrial damage and apoptosis.5.To determine the role of Wnt/β-catenin signaling pathway in DOX-induced cardiotoxicity and wheter DDX3X exert myocardial protective role through regulating Wnt/β-catenin signaling Western blot was used to detect the protein level of β-catenin in H9c2 cardiomyocytes under different experimental conditions mentioned above.Western blot was used to test the protein level of β-catenin,DDX3X,Casp-3 and cleaved Casp-3 in H9c2 cells after DOX treatment for 24 h following DDX3X siRNA transfection and LiCl pretreatment,and Mito-Tracker Red CMXRos and Hoechst 33342 staining were used to detect the level of H9c2 cells mitochondrial damage and apoptosis.Results 1.The level of DDX3X expression was reduced in H9c2 cells treated with DOXThe result of Western blot showed that the ratio of cleaved Casp-3 to Casp-3 was significantly activated in H9c2 cardiomyocytes treated with DOX,and flow cytometric analysis showed that the apoptosis level of H9c2 cells was remarkably increased with DOX treatment,which indicated the in vitro model of DOX-induced cardiotoxicity was successfully constructed;The results of RT-qPCR and Western blot indicated that the level of DDX3X expression was dramatically decreased in H9c2 cardiomyocytes treated with DOX.2.DDX3X knockdown aggravated myocardial injury induced by DOX The result of Western blot showed that DDX3X knockdown increased the ratio of cleaved Casp-3 to Casp-3 in H9c2 cardiomyocytes;The results of flow cytometric analysis,Mito-Tracker Red CMXRos and Hoechst 33342 staining indicated that DDX3X knockdown aggravated H9c2 cells apoptosis induced by DOX;The CCK-8 assay result also suggested that DDX3X knockdown worsened the decrease of cell viability of H9c2 cells induced by DOX.3.The inhibition of DDX3X ATPase activity exacerbated DOX-induced myocardial injury Western blot results suggested that RK-33 pretreatment had no influence on the DDX3X protein level but increased the ratio of cleaved Casp-3 to Casp-3 in H9c2 cells;Mito-Tracker Red CMXRos and Hoechst 33342 staining results indicated that RK-33 pretreatment aggravated H9c2 cells mitochondrial damage and apoptosis induced by DOX.4.The effects of DDX3X overexpression alleviated DOX-induced cardiotoxicity Western blot results indicated that DDX3X overexpression remarkably decrease the ratio of cleaved Casp-3 to Casp-3 in H9c2 cells;The results of flow cytometric analysis,Mito-Tracker Red CMXRos and Hoechst 33342 staining indicated that DDX3X overexpression alleviated H9c2 cells mitochondrial damage and apoptosis induced by DOX.5.Wnt/β-catenin signaling was inhibited in DOX-induced cardiotoxicity and DDX3X could activate Wnt/β-catenin signaling to mitigate myocardial injury induced by DOX The Western blot results suggested that the protein level of β-catenin was reduced in H9c2 cells treated with DOX;DDX3X knockdown and RK-33 pretreatment aggravated the decline of the β-catenin protein level,DDX3X overexpression partly reversed the decrease of the β-catenin protein;LiCl pretreatment following DDX3X knockdown significantly reversed the decline of β-catenin protein,the ratio of cleaved Casp-3 to Casp-3 and cardiomyocytes apoptosis.Conclusion This study was the first time to manifest that DDX3X was involved in the pathological mechanisms of DOX-induced myocardial injury.The level of DDX3X expression was decreased in H9c2 cells with DOX treatment.DDX3X overexpression could alleviate DOX-induced cardiotoxicity.Wnt/β-catenin signaling pathway was inhibited in H9c2 cells treated with DOX,and LiCl pretreatment could activate Wnt/β-catenin signaling,mitigating H9c2 cells mitochondrial damage and apoptosis induced by DOX.DDX3X overexpression significantly reversed the decrease of β-catenin protein induced by DOX.To sum up,DDX3X could positively regulate Wnt/β-catenin signaling,promoting stability and accumulation ofβ-catenin in cytoplasm,and further attenuating DOX-induced cardiomyocytes injury. |
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