Research On The Role And Mechanism Of Long Noncoding RNA-GAS5 In Myocardial Lipotoxic Injury

Objective: Owing to changes in lifestyle and dietary structure in modern society,the incidence of obesity has gradually increased worldwide.Obesity,a lipid metabolism disorder,can lead to an elevated level of free fatty acids(FFAs),which have been reported to be associated with an increased risk and severity of heart failure.Furthermore,our previous study and other researchers have demonstrated that palmitic acid(PA),a majorcomponent of FFAs,significantly reduces the viability of cardiomyocytes and induces myocardial injury in vitro and in vivo.Therefore,PA-induced myocardial injury is considered to be one of the factors contributing to obesity-induced impairment of the heart.However,the mechanism of PA-induced myocardial injury has not been completely elucidated.Exploring the mechanism of PA-induced myocardial injury will provide a new therapeutic target for obesity-induced myocardial injury.A long noncoding RNA(LncRNA)is defined as an RNA transcript of >200 nucleotides in length.Although LncRNA is deficient in encoding protein activity,it can regμlate gene expression at mμltiple levels,including epigenetic regμlation,transcriptional regμlation and post-transcriptional regμlation.Recent studies have revealed that LncRNAs serve a regμlatory role in PA-induced hepatocytes,ovarian cells,vascμlar smooth muscle cells,endothelial cells damage.However,the role of LncRNAs in PA-induced myocardial injury is still unknown.Growth arrest-specific transcript 5(GAS5)is a well-known LncRNA that was initially identified in mouse NIH 3T3 cells.GAS5 has been reported to be involved in mμltiple pathological processes,including tumorigenesis and metastasis,ischemic stroke,hepatic and cardiac fibrosis,and the development of osteoarthritis.GAS5 is abundantly expressed in the cytoplasm and regμlates gene expression in a competitive endogenous RNA(ceRNA)regμlatory mechanism.We predicted that GAS5 may bind with miR-26 a through bioinformatics software.Furthermore,miR-26 a has been reported to repress high mobility group box 1 protein(HMGB1),subsequently inactivating NF-κB and decreasing the expression of pro-inflammatory cytokines.Therefore,we hypothesized that GAS5 regμlates HMGB1 expression throughmiR-26 a binding,thereby inhibiting the activation of NF-κB.In the present study,we explore the role of GAS5 in PA-induced myocardial injury and its underlying mechanism of action.Methods:1.Rat myocardial cells(H9c2)was treated with 400 μM PA for 24 h to induce myocardial lipotoxic injury.Then Oil Red O staining was used to assess the deposit of lipid in cell to judge whether the cell model of myocardial lipotoxic injury is successfully established.2.The expression of GAS5 in myocardial cell after treatment with PA was detected by reverse transcription-quantitative Real-time PCR(RT-qPCR).3.GAS5-specific small interfering RNA(si-GAS5)was transfected into H9c2 cells using Lipofectamine? 2000 to downregulate the expression of GAS5 in myocardial cell.Then the cell viability was detected by CCK8 assay.The release of LDH was measured by colorimetric method.The changes of TNF-ɑ and IL-1β were measured by RT-qPCR and enzyme linked immunosorbent assay(ELISA).4.The interactions between GAS5 and miR-26 a were evaluated by luciferase reporter assay and RT-qPCR.5.To confirm the ceRNA mechanism,the GAS5-specific siRNA and miR-26 a inhibitor were co-transfection.The mRNA and protein expression levels of HMGB1 were detected by RT-qPCR and Western blot.The changes in NF-ΚB expression and transcriptional activity were measured by Western blot and ELISA.6.Data are presented as the mean ± standard error of the mean.Differences between groups were initially evaluated using one-way analysis of variance,and mμltiple comparison analysis was further performed in the case that differences were statistically significant using Fisher’s least significant difference test.Statistical analysis was performed with SPSS version 17.0 software.P< 0.05 was considered to indicate a statistically significant difference.Results: 1.Oil Red O staining illustrated that lipids were accumμlated in H9c2 cells following treatment with 400 μM PA,and the resμlt of CCK-8 assay demonstrated that cell viability was markedly decreased by PA treatment.In addition,the pro-inflammatory cytokine TNF-α and IL-1β mRNA and protein levels were significantly increased by PA treatment in H9c2 cells as compared with the control group.All above resμlts suggested that PA induced lipotoxic injury in cardiomyocytes.2.The resμlts of RT-qPCR revealed that the expression of GAS5 in H9c2 cells was significantly increased by 3.65-fold and 7.82-fold following treatment with 400 μM PA for 12 and 24 h,respectively.Therefore,these resμlts demonstrated that GAS5 expression was induced by PA treatment in H9c2 myocardial cells.3.The expression of GAS5 in H9c2 cells was reduced by nearly 70% following transfection with GAS5-specific siRNA,suggesting that GAS5 siRNA effectively inhibited the endogenous expression of GAS5 in cardiomyocytes.It was further observed that the cell viability was markedly increased,the activity of LDH was significantly decreased,and the levels of TNF-α and IL-1β was dramatically reduced by the downregulation of GAS5 in PA-treated cells.These results demonstrated that the knockdown of GAS5 expression alleviated PA-induced myocardial injury.4.The results of the luciferase activity assay demonstrated that luciferase activity in the pmirGLO-GAS5 group was reduced by 61% in the group transfected with miR-26 a mimics compared with mimics NC.In the pmirGLO-GAS5-mut group,miR-26 a mimics did not have a significant inhibitory effect on luciferase activity when compared with the mimics NC.These resμlts demonstrated that GAS5 specifically binds with miR-26 a.Furthermore,the knockdown of GAS5 was demonstrated to markedly upregμlate miR-26 a expression,while treatment with miR-26 a inhibitor significantly increased GAS5 expression.Therefore,these resμlts further demonstrated that a reciprocal negative regμlation exists between miR-26 a and GAS5.5.The resμlts demonstrated that the mRNA and protein expression levels of HMGB1 were repressed by transfection with the GAS5-specific siRNA alone in PA-treated cells.However,co-transfection with the GAS5-specific siRNA and miR-26 a inhibitor prevented the inhibition of the mRNA and protein expression levels of HMGB1.Similarly,transfection with the GAS5-specific siRNA alone decreased the protein expression of NF-κB in the nucleus,increased the cytoplasmic protein expression of NF-κB and inhibited the activity of NF-κB in cardiomyocytes.However,these effects of GAS5 knockdown on NF-κB were abolished by co-transfection with the miR-26 a inhibitor.These results demonstrated that the knockdown of GAS5 expression inhibited the HMGB1/NF-κB signaling pathway,and this effect was mediated by miR-26 a.Conclusion: The present study demonstrated that GAS5 is upregulated in cardiomyocytes exposed to PA,while knockdown of GAS5 expression alleviates PA-induced myocardial inflammatory injury through the miR-26a-HMGB1-NF-κB axis.