| Background and Aim:Long non-coding RNAs(lncRNAs)participate in the pathogenesis of cardiovascular diseases.Besides existing in cells,lncRNAs can express in the circulation.Moreover,they are differentially expressed in the peripheral blood of patients diagnosed with cardiovascular diseases.Meanwhile,the onset of dilated cardiomyopathy(DCM)can be occult(asymptomatic)and there is lack of targeted therapeutic drugs.Heart transplantation is the only effective treatment strategy for heart failure caused by advanced dilated cardiomyopathy.In addition,early diagnosis of dilated cardiomyopathy and early intervention can greatly improve the patients' quality of life and survival.Nowadays,whether circulating lncRNAs serve as DCM biomarkers remains unclear.Therefore,exploring the differentially expressed lncRNAs in the circulation of patients with heart failure caused by dilated cardiomyopathy can present a new theoretical ground for the prevention and diagnosis of DCM.Methods and results:1.Through plasma lncRNAs microarray detection and expression profile analysis of10 control individuals and 10 patients with dilated cardiomyopathy,we obtained 3257 differentially expressed lncRNAs(P < 0.05,fold change > 2).2.In the plasma of 62 controls and 64 patients with dilated cardiomyopathy,real-time quantitative PCR was used to validate the expression of 20 lncRNAs,which were the most significantly expressed in microarray.Then 8 lncRNAs enriched in plasma were successfully screened out,including ENST00000442293,ENST00000531663,ENST00000545794,ENST00000507296,uc002 tkd.3,ENST00000532365,ENST00000567819,HMlinc RNA1548.By comparing the eight lncRNAs with the chip results,five of them were found to be consistent with the changes in the profile,which were ENST00000442293,ENST00000545794,ENST00000507296,ENST00000532365,HMlinc RNA1548.3.By sequencing analysis of three human primary cardiac cells,lncRNA ENST00000507296 and ENST00000532365 could be detected in one of these cells.Then we employed the Spearman correlation analysis between the two lncRNAs expression levels and cardiac function indexes.The results showed that ENST00000507296 expression levels were negatively correlated with left ventricular ejection fraction(LVEF)(P < 0.0001,r =-0.390)and positively correlated with the left ventricular end-diastolic diameter(LVEDD)(P < 0.0001,r = 0.354)and amino terminal brain natriuretic peptide(NT-pro BNP)(P < 0.0001,r = 0.307).The expression levels of ENST00000532365 were positively correlated with LVEF(P < 0.05,r = 0.210)and negatively correlated with LVEDD(P < 0.001,r =-0.309)and NT-pro BNP(P < 0.01,r =-0.269).4.To analyse the relationship of the two lncRNAs expression levels and heart failure,further,we detected the two lncRNAs expression levels in 196 controls and 198 cases of DCM by real-time quantitative PCR.And ENST00000507296 expression levels in DCM group were 5.4 times as the control group(P < 0.001),while ENST00000532365 expression levels were 0.62 times compared with the control group(P < 0.001);Then,through the analysis of receiver operating characteristic(ROC),the area under the curve(AUC)of ENST00000507296 in DCM was 0.78,while the AUC of ENST00000532365 was 0.61 in DCM.5.To analyse the expression levels of the two lncRNAs with the prognosis of patients with DCM,we examined two lncRNAs expression in plasma of 552 patients with DCM,and followed up these patients for five years.After adjusting for age,gender,New York Heart Association grading,NT-pro BNP,LVEF and LVEDD and the history of diabetes,the COX multiple factors regression analysis suggested that a higher expression level of lncRNA ENST00000507296 in the plasma of patients with DCM indicated a higher event rate(hazard ratio,1.373;95% confidence interval,1.011-1.863).Conclusion:In summary,the expression levels of lncRNA ENST00000507296 in plasma of dilated cardiomyopathy patients were significantly elevated.In addition,the higher the expression levels of ENST00000507296 in patients with heart failure indicated the higher likelihood of diagnosis of dilated cardiomyopathy and the higher the incidence of prognosis events.BackgroundNumerous studies have suggested that non-coding RNAs extensively exist in cells and tissues of the cardiovascular system and play the crucial biological functions.Their expression levels are varied with stress conditions and they have a great impact on the physiological process of heart and vascular diseases.There were amount of aberrantly expressed micro RNAs(mi RNAs)in heart tissues of patients with heart failure(HF),and mi R-320 presented statistical difference in mutiple sequencing samples of HF.However,it has not attracted the interest of researchers owing to the minor fold change.However,our previous studies have suggested that mi R-320 is involved in the process of extensive cardiovascular diseases.Hence,does mi R-320 vary consistently in different types of heart in the progress of HF? This project aimed to explore the difference and mechanism of mi R-320 in different cell types in the pathological process of HF,which might provide a new idea for the treatment of HF.Methods and results1.Mi R-320 was significantly increased in myocardial tissues and plasma of patients with HF.Through real-time quantitative PCR(RT-q PCR),mi R-320 in heart tissues of patients with HF was as 2.37 times as in the control group(P = 0.034).Meanwhile,mi R-320 was also increased in plasma of HF group(1.29 times as the control group,P < 0.001).In addition,its expression levels were negatively correlated with left ventricular ejection fraction(P < 0.001,r =-0.5959).2.Mi R-320 was mainly located in cardiomyocytes(CM)and fibroblasts(CF)of the heart.In the transverse aortic constriction(TAC)-induced mice,mi R-320 expression was increased in CM and decreased in CF.By RNA immunofluorescence in situ hybridization,we found that mi R-320 was mainly distributed in CM and CF of the heart.After TAC operation,mi R-320 expression was enhanced in CM and reduced in CF.However,mi R-320 barely existed in endothelial cells and did not significantly change in response to TAC stress.Through the Langendorff system to isolate CM and CF,the results further confirmed that mi R-320 of CM began to boost on the third day of TAC and kept a high level until the seventieth day,while mi R-320 in CF began to reduced on the third day of TAC and maintained a low level until the seventieth day of TAC.3.Mi R-320 overexpression in CM exacerbated cardiac dysfunction,whereas overexpression of mi R-320 in CF alleviated cardiac fibrosis and hypertrophy.Recombinant adeno-associated virus 9 with CM-specific promoter(c TNT)and CF-specific promoter(FSP1)were used to manipulate mi R-320 expressions in vivo.Echocardiography and Miller catheter data demonstrated that increased mi R-320 in CM aggravated the heart dysfunction induced by TAC,while augmented mi R-320 in CF improved the TAC-induced cardiac dysfunction.4.Enhanced mi R-320 in CF could secrete a series of proteins to alleviate cardiomyocyte hypertrophy,whereas mi R-320 in CM did not affect the proliferation of fibroblasts.In vitro co-culture assay suggested that overexpression of mi R-320 in CF ameliorated Ang II-induced CM hypertrophy.And this effect was not caused by the direct secretion of mi R-320.Then we performed the protein mass spectrometry profile,which indicated that increased mi R-320 in CF could secrete mutiple proteins to be involved in the hypertrophy pathway of CM.Ed U staining showed that enhanced mi R-320 in CM did not affect the proliferation of CF.5.In CM,the transcription factor ELF1 was increased,which led to the increase of Ago2 expression and the decrease of mi R-320 degradation.In CF,the transcription factor STAT1 was decreased,which caused the reduction of Ago2 and the augmented degradation of mi R-320.Western blot assay and RT-q PCR indicated that Argonaute 2(Ago2)was consistent with mi R-320 in CM and CF after TAC operation.Through single cell sequencing,bioinformatics prediction and luciferase reporter gene assay,ELF1,a transcription factor in CM,was increased after TAC,which caused the increase of Ago2 in CM.STAT1,a specific transcription factor in CF,was reduced after TAC,which led to the decrease of Ago2 in CF.Actinomycin D assay and luciferase reporter gene assay suggested that Ago2 in CM and CF modulated the expression of mi R-320 via affecting the degradation of mi R-320.6.In CM,mi R-320 aggravated cardiac hypertrophy by inhibiting the expression of member 3 of the Plecker substrate protein M family(PLEKHM3),while mi R-320 inhibited the proliferation of CF by targeting the interferon-induced transmembrane protein 1(IFITM1).At last,we performed RNA immunoprecipitation(RIP)sequencing by binding Ago2 protein in CM and CF,RIP-PCR validation and luciferase report gene assay.The results indicated that mi R-320 in CM targeted PLEKHM3 and inhibited IFITM1 in CF,respectively.Increased mi R-320 in CM caused the decrease of PLEKHM3 expression,thus aggravating CM hypertrophy.However,decreased mi R-320 in CF reduced the inhibitory effect on IFITM1,leading to the proliferation of CF.ConclusionOur results demonstrated that mi R-320 expression was increased in CM and decreased in CF during the process of HF.The increased expression of transcription factor ELF1 in CM led to the upregulated expression of Ago2,which reduced the degradation rate of mi R-320 in CM.Then mi R-320 was able to inhibit the expression of PLEKHM3,thus exacerbating cardiac hypertrophy.The decrement of transcription factor STAT1 in CF resulted in the reduced expression of Ago2,which caused the increment degradation of mi R-320 in CF.Hence mi R-320 could not inhibit the expression of IFITM1,increasing the proliferation of CF.In these two cells,the interaction of mi R-320 contributes to the progression of HF.Based on mi R-320,we explored the distinct signaling pathways in different cell types during the pathological process of HF,which provided a novel direction for the treatment of HF. |
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