| Background and objectiveRAS is an important component of cardiovascular physiology and plays a very important role in the pathological process of various cardiovascular diseases.AngⅡ is the main effect peptide of RAS,and plays a key role in the blood pressure and cardiovascular state regulation.Local production of AngⅡ in the heart contributes to the occurrence and development of a variety of heart diseases,such as diabetic cardiomyopathy,ischemia-reperfusion injury,and ventricular remodeling after myocardial infarction.Nrf2 is an important regulatory factor in the oxidative stress pathway.Activation of Nrf2 can effectively protect against oxidative stress induced cardiac injury and cardiac dysfunction,such as myocardial ischemia-reperfusion injury,myocardial infarction,and diabetic cardiomyopathy.Our previous study also found that in the middle and late stage of myocardial injury caused by AngⅡ,the oxidative damage of myocardial tissue was aggravated with the decreased expression of Nrf2 in myocardial tissue,while the high expression of Nrf2(Nrf2-TG)and exogenous administration of Nrf2 agonist sulforaphane could effectively resist the oxidative stress and injury of myocardial caused by AngⅡ.These studies,however,confirm the key role of Nrf2-mediated antioxidant defense system in the development and progression of AngⅡ myocardial injury.However,it is still unclear what causes the decreased expression of myocardial Nrf2 and cardiac function under AngⅡ stimulation.LncRNAs are a class of functional RNA molecules whose transcripts are more than 200 nucleotides long and can’t encode proteins.They regulate gene expression at the epigenetic,transcriptional and post-transcriptional levels and gene translation levels.MiRNAs are single-stranded RNA molecules of 19-25 nucleotides in size that negatively regulate the expression of target genes by recognizing specific sites in the 3’UTR region of target mRNA and binding to them.In recent years,more and more evidence has shown that the antioxidant function of Nrf2 is regulated by lncRNAs and miRNAs.Therefore,this study tried to explore the mechanism of AngⅡ regulating myocardial Nrf2 from the level of lncRNAs and miRNAs.High-throughput transcriptome sequencing technology was used to screen and analyze differential lncRNAs and miRNAs in AngⅡ-stimulated myocardial tissues.Combined with literature reports,we identified the miRNA that may regulate Nrf2 and verified it through cell experiments,providing a new molecular target for the diagnosis and prevention of myocardial oxidative damage and cardiomyopathy caused by AngⅡ,and laying a theoretical and experimental foundation for the prevention and treatment of clinical cardiomyopathy.MethodsIn the in vivo experiment,the chronic myocardial injury model induced by AngⅡwas established according to the method reported by our research group.8-week-old male C57/BL mice were subcutaneously injected 0.5mg/kg AngⅡ every other day for 2 months(2M),and the observation was stopped until 6 months(6M).The control group was subcutaneously injected with the same dose of normal saline.Cardiac function was monitored by echocardiography at 2 months(2M),4 months(4M)and 6 months(6M),and record the heart weight and body weight.At the same time,the cardiac tissues of mice were collected,and differentially expressed lncRNAs and miRNAs were analyzed by high-throughput transcriptome sequencing in myocardial tissue of 2M mice.The expressions of lncRNAs and miRNAs differentially in the myocardial tissues of 2M,4M and 6M mice were further verified by qRT-PCR.Relevant indicators were detected using qRT-PCR and Western Blot methods,including:expression of 4-HNE,Nrf2 and HO-1.In vitro,AC16 cardiomyocytes were transfected with miR-144-3p inhibitor and 100 nmol/L AngⅡ was given to the cells for 48 h.The cells were collected and tested as follows:①qRT-PCR and Western Blot were used to detect the expression of Nrf2 after treatment with miR-144-3p inhibitor of different concentrations;②measure the cell area of AC 16 cardiomyocytes.③the expression of 3-NT and 4-HNE was detected by Western blot.④The expression of Nrf2 and HO-1 was detected by Western blot and qRT-PCR.ResultsIn vivo test showed that 35 lncRNAs were differentially expressed at the gene level in the myocardial tissue damaged by 2M AngⅡ.Verifying the 10 of the known differential IncRNAs,it was found that the expressions of GM11967,B130046B21RIK and SORBS2OS in the injured myocardium tissues of 2M,4M and 6M were consistent with the sequencing results,and the expressions of Gm20634,Gm17745,AC157931 and Xist in 2M were consistent with the sequencing results,while they were opposite in 4M and 6M.A total of 32 differentially expressed miRNAs were screened from the myocardium of 2M mice,and 9 of the them were selected for verification in the myocardial injury tissues of AngⅡ.It was found that the expressions of miR-7a-1-3p,miR-201-5p,miR-421-3p and miR-1955-5p in the myocardium tissues of 2M,4M and 6M mice were consistent with the sequencing results.MiR-194-2-3 p was contrary to the sequencing results.The expression of miR-5099,miR-205-5p,and miR-144-3p in 2M injured myocardial tissue was consistent with the sequencing results,while the expression of miR-7a-5p was contrary to the sequencing results,and the expression of miR-7a-5p in 4M and 6M was reversed.The binding of the differentially down-regulated miRNAs to Nrf2 was predicted on the TargetScan website,and the binding sites between miR-144-3p and the 3’UTR of Nrf2 were found.In vitro test showed that AngⅡ stimulation increased the surface area of AC 16 cells,increased the expression of 4-HNE,and decreased the expression of Nrf2 and downstream antioxidant gene HO-1.After miR-144-3p inhibitor was used to inhibit miR-144-3p,the expressions of Nrf2 and HO-1 were significantly increased,and the surface area of AC 16 cells and the expression of 4-HNE were significantly decreased compared with the AngⅡ stimulated group.In vitro tests showed that after knockdown of miR-144-3p,compared with the AngⅡ stimulated group,the cell surface area of AC 16 cardiomyocytes decreased,the expression of 4-HNE decreased,and the expression of Nrf2 and downstream antioxidant gene HO-1 increased.Conclusion1.Based on high-throughput sequencing and validation results,seven lncRNAs,including Gm11967,B130046B21RIK,SORBS2OS,Gm20634,GM17745,AC157931 and Xist,were screened out.As well as miR-194-2-3p,miR-5099,miR-7a-5p,miR-7a-1-3p,miR-201-5p,miR-205-5p,miR-144-3p,miR-421-3p and miR-1955-5p,nine miRNAs may be involved in the occurrence of angiotensin induced myocardial injury.2.AngⅡ may promote miR-144-3p to inhibit the antioxidant function of Nrf2,aggravating oxidative stress and leading to myocardial injury. |
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