| Cardiovascular disease is not only the leading killer of mankind but also a major cause of morbidity and mortality in the whole world including China.Its occurance and development has brought patients and society heavy economic and social burden.Myocardial cells are terminally differentiated cells and have very limited ability to regenerate and repair in damaged myocardium.A large reduction in cardiomyocytes can lead to a series of changes in the heart structure and eventually cause cardiac dysfunction.Three forms of cell death,apoptosis,necrosis and autophagy,all appear in cardiovascular diseases such as heart failure,myocardial infarction,cardiomyopathy,etc.Compared with the latter two,apoptosis,as a programmed and highly regulated cell death,plays a key role in the pathogenesis of various cardiovascular diseases.Angiotensin ?(Ang ?)is an key mediator of the renin-angiotensin system,which can be activated in a variety of cardiovascular diseases and cause myocardial apoptosis.How to reduce the Ang ?-induced myocardial apoptosis is an important treatment target of cardiovascular diseases.Ghrelin is a peptide hormone isolated from the stomach.It is also found to express in the cardiovascular system.Ghrelin can improve cardiac function,reverse ventricular remodeling and inhibit the apoptosis of cardiomyocytes,thus can be regarded as a novel pharmacological target.Recent studies have shown that ghrelin also regulates microRNAs(miRNAs).The pathogenesis of cardiovascular disease is very complex,involving multiple pathways and various molecules,among which the research on endogenous microRNAs(miRNAs)is at the forefront.MiRNA is a kind of highly conserved,single-stranded,small non-coding RNA.It mainly through complete/incomplete complementary pairing to promote mRNA degradation and/or inhibit protein translation,thus negatively regulating the expression of its target genes.Numerous studies have confirmed that miRNA is extensively involved in the regulation of cardiovascular disease and cell pathogenesis.In our previous mi RNA expression profile gained from a rat post myocardial infarction heart failure model,we selected three significantly changed miRNAs,mi R-122-5p,miR-208a-3 p and miR-208b-5 p,which are closely related to cell apoptosis,fibrosis and other pathologic process.MiR-122-5p is a liver-specific microRNA,which is also expressed in the heart.It only costs a small amount of miRNA to cause a global genome alteration.More and more researchers have confirmed the regulatory role of mi R-122-5p in the cardiovascular system.MiR-208 family are cardiac-specific microRNAs.They have very strict spatial and time expression pattern.The family members,miR-208 a and miR-208 b are encoded by Myh6 gene and Myh7 gene respectively.In addition,miR-122-5p and miR-208a-3p can also be used as biomarkers for clinical cardiovascular disease,such as acute myocardial infarction and acute heart failure.In order to study the role of miR-122-5p and miR-208 family in ghrelin inhibiton of Ang ?-induced myocardial apoptosis,we first used MTT assay,flow cytometry,Caspase 3 activity detection kit to verify the protection of ghrelin on Ang ? injury.Then,we used qRT-PCR to detect the expression of miRNA and apoptotic classical molecules in H9c2 cells under different administration.Next,miRNA mimics and inhibitors were transfected into cells followed by Ang ? and ghrelin stimulation to observe the changes in apoptotic percentage.In addition,we use Rat Apoptosis RT2 Profiler? PCR Array to detect which signaling pathway and related genes were affected by those miRNAs.Because of the complexity of miRNAs regulation mechanism,finding the target genes of miRNAs in certain situations is more important.In combination with biologically informatic prediction,qRT-PCR,western blot and dual-luciferase reporting system,we proved that Sestrin-2(Sesn2)is the target gene of mi R-122-5p.Finally,we constructed the pEX-4-Sesn2 overexpression vector and transfect it into H9c2 cells followed by Ang ? and ghrelin stimulation to observe the changes in apoptotic percentage.The following results are obtained from this study:1.MTT assay showed that cell viability changed with different concentration and stimulation time of Ang ? and decreased to the worst when cells were treated with 100 nM Ang ? for 24 h.Flow cytometry showed that Ang ? could significantly increase the percentage of myocardial apoptosis,which could be reversed when pretreated with ghrelin;2.qRT-PCR results showed that Ang ? could significantly increase the expression and activity of Bax-2 and Caspase-3 while significantly reduce the expression of Bcl-2.The expression of miR-122-5p could also be significantly up-regulated,and the expression of miR-208 family were significantly down-regulated.The regulatory effect of ghrelin is just the opposite;3.Overexpression of miR-122-5p could result in increased apoptosis in the culture medium group,Ang ? group and Ang ?+ghrelin group.Inhibition of miR-122-5p resulted in decreased apoptosis in Ang ? group and Ang ?+ghrelin group.Overexpression of miR-208a-3p resulted in decreased apoptosis in Ang ? group and Ang ?+ghrelin group.Overexpression of miR-208b-5p resulted in decreased apoptosis in Ang ? group.Inhibition of miR-208 families resulted in increased apoptosis in the culture medium group,Ang ? group and Ang ?+ghrelin group;4.The results of PCR Array showed that miR-122 up-regulated five pro-apoptotic genes and two anti-apoptotic genes.Pro-apoptotic genes include Casp3,Casp8,Fas,Faslg and Tnf.The down-regulation of miR-208 family affected multiple apoptosis-related genes and various apoptotic signaling pathway including Fas signaling pathway,MAPK signaling pathway and NF-KAPPA B signaling pathway;5.The miRNA-mRNA library was used to preliminarily predict the target genes of miR-122-5p.Then qRT-PCR and western blot were conducted to further verify that miR-122-5p can reduce the mRNA and protein expression levels of Sesn2.Finally,wild and mutant Sesn2 3 'UTR were inserted into pmiR-RB-REPORT? dual luciferase report vector followed by co-transfection with miR-122-5p negative control and mimics.The results showed that the relative luciferase activity significantly decreased in pmiR-RB-REPORT-Sesn2-mut+miR-122-5p mimics group;6.Western blot results showed that SESN2 protein was decreased in Ang ? group,and recovered after the addition of ghrelin.Flow cytometry showed that overexpression of Sesn2 resulted in decreased apoptosis in Ang ? group and Ang ?+ghrelin group.Conclusion:1.Bcl-2/Bax balance,Caspase-3,mi R-122-5p and miR-208 families were all involved in ghrelin inhibition of Ang ?-induced H9c2 cell apoptosis;2.MiR-122-5p has pro-apoptotic effect and miR-208 family has anti-apoptotic effect.Multiple apoptotic pathways were influenced by miR-122-5p and miR-208 families;3.Sesn2 is the target gene of miR-122-5p;4.Sesn2 has anti-apoptotic effect.Increased Sesn2 can reduce the cell apoptosis induced by Ang ? and enhance the protective effect of ghrelin.This study confirmed that miRNA-122-5p/Sestrin-2 and miR-208 family involved in ghrelin protection of Ang ?-induced cardiomyocyte apoptosis for the first time.And it highlights the potential of microRNA-122 and its target,Sestrin-2,as a therapeutic target for against apoptosis in cardiovascular diseases. |
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