Study On The Effets And Mechanism Of Mmu＿circ＿0000021 On Microvascular Dysfunction Induced By Myocardial Ischemia/Reperfusion Injury Via Regulating MiR-143-3p By Targeting NPY

For patients with acute ST-segment elevation myocardial infarction(STEMI),active reperfusion therapy strategies including thrombolysis or direct percutaneous coronary intervention(PPCI)can be effective to improve the long-term prognosis of patients with STEMI.However,the microcirculation level cannot be fully recovered,the phenomenon of ischemic myocardial tissue without effective reperfusion is called "no-reflow phenomenon".Neuropeptide Y(NPY)is a peptide substance containing 36 amino acids widely present in the body.In recent years,studies have found that NPY acts on the Y1 receptor to increase the intracellular calcium ion concentration;it can trigger cardiomyocytes and vascular endothelium.Therefore,we speculate that NPY may cause an increase in the concentration of calcium ions in myocardial cells and the excitation-contraction coupling effect.It has a vasoconstrictive effect on the coronary microcirculation system,participates in the development of myocardial I / R microcirculation disorders,and increases no reflow.MicroRNA(miRNA)is a type of small molecule RNA of about 21 to 25 nucleotides.More and more evidence shows that microRNA plays an important role in the process of myocardial I / R injury.The main mechanism is that microRNA Downstream target genes combine to play a negative role in regulating target gene expression in myocardial I / R injury.In this study,we used bioinformatics technology and target gene prediction software to predict the target matching relationship between microRNA and NPY genes.We selected targets for high software intersections and predicted miR-143-3p.There may be a target relationship between 3p and mouse NPY gene.Circular RNA(circular RNA,circ RNA)is a type of non-coding RNA widely present in mammals,mainly involved in gene regulation in vivo.At present,it has been found that multiple circular RNAs themselves contain at least one miRNA binding site.Therefore,they can be used as RNA "sponges" to adsorb miRNAs,thereby being suppressed by miRNAs through the mechanism of competitive endogenous RNAs(ce RNAs).Screening of differentially expressed circular RNA based on circular linear RNA sequencing and further data analysis and starbase database prediction revealed that circular RNA mm9＿circ＿008757(mmu＿circ＿0000021),mm9＿circ＿01502(mmu＿circ＿0001098)and mmu＿miR-143-3p may have a targeted matching relationship.Therefore,this study intends to confirm whether the circular RNAmmu＿circ＿0000021 can be used as a "sponge" to adsorb miR-143-3p,and further regulate the NPY targeted protein resulting in an increase in the concentration of calciumions in cardiomyocytes,and to induce myocardial microvascular contraction through the excitation-contraction coupling effect with participating in the occurrence and development of myocardial I / R microcirculation disorder,aggravating no reflow.In order to explain this hypothesis,this article is divided into four parts.The first part is based on the mouse myocardial ischemia / reperfusion model to do circular RNA sequencing to screen the differential genes and perform data analysis.In the second part,to detect the NPY,miR-143-3p,mmu＿circ＿0000021 and mmu＿circ＿0001098mRNA in mouse myocardial ischemia / reperfusion model and mouse cell hypoxia / reoxygenation model expression.In the third part,the targeting relationship between miR-143-3p and NPY and the interaction between miR-143-3p and ring-shaped RNA were confirmed.In the fourth part,it was confirmed in the mouse myocardial ischemia / reperfusion model that mmu＿circ＿0000021 mediates miR-143-3p targeting NPY,which affects myocardial ischemia / reperfusion microcirculation disorder and improves cardiac function.PART Ⅰ Circular RNA de-linear sequencing data analysis in mouse myocardial ischemia / reperfusion model Objective:Circular RNA de-linear sequencing and data analysis paved the way for subsequent experiments Methods:First,selecting four groups of mouse myocardial samples and four groups of ischemia / reperfusion model samples to test the total RNA;second,taking 3μg of each sample RNA is constructed in the lnc RNA library,removing the ribosomal RNA(r RNA)from the sample,and selecting different index tags to build the library according to the operating instructions of the NEB Next Ultra Directional RNA Library Prep Kit for Illumina(NEB,Ispawich,USA);Finally,the constructed library is sequenced with Illumina,and then the sequencing results are divided into three parts for data analysis: circular RNA differential analysis,differential circular RNA host gene function analysis and miRNA molecular sponge analysis.Results:In the sequencing results,we found that there were 135 significantly differentially expressed circular RNA,of which 81 were up-regulated and 54 were down-regulated,providing a basis for subsequent experiments.Among them,mmu＿circ＿0000021 and mmu＿circ＿0001098 have a high correlation with the target gene NPY in sequencing results.Therefore,in order to further elucidate the related functions of differential circular RNAs,we made a biological information correlation analysis,which further showed the necessity of the study.Conclusion : 1.There are multiple circ RNAs with significant differential expression in the de-linear sequencing of circ RNA.2.There may be a circular RNA that has a higher relationship with the target protein NPY.PART Ⅱ The expressions of NPY,miR-143-3p,mmu＿circ＿0000021 is detected in myocardial ischemia / reperfusion model and hypoxia /reoxygenation modelObjective:To detect the expressions of NPY,miR-143-3p,mmu＿circ＿0000021and mmu＿circ＿0001098 in MI / RI and H / R.Methods:First,through bioinformatics technology,using target gene prediction software Target Scan,miRanda to predict the target matching relationship between micro RNA and NPY and using starbase database to predict the discovery of circle RNA mm9_circ＿008757(mmu＿circ＿0000021),mm9_circ＿015028(mmu＿circ＿0001098)may have a targeted matching relationship with the predicted micro RNA;secondly,16 C57 BL / 6 mice were randomly divided into 2 groups(8 per group),sham operation(Sham)group and Ischemia/ reperfusion(I / R)group;similarly,neonatal mice were randomly divided into2 groups(8-10 per group),normal group and hypoxia / reoxygenation(H / R)group.The MI / RI model of C57 BL / 6 mice was constructed by ligating and releasing the left anterior descending coronary artery of mice.The Sham group was threaded without ligating the left anterior descending coronary artery,while the I / R group ligated the left anterior descending coronary artery for 45 minutes and then loosened for 3 hours;for cell experiments,the normal group of cardiomyocytes was changed to serum-free the basic medium and the H / R group was changed with the same operation and placed in a hypoxia chamber(Hypoxia Chamber)for 6 hours of hypoxia,and then after changing the serum-free the basic medium in a biochemical incubator to reoxygenate for 6hours to construct a cardiomyocyte H / R model.Finally,the samples of animal models and cell models are collected for subsequent experiments.HE staining is used to observe the damage of myocardial tissue,and the biochemical index detection is used to detect the samples of CK,CK-MB and LDH.RT-qPCR was used to detect the expression of NPY,miR-143-3p,mmu＿circ＿0001098 and mmu＿circ＿0000021 mRNA in myocardial tissues and cardiomyocytes.Results:Target Scan and miRanda predicted that miR-143-3p may have a target matching relationship with NPY target protein;and after bioinformatics analysis and starbase prediction,mm9_circ＿008757(mmu＿circ＿0000021),mm9-circ-015028(mmu＿circ＿0001098)and miR-143-3p may have a targeted matching relationship.HE staining results showed that the myocardial fibers in the Sham group were neat and orderly,with no abnormalities in the nucleus,no myocardial cell necrosis,no interstitial edema,and no neutrophil infiltration.The cell cytoplasm is homogeneously red stained,a large number of contraction bands are formed,and a large number of neutrophils are infiltrated.Biochemical results showed that compared with the Sham group,the serum CK,CK-MB,LDH content in the I / R group increased significantly(P <0.05).RT-qPCR results showed that compared with Sham group,NPY expression in I / R group was significantly increased(P <0.05),miR-143-3p expression was significantly reduced,mmu＿circ＿0000021 was significantly increased(P <0.05),and mmu＿circ＿0001098 was not significantly changed(P <0.05): Similarly,,the same results were obtained for the normal group and the H / R group in the cell experiment.Conclusion:1.Myocardial injury occurs after myocardial / ischemic reperfusion in C57 BL / 6 mice.The contents of CK,CK-MB and LDH in serum are significantly increased,and the contents of CK,CK-MB and LDH in cell supernatant are significantly increasing similarly 2.The expression of NPY,miR-143-3p and mmu＿circ＿0000021mRNA increased in MI / RI and H / R.PART Ⅲ Confirmation of the targeting relationship between miR-143-3p and NPY and whether miR-143-3p can bind to circular RNAObjective:To confirm whether NPY is the target protein of miR-143-3p and to certify the combination of miR-143-3p and circular RNA at the same time.Methods:First,the dual luciferase reporter gene experiment verified whether NPY was the target protein of miR-143-3p;second,miR-143-3p mimic,miR-143-3p inhibitor,and negative control were used to transfect the cardiomyocytes.Results:The results of the dual luciferase reporter gene experiment showed that compared with the NC group,mmu-miR-143-3p significantly reduced the expression of luciferase of m-Npy-3UTR-WT(P <0.001.After mutation,it was compared with the NC group.And then mmu-miR-143-3p failed to down-regulate the expression of luciferase of m-Npy-3UTR-MUT(P> 0.05)by contrast.RT-qPCR results showed that with comparing negative control the expression of miR-143-3p was significantly increased in the mimic group(P<0.05),and the expression of NPY mRNA was significantly reduced(P <0.05);while the expression of miR-143-3p in the inhibitor group was significantly reduced(P <0.05),NPY mRNA expression was significantly increased(P<0.05).Conclusion:1.NPY and miR-143-3p have been verified to have a targeting relationship in 293 T cells,miR-143-3p and mmu＿circ＿0000021 have the possibility of binding site.2.Low expression of miR-143-3p can increase the NPY expression of neonatal C57 BL / 6 mouse cardiomyocytes,while high expression of miR-143-3p can reduce the expression of NPY in cardiomyocytes.PART Ⅳ Confirmation of the circular RNA mediates miR-143-3p targeting NPY and regulates cardiomyocyte H/R injuryObjective:To confirm in vitro that mmu＿circ＿0000021 mediates miR-143-3p targeting NPY,regulates intracellular calcium concentration and thus affects cardiomyocyte H/R injury.Methods: Adeno-associated virus experimental group are constructed in the hypoxia-reoxygenation model.Similarly,through adenovirus transfection of NPY(overexpression)and transfection Stained with NPY-scramble control(sc)(negative control)in this way.Different groups was constructed in the H/R model,observed with an inverted fluorescence microscope and analyzed with a multi-functional microplate reader to relatively quantify the intracellular calcium ion concentration.Results: The results of the multifunctional microplate reader showed that the fluorescence value of the H/R group was higher than that of the Normal group(P<0.05),but interfering with mmu＿circ＿0000021-siRNA in the loop,the fluorescence value of the mmu＿circ＿0000021-siRNA+HR group was less than mmu＿circ＿0000021-ir+ H/R group(P<0.05);mmu＿circ＿0000021-siRNA+NPY(overexpression)+H/R group fluorescence value was found to be higher than mmu＿circ＿0000021-siRNA+NPY-sc(negative control)+H/R group(P<0.05).The above-mentioned median fluorescence value is directly proportional to the intracellular calcium ion concentration.This also means the higher the fluorescence value,the higher the calcium ion concentration.Conclusion: Confirmed mmu＿circ＿0000021 mediates miR-143-3p targeting NPY axis in the experiment and the main mechanism is to affect the H/R injury of cardiomyocytes by regulating the calcium ion concentration.PART Ⅴ Confirmation of the circular RNA mmu＿circ＿0000021 mediates miR-143-3p targeting NPY in the mouse myocardial ischemia / reperfusion model by affecting myocardial ischemia / reperfusion microcirculationObjective:To confirm that mmu＿circ＿0000021 mediates miR-143-3p targeting NPY and to affect myocardial ischemia / reperfusion microcirculation disorder simultaneously.Methods:HE staining was used to observe the aggregation of erythrocytes in myocardial tissues;TTC staining was used to observe the area of??myocardium without reflow and infarction;Microscopic technique was used to observe the ultrastructural changes of microvessels in myocardial tissue.Finally,the expression of myocardial tissue mmu＿circ＿0000021,miR-143-3p,NPY mRNA was determined by RT-qPCR method.Western Blot method was detected NPY protein expression in each group.Results:The mouse myocardial ischemia-reperfusion model was divided into 4groups: normal group,control group,experimental group(IR + AAV9-shRNA)and negative control group(sham + AAV9-NC).We further examined the protein expression of NPY in each group,and found that the expression of NPY protein in the experimental group was significantly less than that in the negative control group or control group(P> 0.05).The results of gelatin ink staining showed that the number of microvessels in the experimental group was improved(P> 0.05).TTC staining showed that AAV9-shRNA can partially relieve myocardial remodeling after myocardial ischemia-reperfusion and quantitative analysis(P> 0.05).Using cardiac ultrasound to detect the cardiac function of mice,it was found that AAV9-shRNA is beneficial to cardiac function recovery and quantitative analysis after myocardial ischemia-reperfusion.Electron microscope observation(EM)revealed that irregular endothelial swelling and luminal stenosis appeared in the heart microvessels after IR injury in WT mice but in IR-injured hearts injected with AAV9-shRNA the microvessels are partially relieved.To sum up,the expression of mmu＿circ＿0000021 reduced in vivo can affect the structural changes of microcirculation and improve cardiac function.Conclusion:mmu＿circ＿0000021 mediates miR-143-3p to target NPY protein,which improves microcirculation disorder,thereby reducing myocardial ischemia / reperfusion injury in mice and improving cardiac function.