| Background: Coronary artery disease(CAD)is the leading cause of death worldwide,because it blocks the basic blood flow to the heart muscle,leading to myocardial ischemia or myocardial infarction(MI).Although reperfusion methods such as drug thrombolysis,interventional therapy or surgical bypass can be used to achieve revascularization,some patients still have insufficient or no myocardial perfusion due to coronary microcirculation dysfunction.Therefore,how to promote angiogenesis after MI to re-establish the blood supply of ischemic myocardium is particularly important.The adult mammalian heart has weak regenerative capacity,but this weak regenerative capacity is usually not enough to produce clinically significant myocardial repair after heart injury.The difference is that the heart of newborn mice has a strong ability to repair itself when it is injured.One of the key factors for myocardial regeneration is the revascularization of damaged tissues.In a normal heart,there is a capillary next to almost every cardiomyocyte,and the ratio of endothelial cells to cardiomyocytes is 3:1.It shows that the coronary microvascular network is established in the early stage of neonatal.Therefore,deconstructing the signal molecules and related pathways that regulate angiogenesis in the damage and repair of neonatal mice has a key impact on improving the prognosis of adult patients with ischemic heart disease.In recent years,more and more studies have shown that non-codingRNA(ncRNA)is an important participant in the occurrence and development of cardiovascular diseases.At present,it has been reported in the literature that a variety of microRNA(miRNA)and long non-codingRNA(LncRNA)play an important role in angiogenesis in the neonatal stage of mice,and another member of the ncRNA family ring It has not been reported whether circularRNA(CircRNA)is involved in the regulation of angiogenesis in the neonatal stage.Through high-throughput sequencing technology to analyze the expression of CircRNA in different developmental stages or physiological and pathological conditions of the heart,it was found that there are a large number of differentially expressed CircRNA in the hearts of newborn and adult mice,and the expression abundance of CircRNA under different physiological and pathological conditions is different,indicating that CircRNA It has a very close relationship with heart development,physiology and pathological processes.In this study,we screened the circularRNA CircERBB2 IP and explored its mechanism of action by screening and analyzing the CircRNA expression profile of neonatal and adult mouse hearts.Part I Screening of CircRNA and its effect on myocardial regeneration in neonatal miceObjective:1、 Screen,verify and identify differentially expressed CircRNA in the hearts of newborn and adult mice and analyze their basic characteristics.2、 In vivo experiments observe the effect of knocking down CircERBB2 IP on myocardial regeneration in neonatal mice.Methods:1 、 Screening and identification of circularRNA: CircRNA expression profile screening and analysis in the hearts of newborn and adult rats were used to screen out circularRNA CircERBB2 IP.q RT-PCR detected CircERBB2 IP at different time points after birth in heart tissue(P1,P7,adult rats).)Expression situation.By designing q PCR primers spanning the CircRNA specific linker sequence,the amplification curve and melting curve were verified.Sanger sequencing to determine the circularization site and the sequence of the circularization site.RNase R(RNase R)detects the stability of CircRNA,and cytoplasmic and nuclear isolation experiments determine the subcellular location of CircRNA.2 、 Establish a neonatal myocardial infarction model by ligating the anterior descending branch of the left coronary artery,and inject the ADV vector or empty vector expressing shRNA targeting CircERBB2 IP into the left ventricular myocardium,and observe the effect of knocking down CircERBB2IP on the heart function,The degree of fibrosis,the proliferation of cardiomyocytes and the effect of angiogenesis.Results:1、 CircERBB2 IP was screened from the CircRNA expression profile in the heart of newborn and adult rats.The results of q RT-PCR confirmed that the expression of CircERBB2 IP in mouse heart tissue was significantly reduced during the development of the heart.2、 The PCR amplification results indicate that the melting curve of the CircRNA primer is a single peak,the Tm value is within the normal range,the electrophoresis band is single,the molecular weight is correct,the Sanger sequencing result is a single peak,and the circularization site is correct.After RNase R is digested and degraded,the mRNA level of linear ERBB2 IP is significantly reduced,and CircERBB2 IP can resist RNase R.Cytoplasmic and nuclear isolation experiments show that CircERBB2 IP is mainly located in the cytoplasm.3、 In the neonatal myocardial infarction model,compared with the control adenovirus group(ADV-sh NC),the ADV-sh CircERBB2 IP group mice showed:decreased cardiac function,increased cardiac fibrosis,and increased scar area.Knockdown of CircERBB2 IP has no effect on the proliferation of cardiomyocytes,but it significantly reduces the density of CD31+ capillary vessels and α-SMA+ arterioles around the infarcted myocardium.Conclusion:1、 During the development of the mouse heart,the expression of CircERBB2 IP was significantly reduced.2、 Knockdown of CircERBB2 IP is not conducive to myocardial regeneration and weakening of angiogenesis after myocardial infarction in neonatal mice.Part II CircERBB2 IP regulates the biological behavior of myocardial microvascular endothelial cells(CMECs)to improve heart repair after myocardial infarctionObjective:1、 To observe the effect of CircERBB2 IP on heart repair after myocardial infarction of adult mice in vivo experiments.2、 To observe the effects of CircERBB2 IP on the proliferation,migration and tube formation of CMECs in vitro.Methods:1、Construct a recombinant adeno-associated virus type 9(AAV9-CircERBB2IP)with cardiac specific overexpression of CircERBB2 IP,establish a mouse myocardial infarction model,tail vein injection overexpresses CircERBBIP in mouse hearts and observe the effects of overexpression of CircERBB2 IP on cardiac function,degree of fibrosis and angiogenesis in mice after myocardial infarction influences.2、Cultivation of CMECs: Enzymatic digestion combined with differential adhesion method was used to cultivate CMECs.Immunofluorescence(IF)was used to identify CMECs markers CD31 and v WF.3、To study the effect of CircERBB2 IP on the biological behavior of CMECs: use adenovirus system overexpression or knock down CircERBB2 IP in CMECs,use EDU staining to detect cell proliferation,flow cytometry to detect cell cycle,transwell cell migration test to detect cell migration,Tubule formation experiment to observe the tubule formation of CMECs.Results:1、 In the mouse myocardial infarction model,compared with the control adeno-associated virus group(AAV9-Vector),the mice in the AAV9-CircERBB2 IP group showed improved cardiac function,reduced cardiac fibrosis,decreased scar area,and infarction CD31+ capillary density andα-SMA+ arteriole density increased in the border zone.2、 CMECs cultured by the enzyme digestion method combined with the differential adherence method,the cells adhered after 24 hours,began to grow on the third day,and grew into a dense cell layer in 6-7 days,which looked like paving stones.IF identified the positive expression of its markers CD31 and v WF.3、 In vitro experiments confirmed that up-regulating the level of CircERBB2 IP in CMECs promotes the proliferation,migration and tubule formation of CMECs;while knocking down CircERBB2 IP will inhibit their proliferation,migration and tube formation ability.Conclusion:1、 Overexpression of CircERBB2 IP after myocardial infarction in adult mice promotes cardiac repair and angiogenesis.2、 CircERBB2 IP promotes the proliferation,migration and formation of CMECs.Part III Study on the mechanism of CircERBB2 IP promoting the proliferation,migration and tube formation of CMECsObjective: Explore the mechanism of CircERBB2 IP promoting the proliferation,migration and tube formation of CMECs.Methods:1、 Jointly use Jaspar and UCSC and other bioinformatics software to predict the upstream transcription factor that regulates CircERBB2 IP,and verify the interaction between the transcription factor GATA4 and CircERBB2 IP promoter through the dual luciferase gene report experiment and Ch IP experiment,and through q RT-PCR to verify whether the transcription factor GATA4 promotes the transcription of CircERBB2 IP.2、 Use Circ MIR 1.0 and Starbase and other bioinformatics software to predict the miRNA that CircERBB2 IP may bind,and verify the combination of CircERBB2 IP and miR-145a-5p through the dual luciferase gene report experiment.Observe CircERBB2 IP and miR-by the cytoplasmic nucleus separation experiment.Whether the subcellular localization of 145a-5p is consistent.3、 Construct miR-145a-5p mimics or inhibitors and act on CMECs to observe the effect of miR-145a-5p on the biological behavior of CMECs.4、 Co-transfect CMECs with CircERBB2 IP overexpression vector and miRNA mimics,and use experiments to investigate whether the interaction between the two is functional.5、 Use bioinformatics software such as Target Scan and miRanda to predict the possible downstream target genes of miR-145a-5p,and verify the expression of target genes through dual luciferase gene report experiment and Western Blot experiment.Results:1、 Through the analysis of bioinformatics software,we found that the transcription factor GATA4 can target and regulate the transcription of CircERBB2 IP.Overexpression of GATA4 can significantly up-regulate the activity of the CircERBB2 IP promoter reporter gene.Ch IP experiments confirmed the combination of GATA4 and CircERBB2 IP promoter.The results of q RT-PCR showed that overexpression of GATA4 can promote the transcription of CircERBB2 IP.2、 miR-145a-5p is the target miRNA molecule of CircERBB2 IP.miR-145a-5p significantly affects the luciferase activity of CircERBB2 IP.The subcellular localization of miR-145a-5p and CircERBB2 IP are consistent,and are both located in the cytoplasm.3、 miR-145a-5p inhibits the proliferation,migration and tube formation of CMECs.4、 Smad5 is the regulatory gene of miR-145a-5p.The dual luciferase reporter gene experiment confirmed that miR-145a-5p mimics can reduce the luciferase activity of Smad5 3’-UTR,overexpress or inhibit miR-145a-5p It can reduce or increase the protein expression level of Smad5.5、 miR-145a-5p mimics can partially reverse the proliferation,migration and tube formation of CMECs due to overexpression of CircERBB2 IP.6、 Overexpression of CircERBB2 IP can up-regulate the protein expression level of Smad5,but this effect can be partially reversed by miR-145a-5p mimics.Conclusion: CircERBB2 IP regulates the expression of Smad5 through miR-145a-5p to promote the proliferation,migration and tubule formation of CMECs. |
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