| Objective: Coronary Slow Flow(CSF)refers to the phenomenon that coronary arteriography(CAG)confirms that there is no obvious stenosis of the coronary arteries,but the blood flow in the coronary arteries slows down.Previous studies have confirmed that cardiac function has been impaired in patients with CSF,and more severe cases can lead to fatal arrhythmia and other malignant cardiovascular events.Therefore,CSF patients should be given early intervention.As the current research on the mechanism of CSF is rare,the mechanism is unclear,and there is currently no standardized treatment method in clinical practice.At present,the diagnosis of CSF relies on CAG,because of its invasiveness and high cost,the diagnosis and follow-up of CSF are difficult to progress.It is currently believed that decreased vascular endothelial function may be the main cause of CSF.Endothelin-1(ET-1)is a vasoconstrictor.When endothelial function is impaired,ET-1 levels increase.Brachial artery blood flow-mediated vascular endothelial dilatation(Flow mediated dilatation,FMD)reflects the vasodilatation function,and is a commonly used noninvasive method of vascular endothelial function detection in clinical practice.Long non-coding RNA(LncRNA)plays an important role in the occurrence and development of cardiovascular diseases.Lung adenocarcinoma transcript 1(MALAT1)was first reported in non-small cell lung cancer.MALAT1 also plays an important role in endothelial dysfunction in cardiovascular diseases.It can participate in the regulation of cell functions such as endothelial cell proliferation,apoptosis,and death.Micro RNA(miRNA)can be targeted to bind to the untranslated region(UTR)at the 3'end of the target gene,thereby inhibiting the function of the target gene and regulating cell function.miRNAs are involved in the occurrence and development of cardiovascular diseases and have become new targets for the treatment of cardiovascular diseases.It has been reported that miR-181b-5p is involved in regulating the occurrence and development of cardiovascular diseases,such as coronary heart disease,atherosclerosis(AS),aneurysm,and myocardial hypertrophy.HOXD9 is a member of the Homeobox Genes(HOX Genes)family,as a transcription factor involved in the regulation of the transcriptional regulation of disease signal molecules.In the cardiovascular field,reports on HOXD9 are relatively rare.Some studies believe that it plays a role in regulating the function of human microvascular endothelial cells or human umbilical vein endothelial cells.The transcription factor Myocyte enhancer factor 2A(MEF2A)is a member of the MEF2 family.It can act as an active factor that maintains the stability of vascular endothelial function,participates in endothelial inflammation,and affects the integrity of endothelial cells.Endothelial cell apoptosis and proliferation and other functions.This study aims to clarify the mechanism of CSF,find molecular biological targets,and provide biomarkers for CSF diagnosis and treatment.Methods: Part 1: 41 patients with CSF diagnosed by coronary angiography as the case group,and 37 patients with age and sex matching during the same period as the control group are collected.TIMI frame count(TFC)was used to evaluate coronary blood flow velocity.Conventional echocardiography,tissue Doppler and two-dimensional speckle tracking techniques were used to evaluate the left ventricular systolic and diastolic function of the subject;FMD was used to evaluate endothelial function.ELISA and RT-PCR methods were used to detect the expression of plasma ET-1,MALAT1 and miR-181b-5p.Part 2: Construction of HUVECs induced by CSF under oxygen glucose deprivation conditions,RT-PCR detection of the objective MALAT1,miR-181b-5p,MEF2 A and ET-1 expression levels;Western blot detection of ET-1 and MEF2 A expression levels;construction of MALAT1 expression Silent adenovirus,overexpression Synergistic Activation Mediator(SAM)dual vector lentivirus,miR-181b-5p overexpression and expression silenced adenovirus,MEF2 A overexpression and expression silenced adenovirus,respectively infected CSF-HUVECs,and obtained MALAT1 expression silence and Overexpressed CSF-HUVECs,miR-181 b overexpression and silent CSFHUVECs,MALAT1 and miR-181 b double overexpressed CSF-HUVECs,MEF2 A overexpression and silent CSF-HUVECs,MEF2 A and miR-181 b double Overexpressed CSF-HUVECs.Observe the fluorescence of the cells under a fluorescence microscope to confirm the transfection efficiency.CCK8 and EdU experiments to detect cell proliferation ability;flow cytometry to detect cell apoptosis;dual luciferase reporter gene system verifies that miR-181b-5p and MEF2 A,miR-181b-5p and MALAT1 have targeted binding effects;chromatin Coimmunoprecipitation experiments verified that MEF2 A has a binding effect with the ET-1 promoter region.The third part: construction of CSF-induced HUVECs,RT-PCR detection of the target HOXD9 expression level;Western blot detection of HOXD9 expression level;construction of HOXD9 silenced CSF-HUVECs,HOXD9 expression silence and MALAT1 overexpression CSF-HUVECs.CCK8 and EdU experiments to detect cell proliferation ability;flow cytometry to detect cell apoptosis;Western blot to detect the expression of ET-1;ChIP experiment verified that HOXD9 has a binding effect with the MALAT1 promoter region.Results: 1.The expression of MALAT1 and ET-1 in the peripheral blood plasma of CSF patients is increased,and miR-181b-5p is decreased;in CSF patients,FMD is decreased,GLS is decreased,and MV-E is decreased;MALAT1,miR-181b-5p and ET-1 are in The expression in the peripheral blood of CSF patients is correlated with the coronary blood flow velocity of CSF patients,and can be used as a predictor of CSF.2.Compared with normal cultured HUVECs,CSF-HUVECs decreased proliferation,increased apoptosis,and increased ET-1 expression.3.MALAT1 is highly expressed in CSF-HUVECs.Silencing MALAT1 can increase the proliferation of CSF-HUVECs,decrease apoptosis,and decrease ET-1.4.The expression of miR-181b-5p is low in CSF-HUVECs;overexpression of miR-181b-5p can increase the proliferation of CSF-HUVECs,decrease apoptosis,and decrease the expression of ET-1.5.MALAT1 can be combined with miR-181b-5p.Overexpression of MALAT1 can reverse the protective effect of miR-181b-5p on endothelial cells,reduce proliferation,increase apoptosis,and increase ET-1 expression.6.MEF2 A is highly expressed in CSF-HUVECs.Silencing MEF2 A increases the proliferation of CSF-HUVECs,reduces apoptosis,and decreases ET-1 expression;overexpression of MEF2 A inhibits proliferation and promotes apoptosis and ET-1 expression.7.MEF2 A binds to the ET-1 promoter region to regulate its transcription.10.MEF2 A and miR-181b-5p target binding.8.Silencing miR-181b-5p significantly increased the expression of MEF2A;up-regulating miR-181b-5p inhibited the expression of MEF2 A.Silencing MALAT1 significantly inhibits the expression of MEF2A;further up-regulating miR-181b-5p can restore the expression of MEF2 A.Overexpression of MEF2 A on the basis of overexpression of miR-181b-5p can reverse the increase of proliferation induced by overexpression of miR-181b-5p,the decrease of apoptosis and the decrease of ET-1expression.9.HOXD9 is up-regulated in CSF-HUVEC.Silencing HOXD9 can increase proliferation,reduce apoptosis,and reduce ET-1 expression;further overexpression of MALAT1 can reverse the effect of silencing HOXD9.10.HOXD9 regulates the transcription of MALAT1 and participates in regulating the endothelial function of CSF-HUVECs.Conclusion: 1.This study confirms that the endothelial function of CSF patients is reduced;MALAT1 and ET-1 are elevated in the peripheral blood of CSF patients,and miR-181b-5p is reduced in the peripheral blood of CSF patients,and it is correlated with coronary blood flow velocity;MALAT1,miR-181b-5p and ET-1 can be used as biomarkers to predict CSF.2.MALAT1 can bind miR-181b-5p through sponge adsorption,thereby weakening the negative regulation of miR-181b-5p on the target gene MEF2 A,up-regulating the expression of MEF2 A,and further promoting downstream ET-1 transcription,affecting CSF vascular endothelium Features.3.HOXD9 transcription regulation MALAT1 affects the function of CSF vascular endothelial. |
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