The Mechanism Of NEAT1/miR-148b-3p/ICAM-1 Pathway For Regulating Vascular Endothelial Cells Inflammatory Response In Coronary Slow Flow

Objective: Coronary slow flow(CSF)is an angiographic phenomenon characterized by delayed coronary opacification with normal or near-normal epicardial coronary arteries.Clinical studies have shown that 80–90% of CSF patients experience recurrent chest pain,and 33% of patients require subsequent hospitalized for treatment,which seriously affects their quality of life.In addition,2.5% of patients have a poor prognosis.There is an increased risk of ventricular arrhythmia,cardiovascular mortality and critical events such as sudden death.At the same time,objective evidence such as echocardiography found that the systolic function of the left and right ventricles was reduced in patients with CSF.Therefore,clinicians should pay close attention to patients with CSFThe pathogenesis of CSF is unclear,and it is currently believed to be related to factors such as inflammation,reduced vascular endothelial function,abnormal microvascular reserve function,early atherosclerosis lesion and genetic factors.Among these factors,the role of inflammation in the occurrence and development of CSF is recognized by most researchers,although the mechanism remains to be established.Due to the unclear pathogenesis of CSF,biomarkers closely related to the development,diagnosis and treatment of CSF have not been identified for use in clinical practice.Leukocyte exudation is one of the most important features of the inflammatory process,which includes the adhesion of leukocytes to vascular endothelial cells.Intercellular adhesion molecule-1(ICAM-1)plays an important role in the adhesion of leukocytes and the migration of transendothelial cells,which can enhance the adhesion between leukocytes,inflammatory cells and endothelial cells and promote endothelial cell activation.Micro RNAs(miRNAs)can be targeted bind to the 3’untranslated region of target genes,and form RNA-induced silencing complexes by binding to Argonaute 2(Ago2)protein to negatively regulate their expression,thereby regulating changes in cell function.Long non-coding RNAs(lnc RNAs)can serve as a “miRNA molecular sponge”to participate in the formation of a competitive endogenous RNA network,and regulate gene expression by combining with miRNAs.The first part of this study firstly determined the plasma levels of soluble ICAM-1,miR-148b-3p and NEAT1 in CSF patients,and explored the correlation between these molecules and clinical factors in CSF patients.In the second part of this study,we used the oxygen-glucose deprivation(OGD)cell model to simulate the functional changes in CSF endothelial cells,and to explore the interaction between ICAM-1,miR-148b-3p and NEAT1 at the cellular level as well as the possible mechanism underlying its effect in CSF cells.In this study,we aimed to provide anew theoretical and experimental basis for the occurrence and development of CSF,and to provide possible biomarkers for the diagnosis and treatment of CSF.Methods:Part 1: The subjects were divided into CSF group and control group by corrected thrombolysis in myocardial infarction frame count(c TFC).The functional status and quality of life were evaluated by seattle angina questionnaire(SAQ).Echocardiography was used to evaluate the left ventricular systolic and diastolic function.ELISA and quantitative real-time PCR(qRT-PCR)methods were used to detect the plasma levels of soluble ICAM-1,miR-148b-3p and NEAT1.Part 2: Human umbilical vein endothelial cells(HUVEC)cultured under OGD conditions were used to as an in vitro model of CSF cells.Cells overexpressing and/or knocking down NEAT1,miR-148b-3p and ICAM-1 were constructed by cell infection.The expression levels of NEAT1,miR-148b-3p and ICAM-1 in cells were measured by qRT-PCR or Western blotting.Cell proliferation was measured by 5-Ethynyl-2?-deoxyuridine(Ed U)and Cell Counting Kit-8(CCK-8)assay.Cell apoptosis was detected by apoptosis assay.The relationship between NEAT1 and miR-148b-3p was verified by luciferase reporter gene assay and RNA immunoprecipitation(RIP)assay.The relationship between ICAM-1 and miR-148b-3p was verified by luciferase reporter gene assay.Results:1.Compared with the control group,the physical limitation score of SAQ,LVGLS,MV E and MV E/A in the CSF group was significantly lower than that in the control group.2.The plasma soluble ICAM-1,IL-6 and TNF-α levels were significantly increased in CSF patients,and were significantly positively correlated with the mean c TFC.3.Compared with the control group,the plasma miR-148b-3p was in the CSF group was significantly reduced,and was negatively correlated with the mean c TFC and s ICAM-1 levels.The plasma NEAT1 level in the CSF group was significantly higher than that in the control group,and was positively correlated with the mean c TFC and s ICAM-1 levels.4.Circulating plasma s ICAM-1,miR-148b-3p and NEAT1 have good diagnostic ability for CSF.5.Compared with normal cultured HUVECs,the expression of NEAT1 and ICAM-1was upregulated in OGD-treated HUVECs,while miR-148b-3p expression was downregulated.OGD-treated HUVECs inhibits proliferation and promotes apoptosis.6.ICAM-1 knockdown promotes the proliferation of OGD-treated HUVECs and inhibits apoptosis.7.miR-148b-3p promotes the proliferation of OGD-treated HUVECs and inhibits apoptosis.8.miR-148b-3p can target and bind to ICAM-1,and miR-148b-3p reverses the effects of ICAM-1-mediated inhibition of cell proliferation and promotion of apoptosis.9.NEAT1 knockdown promotes the proliferation of OGD-treated HUVECs and inhibits apoptosis.10.miR-148b-3p can target and bind to NEAT1,and NEAT1 reverses the effects of miR-148b-3p-mediated promotion of cell proliferation and inhibition of apoptosis.Conclusion:1.The functional status,the left ventricular systolic and diastolic function of CSF patients are decreased,while the inflammatory response is enhanced.Plasma NEAT1 level,circulating miR-148b-3p level and s ICAM-1 level could be used as biomarkers for the diagnosis of CSF.2.The highly expressed NEAT1 functions as a competitive endogenous RNA in OGD-treated HUVECs.By specifically binding to miR-148b-3p,NEAT1 weakens the negative regulatory effects of miR-148b-3p on the target gene ICAM-1 to upregulates ICAM-1 expression,as well as inhibiting the proliferation of HUVECs and promoting their apoptosis.