MicroRNA-150-5p Regulates The Function And Mechanism Of Vascular Smooth Muscle Cells And Abdominal Aortic Aneurysm Via Targeting EGR2/EPHB2

Background:Abdominal aortic aneurysm(AAA)is a common and serious disease with high mortality and aneurysm rupture,characterized by progressive abdominal aortic dilation.Its fatality rate is as high as 85%,but its genetic factors have not been fully determined.Therefore,early diagnosis,prevention and treatment are particularly important.In addition,the application of bioinformatics in AAA makes it possible to study the mechanism of its occurrence and development.Through AAA full-length transcriptome sequencing,we aimed to elucidate the transcriptome map of AAA and further reveal its molecular mechanism through the ONT MinION platform.Based on full-length transcriptome data,this will help us further understand the genome annotation and gene structure of AAA.Smooth muscle cells(SMCs)play a very important role in the development and progression of vascular diseases such as abdominal aortic aneurysm(AAA).The lesions mainly occur in the abdominal segment of the aorta,and the pathological mechanisms of AAA include elastic tissue degeneration,vascular smooth muscle cell(VSMC)depletion,and rupture.At the same time,VSMC migration will activate related pathways,leading to the pathogenesis of AAA.EGR2 is an early growth response protein.Studies have found that EGR2 is regulated by growth factors and plays an important anti-cancer role.At the same time,it can also play an important regulatory role in cardiovascular diseases such as diabetes by inhibiting the proliferation and migration of VSMCs.However,the role of EGR2 in AAA has not been reported,and this study was devoted to exploring the mechanism of EGR2 in AAA.Previous studies have shown that microRNAs(miRs)are involved in multiple cellular functions and serve as specific targets for the diagnosis and treatment of AAA.However,the role of miRNAs in smooth muscle cells and whether the underlying mechanisms regulate AAA progression remain unclear.Purpose:This study aimed to evaluate the differential expression and clinical value of miR-150-5p in AAA patients,whether EGR2 is regulated by miR-150-5p and its function and mechanism,and to explore the effect of miR-150-5p on vascular smooth muscle cell proliferation and migration play an important role in regulating the occurrence and development of AAA.Methods:The transcriptome features of AAA were identified by AAA full-length transcriptome sequencing analysis,and its possible molecular mechanism was further revealed at the gene level.-PCR,VSMC culture,cell proliferation assay,scratch assay,Transwell assay,RNA pull-down assay,establishment of mouse AAA model,cell transfection,construction of MiR-150-5p mimics and inhibitors,H&E staining,immunohistochemistry,Western blot,immunofluorescence and other experimental procedures to explore the function of miR-150-5p in regulating vascular smooth muscle and AAA through the target EGR2/EPHB2,whether there are other targets of miR-150-5p,such as EPHB2,and its regulation and its function and mechanism.Results:1.Full-length transcriptome sequencing analysis,which proves that the ONT method is suitable for the identification of lncRNAs.The results of DETs analysis showed that there were 7044 differentially expressed transcripts in the two groups,including 4278 up-regulated transcripts and 2766 down-regulated transcripts.2.In the KEGG analysis,organismal systems,human diseases,and environmental information processing were the top three categories that account for the highest proportions.Generally,a total of 4071 annotated DETs were associated with some pathways associated with human disease,in which infectious disease was significantly identified,such as staphylococcus aureus infection,tuberculosis,Epstein-Barr virus infection,and Herpes simplex infection.A total of 1684 DETs might participate in organismal systems-related pathways,in which the Fc gamma Rmediated phagocytosis,natural killer cell mediated cytotoxicity,and intestinal immune network for IgA production were the top three pathways.A total of 1381 DETs were classified as belonging to environmental information processing,in which the PI3K-Akt,NF-kappa B,and calcium signaling pathways were the top three.3.According to the above sequencing results,EGR2 is one of the most significantly differentially expressed genes.4.MiR-150-5p can directly bind to EGR2 and regulate its functional activity,and miR-150-5p is significantly highly expressed in AAA clinical samples.5.MiR-150-5p mimics promote vascular smooth muscle proliferation and migration,while miR-150-5 inhibitors inhibit vascular smooth muscle cell proliferation and migration.6.MiR-150-5p can also regulate its downstream target EPHB2.7.MiR-150-5p inhibitor attenuates the progression of the mouse AAA model.In conclusion,the expression of miR-150-5p was significantly upregulated in human and mouse AAA models compared with human and mouse controls.Overexpression of miR-150-5p induced proliferation and migration of vascular smooth muscle cells,whereas inhibition of its expression led to the opposite result.Based on gene interactions,EPHB2 and EGR2 were identified as direct downstream targets of miRNA-150-5p,and miR-150-5p inhibitor attenuated progression in a mouse model of AAA.Conclusion:The third-generation nanopore-based RNA sequencing was introduced to explore the regulatory mechanism of AAA,and the regulatory effect of RNA on AAA was analyzed from the gene level.Inhibition of miRNA-150-5p could exert protective effects in AAA by targeting EPHB2 and EGR2.Therefore,the current study provides more insights into the mechanism of miRNA-150-5p and AAA,and provides a theoretical basis for nucleic acid drugs to treat AAA.