Long Non-coding RNA PGM5-AS1 Inhibits Migration Of Endothelial Cells By Promoting Phosphoglucomutase 5

Background and ObjectiveAtherosclerosis is the main cause of coronary heart disease,ischemic cerebrovascular disease,and peripheral artery disease.Although the pathogenesis of atherosclerosis is not completely clear,it is generally believed that endothelial cell dysfunction caused by stimulation such as hemodynamics,hypoxia,and endotoxin is the initial factor of atherosclerosis.It is of great significance to find new targets to prevent and treat the occurrence and development of atherosclerosis by regulating the function of endothelial cells.Long non-coding RNA is defined as a non-coding transcript larger than 200nt.Many studies have shown that long non-coding RNA plays an important biological role and participates in regulating the occurrence and development of human diseases.Multiple studies have proved that long non-coding RNA affects the occurrence and development of atherosclerosis by regulating endothelial cell migration and angiogenesis.PGM5 is a member of the phosphoglucomutase family,which encodes proteins that play an important role in intracellular carbohydrate metabolism.Previous studies on PGM5 have focused on tumor and muscle differentiation.Many recent studies have found that PGM5 can be used as a new tumor suppressor and has been proved to inhibit the proliferation,migration,and invasion of colorectal cancer cells in vitro.However,there are no reports on the relationship between PGM5 and atherosclerosis.Materials and methods1.Long non-coding RNA PGM5-AS1 promotes the expression of its adjacent coding gene PGM5.1.1 Long non-coding RNA microarray profilingTotal RNA from three atherosclerotic plaque and three normal arterial wall was extracted with TRIzol reagent.According to the manufacturer’s plan,the Agilent Array platform is used for data analysis.All the original microarray data were stored in NCBI’s Gene expression Omnibus database(GEO GSE97210).1.2 Bioinformatics Analysis.To explore the position of PGM5-AS1 in the genome and the proteins that may function.1.3 Construction and transfection of lentivirus overexpressing PGM5-AS1.To explore the regulatory effect of PGM5-AS1 on PGM5 and the effect of PGM5-AS1 on endothelial cell migration and angiogenesis.1.4 Real-time fluorescence quantitative PCR and Western blotting.The expression level of the target gene at the transcriptional and translational level was detected.2.Long non-coding RNA PGM5-AS1 and PGM5 inhibit endothelial cell migration and angiogenesis.2.1 Construction of Recombinant plasmid overexpressing PGM5(pcDNA-PGM5),Cell Migration,and Angiogenesis.The effects of PGM5 on vascular endothelial cell migration and angiogenesis were detected.3.Long non-coding RNA PGM5-AS1 inhibits MMP9 expression and cell migration through PGM5.3.1 Construction and transfection of PGM5 small interference RNA(siRNA-PGM5).It is proved that PGM5 mediated the inhibition of MMP9 expression and cell migration by PGM5-AS1.Results1.The expression of PGM5-AS1 and PGM5 decreased in atherosclerotic plaques.Three cases of normal intima and atherosclerotic plaque were selected for gene chip analysis.The expression of PGM5-AS1 and its adjacent gene PGM5 in atherosclerotic plaque decreased by 16.01 and 27.7 times,respectively.2.In vascular endothelial cells,overexpression of PGM5-AS1 could increase the expression of PGM5.Bioinformatics analysis showed that PGM5-ASI and PGM5 were located on chromosome 9 and adjacent to each other.PGM5-AS1 overexpression lentivirus vector was transfected into vascular endothelial cells.The results of real-time fluorescence quantitative PCR and immunoblotting showed that the mRNA and protein levels of PGM5 were up-regulated.3.Overexpression of PGM5-AS1 and PGM5 inhibited endothelial cell migration and angiogenesis.Human umbilical vein endothelial cells stably overexpressed PGM5-AS1 were transiently transfected with PGM5 overexpression recombinant plasmid or empty vector(pcDNA-Mock).The cells were divided into an experimental group and a control group.Cell migration and angiogenesis experiments were performed.The results showed that the angiogenesis and cell migration ability of vascular endothelial cells was inhibited after overexpressing PGM5-AS1 and PGM5.4.PGM5-AS1 affected the migration of vascular endothelial cells and suppressed the expression of MMP9 by promoting the expression of PGM5.After the total protein and mRNA of endothelial cells overexpressing long non-coding RNA PGM5-AS1 and PGM5 were extracted,the results of real-time fluorescence quantitative PCR and Western blotting showed that the expression of MMP9 decreased.siRNA-PGM5 was added to or not added to endothelial cells with stable overexpression of PGM5-AS1,and protein and mRNA were extracted for western blot and cell migration experiments.The results showed that the expression of MMP9 was increased and the migration ability of endothelial cells was enhanced.