| Backgroud and objectiveAtherosclerosis is a class of cardiovascular disease(CVD)characterized by changes in systemic vascular structure and function,mainly involving the coronary arteries.It is considered as the leading cause of mortality and morbidity worldwide.Although many altered pathways and aberrantly expressed genes involved in atherogenesis were identified,the precise molecular mechanisms for atherosclerosis are not entirely clear.Arterial endothelial dysfunction is considered as an important factor contributing to atherogenesis.Long noncoding RNA(IncRNA)is a recently discovered new RNA molecules with a broad spectrum of biological effects.They are a heterogeneous class of transcripts that regulate cellular processes by controlling gene expression at various levels,including transcription,epigenetic,posttranscription and even translation.Several lines of evidence have revealed that lncRNAs are pivotal regulators of vascular smooth muscle cells(VSMCs)and endothelial cells(ECs)cellular function,which raise the possibility that lncRNAs are potential regulators of atherogenesis.Therefore,researches of lncRNA in AS and related CVD to explore the relevant mechanism are of great significance for prevention and therapy of AS.In the present study,we identified a novel lncRNA-RP11-714G18.1 that was significantly downregulated in human aorta atherosclerotic lesions,which is mostly accompanied with reduced expression of LRP2BP.UCSC genome database and luciferase reporter analysis showed that RP11-714G18.1 could directly target coding sequence(CDS)of LRP2BP and promote the expression of LRP2BP.Further functional studies have revealed that RP11-714G18.1 could regulate vascular cell function,and remarkably inhibited MMP1 mediated cell migration through targeting LRP2BP in VSMCs and ECs.Totally,RP11-714G18.1 exerts a remarkable atherprotective role in atherosclerosis via RP11-714G18.1/LRP2BP/MMP1 pathway.Based on our findings,manipulation of the lncRNA-RP11-714G18.1/LRP2BP/MMP1 pathway may open up a novel avenue for treating atherosclerosis and related cardiovascular disorders.Contents and methods1.Expression of LncRNA RP11-714G18.1 and LRP2BP in tissue cells and clinical diagnostic value of LRP2BP1.1 Detect the expression levels of lncRNA RP11-714618.1 and LRP2BP in atherosclerotic plaque tissues and normal artery intima by qRT-PCR.1.2 Explore the cellular sources of LRP2BP in human plaques by confocal microscopy.1.3 Explore the correlation between levels of LRP2BP and traditional atherosclerosis-related serological biomarkers in patients with atherosclerosis.2.The gene expression regulation of RP11-714G18.1 to LRP2BP2.1 Explore position relation between IncRNA RP11-714G18.1 and LRP2BP through UCSC and Pubmed genome database.2.2 Construct cell lines that stably overexpressing RP11-714G18.1,explore the regulation of gene expression of RP11-714618.1 to LRP2BP in cells.2.3 Explore the interaction between RP11-714G18.1 and LRP2BP via dual luciferase assays.3.The effects of RP11-714G18.1 on cellular function in HUVECs and HA-VSMCs3.1 The effects of RP11-714G18.1 in HUVECs migration,adhesion,NO generation,and angiogenesis ability.3.2 The effects of RP11-714G18.1 in HA-VSMCs migration,apoptosis and cell cycle progression.4.The underlying mechanism of the inhibitory role of RP11-714G18.1 on cell migration4.1 Construct recombinant plasmid that overexpressing LRP2BP and tranfected into cells,explore the effects of LRP2BP in cell migration capacity of HUVECs and HA-VSMCs.4.2 Effects of RP11-714G18.1 and LRP2BP on expression of migration related key genes4.3 Effects of small interferon RNA-LRP2BP on mRNA and protein expression level of LRP2BP in HUVECs and HA-VSMCs.4.4 Effects of knocking down of LRP2BP on MMP1 expression and cell migration ability in RP11-714G1 8.1 overexpressing cellsResults1.Expression levels of lncRNA RP11-714G18.1 and LRP2BP in atherosclerotic plaque tissues were significantly lower than those in normal arterial intima.Serum levels of LRP2BP were positively correlated with HDL,but were negatively associated with CTnI levels in patients with atherosclerosis.1.1 Expression levels of lncRNA RP11-714G18.1 and LRP2BP in atherosclerotic plaque tissues were significantly lower than those in normal arterial intima1.2 LRP2BP could be detected in endothelial cells,smooth muscle cells,and macrophages,and its expression level was lower in cells from advanced atherosclerotic plaque tissues than those from normal arterial intima.1.3 Serum levels of LRP2BP were positively correlated with HDL,but were negatively associated with CTnI levels in patients with atherosclerosis.2.RP11-714G 18.1 promoted LRP2BP expression by directly targeting its CDS2.1 RP11-714G18.1 and LRP2BP were both located on chromosome 4 with the opposite transcription direction2.2 Cell lines that stably overexpressing RP11-714G18.1 were successfully constructed,and overexpressing RP11-714G18.1 could promote mRNA and protein exoression levels of LRP2BP.2.3 RP11-714G18.1 promote LRP2BP expression by directly targeting its CDS3.RP11-714G81.1 regulated cellular function via suppressing migration of HUVECs and HA-VSMCs,adhesion of HUVECs,neoangiogenesis,and apoptosis of HA-VSMCs,while promoting NO production from HUVECs,facilitating cell cycle progression of HA-VSMCs.3.1 RP11-714G81.1 suppressed HUVECs migration,adhesion,neoangiogenesis,while promoting NO production from HUVECs.3.2 RP11-714G81.1 suppressed HA-VSMCs migration,apoptosis,while facilitating cell cycle progression of HA-VSMCs.4.RP11-714G18.1 suppressed cell migration via directly targeting its nearby gene LRP2BP in HUVECs and HA-VSMCs4.1 Recombinant plasmid that overexpressing LRP2BP were successfully constructed and transiently transfected into cells,LRP2BP suppressed migration of HUVECs and HA-VSMCs.4.2 Both RP11-714G18.1 and LRP2BP could significantly reduce MMP1 expression in either HUVECs or HA-VSMCs4.3 Both protein and mRNA expression levels of LRP2BP were significantly decreased in cells transfected with LRP2BP-shRNA.4.4 The suppression of cell migration via RP11-714G18.1 transfection was completely compensated by shRNA-mediated silencing of LRP2BP in HA-VSMCs and HUVECs.Conclusions1.Expression levels of IncRNA RP11-714G18.1 and LRP2BP in atherosclerotic plaque tissues were significantly lower than those in normal artery intima.Serum LRP2BP concentration was positively correlated with HDL,while was negatively associated with CTnI levels in patients with AS;2.RP11-714G18.1 promoted LRP2BP expression by directly targeting its CDS;3.RP11-714G18.1 regulated vascular cell function via suppressing migration of HUVECs and HA-VSMCs,adhesion of HUVECs,apoptosis of HA-VSMCs,and neoangiogenesis,while promoting NO production from HUVECs,facilitating cell cycle progression of HA-VSMCs.4.RP11-714G18.1 suppressed MMP1 expression as well as vascular cell migration via directly targeting LRP2BP,the RP11-714G18.1/LRP2BP/MMP1 pathway maybe essential for the regulation of vascular dysfunction. |
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