| BackgroundPopulation aging,with its associated organ and tissue-level homeostasis imbalance and dysfunction,has become a major societal issue in the 21st century.Vascular aging is a major contributor to morbidity and mortality in the older population.It causes arterial stiffness,calcification,which leads to progressive development of chronic diseases,including hypertension and atherosclerosis[1].Vascular aging is mechanistically complex and unclear.It has been reported to be closely associated with circadian rhythm genes[2,3].These genes,controling 24-h physiological oscillation,can be divided into central and peripheral circadian clock genes according to the location.Previous studies have revealed some peripheral circadian clock genes may affect the development of vascular aging[3,4].However,the specific role of circadian rhythm genes in vascular aging remains unclear.Recent studies have revealed that the expression of circadian rhythm genes may be regulated by long non-coding RNAs（lncRNAs）at the post-transcriptional level.Some crucial lncRNAs have been identified to play significant roles in vascular aging and act as potential therapeutic targets in cardiovascular aging diseases,including atherosclerosis and heart failure[5,6].However,which lncRNA is involved in vascular aging and the functional roles and mechanisms of crucial lncRNAs remain unknown.This study was divided into three stages.The first stage was to investigate the involvement of lncRNAs in aortic aging through sequencing in vascular tissues and a circadian rhythm-related lncRNA was chosen as the target for further research.The second stage explored the transcriptional characteristics of the target lncRNA and its regulatory effect on endothelial cell senescence.The third stage investigated the target lncRNA mechanism of action using tissue-level sequencing and molecular biology experiments.Methods:Part Ⅰ Transcriptome analysis of vascular aging and the expression analysis of lncRNA NEAT1-2021.1 Aortic aging model construction and aging marker detection:The aortic aging C57BL/6J mouse model was induced by administering different doses of D-gal.Survival rate,body weight,blood glucose,and blood lipid levels were measured after eight weeks.Aortic thickness was determined via H&E staining.Vascular elastic and collagen fibers were detected via Masson’s trichrome staining.Vascular contractile and diastolic functions were assessed via echocardiography.P21 and P16 levels were detected via both western blotting and immunohistochemistry,respectively.Levels of molecular of senescence-associated secretory phenotype（SASP）,including IL-6 and CRP,were detected via enzyme-linked immunosorbent assay.Results were used to identify the optimal D-gal concentration for the induction of vascular aging.1.2 Screening for key lncRNAs in vascular aging:Aging aortas from the mouse model were used for high throughput sequencing to explore differentially expressed lncRNAs,followed by GO/KEGG enrichment analysis.The sequencing accuracy of key lncRNAs was verified using real-time fluorescence quantitative polymerase chain reaction（q RT-PCR）.The expression of lncRNA NEAT1-202 was measured using fluorescence in situ hybridization（FISH）.Part Ⅱ Role of lncRNA NEAT1-202 in endothelial cell senescence2.1 Characteristics of lncRNA NEAT1-202:Bioinformatics software,including ENSEMBL,UCSC,and DNAMAN,were used to evaluate the conservation of lncRNA NEAT1-202.Tissue specificity of lncRNA NEAT1-202 was evaluated using q RT-PCR and cell specificity and subcellular localization were assessed using immunofluorescence staining and FISH.2.2 Establishment of an endothelial senescence model and detection of aging markers:An HCAEC senescence model was established using a range of D-gal doses in vitro.P21 and P16 senescence markers were detected in the established model using western blotting and q RT-PCR,respectively.Senescent cells were detected viaβ-galactosidase staining.Endothelial cell（EC）function was quantified by detecting nitric oxide（NO）in cell supernatants.Reactive oxygen species（ROS）were detected with a DCFH-DA probe.Inflammatory factors IL-6 and IL-1βwere detected via q RT-PCR.Cell proliferation and migration were detected using a cell counting kit-8 and scratch assays,respectively.The results of these assays were used to determine the optimal D-gal concentration for causing HCAEC senescence.2.3 Function of lncRNA NEAT1-202 in endothelial senescence:The expression of lncRNA NEAT1-202 was evaluated in the endothelial senescence model.Based on the results,a low lncRNA NEAT1-202 expression cell model was established by Small interfering RNA（si RNA）transfection into ECs in vitro.To assess the effect of lncRNA NEAT1-202 silencing on endothelial senescence,P21 was detected via western blot,senescent cells were detected viaβ-galactosidase staining,ROS was detected using DCFH-DA probe,and P16,IL-6,and IL-1βwere detected via q RT-PCR.Part Ⅲ Mechanism of lncRNA NEAT1-202 in promoting vascular aging3.1 Screening of key m RNAs downstream of lncRNA NEAT1-202:Aortic aging model mice were subjected to RNA sequencing（RNA-seq）to identify differentially expressed m RNA.The transcriptome data for aging aortic pathology and physiology（GSE145972）were combined using R,followed by GO/KEGG enrichment and protein interaction analyses based on differentially expressed genes.3.2 LncRNA NEAT1-202 promotes endothelial cell senescence via ARNTL.To evaluate the effect of ARNTL on HCAEC senescence,endothelial cells with high and low ARNTL expression were established in vitro by transfecting an overexpression plasmid or si RNA,respectively.P21 levels were detected via western blotting,senescent cells were detected viaβ-galactosidase staining,ROS was detected using a DCFH-DA probe,and P16,IL-6,and IL-1βwere detected via q RT-PCR.The regulatory relationship between lncRNA NEAT1-202 and ARNTL in HCAECs was then examined by reducing the expression of lncRNA NEAT1-202 and overexpressing ARNTL.si-NEAT1-202 and oe-ARNTL were co-transfected into an endothelial senescence model and the P21 and P16 levels were detected via q RT-PCR.3.3 Mechanism behind lncRNA NEAT1-202 regulating ARNTL:micro RNA-seq（mi RNA-seq）was performed on aging aortas to screen for mi RNAs displaying differential expression..q RT-PCR was used to detect the expression of candidate mi RNAs in the endothelial senescence model.The effect of lncRNA NEAT1-202 on m RNA expression was then assessed by transfecting si-NEAT1-202 into HCAECs and detecting the expression of candidate mi RNAs via q RT-PCR.Results:Part Ⅰ Transcriptome analysis of vascular aging and expression analysis of lncRNA NEAT1-2021.1 Aortic aging model construction and aging marker detection:After continuous administration of different doses（0,160,320,and 480 mg/kg/d）of D-gal for 8 weeks,mice displayed reduced coat pigmentation（yellowish hue）and weight gain rate and increased cognitive dysfunction.The aged aortas developed thickened walls,enlarged lumens,disorganized arrangement of smooth muscle cells,increased collagen,and decreased elastic fibers.Functionally,the aortas developed narrowed systolic vascular diameter and increased vascular stiffness and P21 and P16 expression.IL-6,CRP,and plasma inflammatory factor levels also gradually increased with time.The results indicated that the vascular aging model was successfully constructed and the severity of vascular aging first increased and then decreased with increasing D-gal stimulation.The optimum D-gal concentration for inducing vascular aging and disease modeling was 160 mg/kg/d.1.2 Screening key lncRNAs in the vascular aging model:A total of 344differentially expressed lncRNAs were identified.Bioinformatics analysis showed that these lncRNAs were mainly enriched in age-related pathways and diseases.In particular,lncRNA NEAT1-202 was downregulated in the senescent HCAECs and its candidate target genes were enriched in aging pathways,including circadian rhythm.Part Ⅱ Role of lncRNA NEAT1-202 in endothelial cell senescence2.1 Transcript characteristics of lncRNA NEAT1-202:Conservation analysis revealed that lncRNA NEAT1-202 was expressed in both mice and humans.In the mouse,lncRNA NEAT1-202 was relatively enriched in the aorta compared with other tissues,expressed in ECs and vascular smooth muscle cells（VSMCs）of the aorta,and highly expressed in cell nuclei.2.2 Establishment of endothelial senescence model and aging marker detection:D-gal treatment increased P21,P16,ROS,IL-6,and IL-1βlevels andβ-galactosidase-positive cell counts,while decreasing NO levels and HCAEC proliferation and migration.A model of endothelial cell senescence was successfully established,and 50mmol/L was determined to be the optimum D-gal concentration for vascular aging and disease modeling.2.3 Role of lncRNA NEAT1-202 in endothelial senescence:P21,P16,ROS,IL-6,and IL-1βlevels andβ-galactosidase positive cells were notably downregulated after lncRNA NEAT1-202 knockdown.Part Ⅲ Mechanism of lncRNA NEAT1-202 in promoting vascular aging3.1 Screening key m RNAs downstream of lncRNA NEAT1-202:Differentially expressed genes involved in physiological and pathological aortic aging were enriched in circadian rhythm functions.The core circadian rhythm genes NPAS2 and ARNTL displayed strong correlations in a Protein-Protein Interactions（PPI）interaction network and were strongly associated with lncRNA NEAT1-202.3.2 lncRNA NEAT1-202 promoted endothelial senescence via ARNTL:Knockdown of ARNTL resulted in decreased P21,P16,ROS,IL-6,and IL-1βlevels andβ-SA-gal positive cells in HCAECs.In contrast,ARNTL overexpression increased P21,P16,ROS,IL-6,and IL-1βlevels andβ-SA-gal positive cells.Knockdown of lncRNA NEAT1-202 in HCAECs decreased the expression of ARNTL.Low lncRNA NEAT1-202 expression was rescued by high ARNTL expression,which increased cellular senescence.The results indicated that lncRNA NEAT1-202 promoted vascular senescence via ARNTL.3.3 Mechanism of lncRNA NEAT1-202 in promoting vascular aging:mi RNA-seq and bioinformatics analyses showed that mi R-320-3p and mi R-455-3p were related to lncRNA NEAT1-202 and ARNTL and were enriched in circadian rhythm regulation.The expression levels of mi R-320-3p and mi R-455-3p were increased in the HCAEC senescence model.lncRNA NEAT1-202 knockdown also elevated mi R-320-3p and mi R-455-3p expression.Conclusions:1.A C57BL/6J aortic aging model was successfully established using D-gal,and lncRNAs were screened through high-throughput sequencing.lncRNA NEAT1-202 was downregulated in the aging aortic mouse model,which was preliminary assessed to be involved in a signaling pathway linking vascular aging to circadian rhythm.2.lncRNA NEAT1-202 was confirmed to be expressed in both humans and mice.It was relatively enriched in the mouse aorta and distributed in the aortic endothelium and smooth muscle,suggesting a possible role in aortic biological processes.lncRNA NEAT1-202 expression was decreased during endothelial cell senescence and its downregulation inhibited endothelial cell senescence at the cellular level;lncRNA NEAT1-202 exerted a protective effect against senescence.3.Crosstalk between vascular aging and circadian rhythm disorders was demonstrated.lncRNA NEAT1-202 promoted vascular aging via ARNTL and its regulation may be related to mi R-320-3p and mi R-455-3p. |
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