| BackgroundWith the improvement of people's living standard and the aging of population structure,the morbidity and mortality of atherosclerosis have increased year by year.Carotid artery stenosis has been widely concerned because of the cerebrovascular events.It develops very slow and is difficult to detect.Most of the time,carotid stenosis was found after the occurrence of clinical symptoms.If it can't be promptly dealt with may cause hemiplegia,aphasia or even death.In recent years,carotid artery stenosis treatment technology has made significant progress,but there are still many patients suffer cerebrovascular events before diagnosis,especially carotid artery stenosis patients with unstable plaque are more prone to cerebrovascular events.Therefore,it is significant to research the pathogenesis and prognosis of carotid artery stenosis,to find out the biomarker associated with carotid plaque stability,to identify possible targets for its treatment.Long-noncoding-RNA(lncRNA)refers to RNA which does not have a coding function with a length greater than 200 nt.In the past,lncRNA was thought have no effect.As research continues,lncRNA was thought to play an important role in regulating the metabolism and pathophysiology.At the same time,some research found that lncRNA has a closely relationship with vascular disease,for example,lncRNA(MALAT1 and Tie-1-AS),expressed in endothelial cells,can regulate vascular proliferation and cell function.Most researches on lncRNA and atherosclerotic disease focused on the occurrence of atherosclerosis,while the mechanism of lncRNA affecting plaque stability has not been reported in the worldObjectiveThe objective of this study was to investigate the differential expressed lncRNA affecting plaque stability,to explore the impact of lnc-RP11-159G9.5.1-2 on plaque stability,and to further elucidate the cell molecular mechanisms of lnc-RP11-159G9.5.1-2 regulating function of vascular smooth muscle cells(VSMCs).Method(1)Clinical sample studies: This study was approved by the Ethical Committee of Changhai hospital and the patient informed consent was obtained.The patients underwent carotid endarterectomy with complain of carotid artery stenosis were included between August 2015-June 2016 and the carotid artery plaque specimens were collected.According to the HE stain results,the plaque specimens has been divided into stable plaque group and unstable plaque group.The lncRNA and mRNA expression profiles were screened by high-throughput microarray.Bioinformatics was used to analyze the selected differential expressed genes and predict its possible functional and biological pathways.(2)Chip results verification: The detected differential expressed lncRNAs by chip screening were verified by real-time PCR.After screening,lnc-RP11-159G9.5.1-2 was selected as the target lnc RNA,and bioinformatics prediction of lnc-RP11-159G9.5.1-2 was carried out by co-expression analysis and GO analysis.(3)Cell molecular studies: Lentivirus packaging and lentivirus transfection was conducted to intervene lnc-RP11-159G9.5.1-2 up-regulation and down-regulation in VSMCs.Then the VSMCs were divided into lnc-RP11-159G9.5.1-2 up-regulated group,lnc-RP11-159G9.5.1-2 up-regulated negative control group,lnc-RP11-159G9.5.1-2 down-regulated group,lnc-RP11-159G9.5.1-2 down-regulated control group and blank control group.By cck8 colorimetric,the effect of lnc-RP11-159G9.5.1-2 on the proliferation of smooth muscle cells was detected.Flow cytometry Annexin V FITC-PI was used to detect the effect of lnc-RP11-159G9.5.1-2 on the apoptosis of smooth muscle cells.Results(1)Clinical samples study: During August 2008-June 2016,8 carotid plaque specimens were collected in patients underwent carotid endarterectomy for carotid artery stenosis in Department of Vascular Surgery,Changhai Hospital.Through high-throughput lncRNA gene chip detection on the lncRNA expression in plaque tissue of patients with stable carotid artery plaque and unstable plaque,212 differential expressed lncRNA were found for the first time,among which 115 lncRNAs were up-regulated and 97 were down-regulated in unstable plagues compared with those in stable plaques.In the GO analysis and KEGG pathway analysis,differentially expressed mRNAs are enriched in regulation of vascular size,platelet aggregation,cellular components dissociation involved in the implementation phase of apoptosis,cell response to stimulation of vascular endothelial growth factor,negative regulation of apoptosis,signal way of vascular endothelial growth factor receptor,up-regulation of endothelial cell chemotaxis and SMC migration and other items.These biological functions related to occurrence and development of carotid plaque and the whole cell cycle process.(2)Chip results verification: Chip results show high expression of lnc-RP11-159G9.5.1-2 in unstable plaque.RT-PCR results confirmed that expression of lnc-RP11-159G9.5.1-2 was significantly higher than that of the stable plaque group(p <0.0001).The relative expression level of lnc-RP11-159G9.5.1-2 was 4.666 ± 1.741 in patients with unstable plaque,while it was 1.000 ± 0.613 in the stable plaque patients.(3)Cell molecular research: lnc-RP11-159G9.5.1-2 can inhibit the proliferation of vascular smooth muscle cells and promote the apoptosis process.ConclusionThe lncRNA expression profile of stable plaque and unstable plaque in patients with carotid stenosis was obtained by high-throughput lncRNA gene chip screening,suggesting high expression of lnc-RP11-159G9.5.1-2 in unstable plaque.Lnc-RP11-159G9.5.1-2 influence the stability of smooth muscle cells by inhibiting the proliferation and promoting the apoptosis process.The results suggested that the high expression of lnc-RP11-159G9.5.1-2 in unstable plaque suppresses the proliferation and promotes apoptosis of smooth muscle cells in plaque fibrous cap,resulting in reduction of SMCs,thinning of plaque fibrous cap and vulnerability to plaque rupture.Eventually the high expression of lnc-RP11-159G9.5.1-2 lead to unstable plaque,high risk of cerebrovascular events and thus affected the progression and prognosis of the disease. |
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