Mechanism Of Lnc-PDHB-1:1 In Regulating Macrophage Function And Affecting Carotid Atherosclerotic Plaque Stability

Part ? The verification and functional exploration of differential lncrna in carotid atherosclerotic plaqueBackgroundCarotid artery stenosis is responsible for up to 30%of the causes of ischemic stroke.With the improvement of the living standards of our people and the aging of the population structure,atherosclerotic carotid stenosis has accounted for 90%of all carotid stenosis.Because of its slow onset and not easy to be detected in the early stage,patients who have clinical symptoms without timely treatment often leave serious sequelae such as aphasia,hemiplegia and even death.The current international clinical diagnosis,treatment experience and scientific research have found that even for plaque tissue samples with the same degree of carotid stenosis have different infiltration of lipid,thrombus and inflammatory cells,and the stability of these plaques are significantly different.Patients'previous cerebral infarction and the incidence of recurrent stroke are closely related to the degree of plaque stability.Long non-coding RNA?lncRNA?refers to RNA that has no coding function and is longer than 200 nt.Although it has been confirmed that lncRNA can lead to the occurrence and progress of atherosclerosis by influencing metabolic regulation,vascular function,immune and inflammatory response,there are relatively few studies on the relationship between lncrna and the stability of atherosclerotic plaque.ObjectiveOur institution has carried out a genomics study of carotid plaques in the early stage and screened out differentially expressed lncRNAs and mRNAs in stable and unstable carotid atherosclerotic plaque.The purpose of this part is to verify the differences of lncrnas in stable and unstable plaques indicated by high-throughput sequencing chip,and to explore the specific molecular mechanism of differential lnc-pdhb-1:1 and synergistic substances in specific cellsMethods1.Under the premise of hospital ethics review and informed consent of patients,standardized procedures were used to collect human carotid atherosclerotic plaque samples.According to pathological criteria,plaque samples were divided into stable plaque group and unstable plaque group.qRT-PCR,PCR and gel electrophoresis experiments were carried out to verify the difference between the stability of the first 9 lncRNAs and the co expression mRNA predicted by the high throughput sequencing chip.2.Bioinformatics searches for evidence of correlation between lncrna and co mRNA.Western blot was used to detect the content of mRNA translation protein in stable and unstable plaque.3.Immunohistochemical localization of the distribution of CO molecules and target molecules in carotid atherosclerotic plaques.Results1.After pathological standard identification,22 cases of stable and unstable plaques were used qRT-PCR,PCR and gel electrophoresis experiments verify 1.5-fold differentially expressed top 9 lncRNAs?lnc-XXbac-BPG116M5.15.4-2:1,lnc-SLC2A14-2:1,lnc-PDHB-1:1,lnc-STARD3NL-6:1,lnc-PTBP3-6:1,lnc-SRPX2-2:1,lnc-METTL6-5:1,lnc-PPM1J-1:1,lnc-POTEG-4:21?in high-throughput RNA chips.The expression of lnc-PDHB-1:1 in unstable plaques was still significantly down-regulated?P<0.05?,which is consistent with the results of the previous gene chip.Co-expression analysis showed that 4 mRNAs?Dnase113,Ndufaf4,Sponl,Mllt11?were correlated with lnc-PDHB-1:1,of which Dnase113 was significantly positively correlated with lnc-PDHB-1:1?P<0.05?.qRT-PCR verified that the expression of Dnase113 was significantly down-regulated in unstable plaques?P<0.05?,which is consistent with the results of the previous gene chip.2.Based on the existing lncrna and gene database?LNCipedia:https://lncipedia.org;UCSC:http://genome.ucsc.edu?,the transcriptional gene of lnc-PDHB-1:1,the base sequence of lnc-PDHB-1:1 and Dnase113 genes were retrieved.The length of the transcriptional gene of lnc-PDHB-1:1 was 3161 bp;Lnc-pdhb-1:1 is a 702bp nucleic acid sequence with two terminal elements,while Dnase113 is a 18747bp nucleic acid sequence with seven exons.By comparing the transcription gene sequence of lnc-PDHB-1:1 with Dnase113 gene sequence,it was found that the transcription gene of lnc-PDHB-1:1 and Dnasell3 was complementary.It was clear that there was a significant correlation between them.Western blot showed that the content of dnase113 protein in unstable plaque was significantly lower than that in stable plaque?P<0.05?.3.Immunohistochemical localization in carotid plaque samples showed that the content of Dnase113 in unstable plaque decreased significantly,while the content of METs in unstable plaque was significantly higher than that in stable plaque.ConclusionLnc-PDHB-1:1 was significantly down-regulated in unstable plaques,and Dnase113,a synergistic substance with positive regulatory relationship,was significantly down regulated in mRNA and protein levels,while Dnase113 could degrade macrophage extracellular traps?METs?.It is suggested that the down-regulation of lnc-PDHB-1:1 may result in the subsequent content changes of dnase113 and METs,which may affect the stability of carotid plaque.Part ? Study on the function and mechanism of lnc-PDHB-1:1 regulating macrophagesObjectiveTo investigate the specific regulatory mechanism between Lnc-PDHB-1:1 and Dnase113,and to clearly influence the effect of Lnc-PDHB-1:1 expression on macrophage function.Methods1.In situ hybridization RNA-Scope technology for intracellular localization of Lnc-PDHB-1:1 and cooperating molecule Dnase113,predicting the possible molecular mechanism between them.2.The RNA pull down experiment specifically settled Lnc-PDHB-1:1,also the RNA and protein bound to it,and verified the binding cooperation relationship by Western Blot and qRT-PCR.3.Packaging interferes lnc-PDHB-1:1 up-regulation and down-regulation lentiviruses.Human macrophages are divided into lnc-PDHB-1:1 up-regulation group,lnc-PDHB-1:1 up-regulation negative control group,lnc-PDHB-1:1 down-regulation group,lnc-PDHB-1:1 down-regulation negative control group and blank control group.Western Blot and qRT-PCR to detect changes in Dnase113 content after intervention with Lnc-PDHB-1:1 expression;After GM-CSF,C5a,and LPS re-intervented macrophages,qRT-PCR was used to detect the expression of Lnc-PDHB-1:1 and Dnase113;Comparison of the content of citrated citH3 in macrophages of different groups by immunofluorescence.4.Dual luciferase report experiment validates the specific regulatory mechanism between Lnc-PDHB-1:1 and Dnase113.Results1.In situ hybridization RNA-Scope experiments showed that the fluorescence signals of lnc-PDHB-1:l and Dnase1113 were localized in the cytoplasm.Some fluorescent signals overlap,suggesting that there may be a binding relationship between them.2.RNA pull down experiment results show that there is no direct binding relationship between lnc-PDHB-1:1 and Dnase113 at the mRNA or protein level.3.After lentivirus intervention in macrophages:while lnc-PDHB-1:1 overexpression,the expression of Dnase113 was also significantly increased?P<0.05?;Interfering with lnc-PDHB-1:1,the expression of Dnase113 was significantly decreased?P<0.05?.4.The results of the dual luciferase report experiment showed that:Lnc-PDHB-1:1 competitively binds miR-6866-5P,which reduces the inhibitory effect of miR-6866-5P on the translation of Dnase113,thereby up-regulating the expression of Dnase113.ConclusionThis section demonstrates that Lnc-PDHB-1:1 competitively binds miR-6866-5P and up-regulates the expression of Dnase113.The overexpression of lnc-PDHB-1:1 in macrophages up-regulated the expression of Dnase113,which may reduce the METs content by affecting the synthesis of citrated citH3 or degrading the synthesized citrated citH3.Part ? The effect and mechanism of lnc-PDHB-1:1 on the stability of carotid plaqueObjectiveTo explore the effect and mechanism of lnc-PDHB-1:1 intervention on the stability of carotid plaque in miceMethods1.The consistency of lnc-PDHB-1:1 in human mouse species was detected by bioinformatics.The products of lnc-PDHB-1:1 primer and the expression of Dnase113 in mouse monocytes were detected by qRT-PCR.2.The male mice of C57BL/6J ApoE?-/-?were used to construct the carotid atherosclerotic plaque model.Packaging adeno-associated virus,which can interfere with the expression of lnc-PDHB-1:1,was injected into tail vein for intervention after operation.The groups were set as:lnc-PDHB-1:1 up-regulated group,lnc-PDHB-1:1 up-regulated negative control group,lnc-PDHB-1:1 down-regulated group,lnc-PDHB-1:1 down-regulated negative control group and blank control group.Carotid atherosclerotic plaque specimens were taken from the mice after modeling,and the pathological HE staining was used to identify the stability.The differences in the occurrence of stable plaques and unstable plaques between different groups were compared.3.Design of mouse lnc-PDHB-1:1 probe and in situ hybridization to verify virus transfection efficiency.4.After interfering with lnc-PDHB-1:1 expression,the content of Dnase113 was detected by qRT-PCR;Dnase113,and citrated citH3 in carotid plaques were labeled by immunohistochemistry,and the contents of different groups were compared.Results1.Based on homology sequence matching and Bioedit software analysis,it was found that the base coincidence rate of mouse-derived Lnc-XR₃83076.3 and human-derived lnc-PDHB-1:1 is as high as 57.7%,and the positions and sequences of key elements are basically consistent.The downstream genes are basically coincident,all adjacent to Dnase113.2.Comparison between groups after successful mouse modeling and viral intervention:lnc-PDHB-1:1 down-regulation group had severe carotid stenosis or even occlusion,and the incidence of unstable plaque was significantly higher than other groups?P<0.05?.3.In situ hybridization results indicate successful virus transfection.4.qRT-PCR verification:while lnc-PDHB-1:1 overexpression,the expression of Dnase113 in mouse carotid plaque was significantly increased?P<0.05?;interference with lnc-PDHB-1:1 expression,the expression of Dnase113 was significantly down-regulated?P<0.05?.Immunohistochemical showed that the content of citrated citH3 in mice unstable carotid plaques increased significantly,and the content of Dnase113 was significantly reduced?P<0.05?.ConclusionInterfering with the expression of lnc-PDHB-1:1 in macrophages,up-regulating Dnase113 and reducing METs content can reduce the incidence of unstable plaques.