Effects Of Circ₀029343/miR-96 Regulation Of SR-BI On Atherosclerotic Lesions In ApoE^-/- Mice

Atherosclerosis?As?is a chronic inflammatory response caused by multiple factors.Dyslipidemia has been identified as an important risk factor.Epidemiological studies have shown that the level of high density lipoprotein cholesterol?HDL-C?is negatively correlated with the risk of atherosclerotic cardiovascular disease.The protective effect of HDL on atherosclerosis is closely related to its key role in the reverse cholesterol transport?RCT?.Some drugs designed to improve HDL-C levels have been developed,such as those to increase apoprotein AI?apoAI?levels,which have shown some beneficial effects in preclinical studies or in patients with coronary heart disease?CHD?.However,the presumption that increased HDL-C levels are beneficial for cardiovascular disease?CVD?is increasingly questioned because in most clinical trials,simply increasing HDL-C levels has not resulted in improved cardiovascular disease.Therefore,drugs designed to restore HDL function may be more effective in the treatment of atherosclerosis than drugs used to raise HDL-C levels.The scavenger receptor class B type I?SR-B??is a polyligand physiological HDL membrane receptor protein whose main function is to facilitate the transport of cholesterol esters from high-density lipoprotein to cells through a non-endocytic mechanism called selective uptake.In addition to promoting the flow of selective cholesterol into cells,SR-B? also promotes the flow of free cholesterol from peripheral tissues?including macrophages?to HDL.SR-B? is expressed in a variety of tissue and cell types,including adipocytes,macrophages and endothelial cells et al.However,in rodents,SR-B? is widely expressed in liver and adrenal glands,macrophages and steroid-producing cells.SR-B? is closely related to AS in liver cells and macrophages.The SR-B? mediated cholesterol flow of macrophages is bidirectional,and the net cholesterol movement depends on the intracellular lipid content and the direction of cholesterol gradient.SR-B? plays an important role in promoting bidirectional cholesterol flux in lipid loaded cells.The inactivation of macrophage SR-B? promotes the development of atherosclerosis in apoE-deficient mice and LDL-deficient mice.However,macrophage SR-B? may have a dual effect on atherosclerosis,as it has been reported to promote the development of early lesions while inhibiting more advanced lesions.These observations suggest that SR-B? plays a dual role in regulating macrophage cholesterol homeostasis.Liver SR-B? mainly participates in the final part of RCT,mediates HDL-CE selective lipid uptake?SLU?into the liver,and promotes the plasma HDL-C clearance.Studies have shown that in SR-B? gene deficient mice(SR-B?^-/-),plasma HDL-C level is decreased due to overexpression of SR-B? in liver and increased due to deletion of SR-B? gene.However,even if the plasma HDL-C concentration is low,the up-regulation of SR-B? can alleviate atherosclerosis formation in LDLR^-/-mice,and SR-B? gene knockout can aggravate atherosclerosis formation.SR-B? selective HDL-cE uptake pathway has been evaluated for its potential in the prevention of AS.MicroRNAs?miRNA?are highly conserved single-stranded non-coding small RNAs?about 22 nucleotides in length?that target 3'non-coding regions?3'UTR?through base pairing,promoting the degradation of the target mRNA or inhibiting its transcription to regulate gene expression.Previous studies have shown that miRNAs play an important regulatory role in HDL metabolism and lipid homeostasis,and have been used for the treatment of cardiovascular diseases including AS.It has been reported that miR-96 is closely related to intracellular lipid homeostasis.High fat diet can induce miR-96 expression in C57BL/6N mice.In addition,miR-96 can regulate expression of Sterol regulatory element-binding proteins?SREBPs?.Bioinformatics indicate that the 3'UTR of SR-B? and miR-96 had conservative binding sites in a variety of biological species.Whether miR-96could inhibit intracellular cholesterol uptake,outflow and RCT by targeting SR-B?,cause intracellular lipid accumulation and lipid deposition in the blood vessel wall,and ultimately lead to the formation of AS.circRNA?circular RNA?is a kind of non-coding RNA reported recently.circRNA can play the role of competitive endogenous RNA?ceRNA?by competing with miRNAs 3'UTR in miRNA response elements?MRE?,eliminating the inhibition of miRNA to target mRNA.Thus,the expression of miRNA target genes can be increased through the post-transcriptional level.It has been confirmed that circRNA regulatory networks are abnormal in the development of a variety of diseases.The previous application of bioinformatics successfully predicted that circ₀029343 could be expressed in AS and bind to miR-96,so how circ₀029343 regulated miR-96 and whether it could regulate the expression of miR-96 target gene through competitive binding of miR-96,affecting the process of AS.However,no relevant literature has been reported so far.In order to study the effect of circ₀029343on AS,THP-1 macrophages,HepG2 cells and apoE^-/--mouse were used to study the mechanism of circ₀029343 as`miRNA sponges and verify the effect of circRNA on AS.Part ?:Prediction of miR-96 target gene and its targeting effect with SR-B? 3'UTRAims:to predict SR-B? 3'UTR targeting binding sites with miR-96 and verify the possibility of targeting binding in THP-1 and HepG2 cells.Methods:1.miR-96 mature sequences were obtained by querying miRBase database,and SR-B? 3'UTR sequences were obtained from GeneBank website.The combination of SR-B? 3'UTR and miR-96 was analyzed through TargetScan and miRanda website.2.Based on the predicted results,wild-type psiCHECK-2-SR-B?-UTR-W and mutant psiCHECK-2-SR-B?-UTR-Mut plasmids were constructed,and they were co-transfected with pMIR96?miR-96?and miR-96 inhibitor?anti-miR-96?to HEK293T cells for luciferase reporter gene activity.3.MiR-96/anti-miR-96were transfected into THP-1 and HepG2 cells,mRNA and protein levels of SR-B? were determined by RT-PCR and Western blot.Results:1.Bioinformatics predictive analysis showed that miR-96 could bind to SR-B? 3'UTR,and the binding free energy between the two was low.2.The relative activity of luciferase was significantly inhibited after the co-transfection of miR-96 with wild-type psiCHECK-2-SR-B?-WT 3'UTR,and increased after transfection with anti-miR-96.After co-transfection with corresponding mutant,relative luciferase activity remained unchanged.3.miR-96 inhibited the mRNA and protein expression of SR-B? in THP-1 and HepG2 cells,while anti-miR-96 transfection showed the opposite results.Conclusions:miR-96 can target SR-B? 3'UTR and inhibit the expression of SR-B? in THP-1 and HepG2 cells.Part ?:Effect of miR-96 on selective HDL-CE uptake in macrophages and liver cellsAims:to observe whether miR-96 can regulate the uptake of Dil-HDL and HDL-CE in THP-1 and HepG2 cells by inhibiting SR-B? expression,and change the intracellular lipid content.Methods:1.Human THP-1 and HepG2 cells were transfected with adenovirus pMIR96 and anti-miR-96.The uptake of Dil-HDL in THP-1 and HepG2 cells was observed by flow cytometry.2.TC measurement kit was used to evaluate the intracellular TC level.The contents of TC,FC and CE in THP-1 cells were determined by HPLC.RT-PCR was used to determine the mRNA levels of insulin induced gene-1?insig-1?,ATP binding cassette transporter G1?ABCG1?and sterol regulatory element-binding protein?SREBP-lc/2?,respectively.Results:1.Compared with the control group,Dil-HDL uptake and selective HDL-CE uptake were significantly reduced in THP-1 and HepG2cells transfected with adenovirus pMIR96.2.TC level in HepG2 cells decreased,while lipid content in THP-1 cells increased.After transfection with anti-miR-96,the above results showed an opposite change.In addition,after adenovirus pMIR96 transfection,the expression of insig-1 and SREBP-lc/2 in THP-1 cells was down-regulated and up-regulated,respectively.Conclusions:1.MiR-96 reduces the lipid uptake of THP-1 and HepG2cells by inhibiting the expression of SR-B? gene.2.The increased lipid content in THP-1 cells may be due to the inhibition of cholesterol efflux and the increased expression of cholesterol synthesis genes.Part ?:Effect of circ₀029343 on selective HDL-CE uptake by targeting miR-96 in macrophages and liver cellsAims:to predict the targeting binding sequence of hsa_circ₀029343and miR-96,and enhance the function of SR-B? by inhibiting miR-96,ultimately affecting the lipid content in THP-1 and HepG2 cells.Methods:1.SCARB1/mir/CIRC interaction network map was obtained from CircNet,circRNA binding with miR-96-5p was obtained from starbase,and the circRNA sequences encoded by SR-B? were queried in deepBase,CircBase and Gene Bank databases.Based on the above predicted results,the wild-type pCK-circ₀029343-WT and the mutant pCK-circ₀029343-Mut recombinant plasmid were respectively constructed to determine the luciferase reporter gene activity.The intracellular localization of circ₀029343 was observed by RNA FISH assay.2.The effect of circ₀029343 overexpression on the HDL uptake was observed by flow cytometry in THP-1 and HepG2 cells.The effect of circ₀029343 on the HDL-CE uptake was observed by isotope ¹²⁵I-TC and[³H]CEt double HDL.The TC level in cells was assessed by TC measurement kit.After interference with circ₀029343,the changes of the above indicators were detected.3.Luciferase activity verified whether circ₀029343 regulated SR-B? expression by targeting miR-96,RNA pull down,and qRT-PCR to detect the binding of circ₀029343 and miR-96.RNA FISH was used to observe the intracellular co-localization of circ₀029343 and miR-96.After co-transfection of circ₀029343 plasmid,pMIR96,Western blot was used to detect the effect of circ₀029343 on the expression of SR-B? in THP-1 and HepG2 cells,as well as the changes in lipid uptake and lipid content.Results:1.Hsa_circ₀029343 can bind to miR-96-5p and is located in THP-1 cytoplasm.2.After overexpression of circ₀029343,the Dil-HDL uptake and selective HDL-CE uptake were significantly enhanced in THP-1and HepG2,and TC level in HepG2 cells was increased,while the lipid content in THP-1 cells was decreased.After interference with circ₀029343,the above results showed an opposite change.3.Circ₀029343 co-locates with miR-96 in THP-1 cytoplasm.After targeted binding of miR-96 by circ₀029343,SR-B? expression,Dil-HDL uptake and HDL-CE selective uptake were significantly increased in THP-1 and HepG2.Conclusions:1.Circ₀029343 increased the expression of SR-B? in THP-1 and HepG2 cells by targeting miR-96.2.Circ₀029343 enhanced SR-B? function by targeting miR-96,increased lipid uptake in THP-1 and HepG2 cells,increased TC level in HepG2 cells,and decreased lipid content in THP-1 cells.Part ?:Effects of circ₀029343 on AS plaque formation by regulating miR-96 in apoE^-/--miceAims:to investigate the effect of circ₀029343 targeting miR-96 on the expression of SR-B?,RCT,lipid level,lipid content in liver,lipid deposition in liver and arterial wall and the progression of AS in apoE^-/--mouse.Methods:1.Sixty apoE^-/-mouse were fed a high-fat diet,and were randomly divided into four groups in functional experiment of miR-96:adenovirus miR-96 negative control?miR-NC?,adenovirus miR-96 group?miR-96?,miR-96 antagomir control?AV-NC?,and miR-96 antagomir group?AN?.Expression of aortic and liver tissue SR-B? was detected by RT-PCR,Western blot and immunohistochemistry.Efficiency of RCT was measured by³H-cholesterol radioactivity.Plasma and liver tissue lipid level were evaluated enzymatically.aortic sinus and liver tissue lesions,lipid deposition in organization and the level of collagen fibers were observed with HE,oil red O and Masson trichromatic stain.2.The mice were divided into four groups in functional experiment of circ₀029343:adenovirus miR-96 negative control?miR-NC?,adenovirus miR-96 group?miR-96?,miR-NC and circ₀029343co-transfection group?miR-NC+circ₀029343?,miR-96 and circ₀029343co-transfection group?miR-96+circ₀029343?.All test indicators are the same as the above methods.Results:1.Compared with the control group,mices in miR-96 group showed down-regulated SR-B? expression,RCT efficiency in aorta and liver tissues,elevated lipid levels in liver tissues and plasma,and increased lesions and lipid deposition in aortic sinus and liver tissues,while the above indicators showed the opposite results in AN group mice.2.Circ₀029343antagonised the function of miR-96 in mice,improved RCT efficiency,reduced lipid levels in plasma and liver tissue,alleviated lesions and lipid deposition in aortic sinus and liver tissue.Conclusions:1.miR-96 reduced RCT efficiency,aggravated lipid deposition in blood vessel wall and liver tissue,and accelerated AS process in apoE^-/-mouse.2.Circ₀029343 antagonizes the above functions of miR-96 in apoE^-/-mice,which may be related to the enhancement of SR-B? function.