CCDC80 Down-regulated LPL Expression In Vascular Smooth Muscle Cells And The Effects And Mechanisms On Atherosclerosis

Atherosclerosis?AS?is a major cause of coronary atherosclerotic heart disease,cerebral infarction,and peripheral vascular disease.The pathogenesis of AS is very complex,mainly including vascular endothelial cell injury,abnormal lipid metabolism,inflammation and immunity.Abnormal lipid metabolism is the pathological basis of AS,including hypercholesterolemia,hypertriglyceridemia,low-density lipoprotein metabolism abnormalities and high-density lipoprotein metabolism abnormalities.Studies have shown that hypertriglyceridemia is an important risk factor for metabolic syndrome-related diseases such as coronary heart disease,hypertension,and diabetes.Therefore,studying the mechanism of regulating plasma triglyceride metabolism is of great significance for the prevention and treatment of AS.Lipoprotein lipase?LPL?is a secreted glycoprotein that is hydrolyzed by triglyceride-rich lipoproteins such as very low-density lipoprotein?VLDL?,chylomicron?CM?,reducing plasma triglyceride?TG?levels.Study found that plasma TG levels were significantly elevated,and AS plaques appeared in the aortic roots in LPL knock-out mice.LPL overexpressing in muscle tissue,heart and adipose tissue significantly reduced plasma TG levels and reduced AS lesion area.It is suggested that LPL is a key protein regulating plasma TG metabolism and can effectively prevent the development of AS.Recent studies have shown that LPL methylation levels are increased in patients with metabolic syndrome,and LPL methylation levels are positively correlated with plasma TG levels and metabolic abnormalities.Therefore,finding a mechanism to inhibit LPL methylation modification is of great significance for regulating plasma TG metabolism.Coiled-coil domain-containing protein 80?CCDC80?is a protein expressed and secreted by fat cells,smooth muscle cells and so on.Research has found that CCDC80 can promote the occurrence and development of pulmonary hypertension and increase the risk of obesity-related metabolic diseases.Studies have shown that plasma CCDC80 levels are significantly increased in obese patients,and positively correlated with fasting plasma TG levels and atherosclerosis?intimal to medial thickness ratio?,suggesting that CCDC80 may be involved in the development of AS.Therefore,it is of great significance to study the effect and mechanism of CCDC80 on LPL-catalyzed TG hydrolysis and AS development.The underlying mechanism of regulating the expression of CCDC80 has not yet been elucidated.Large of studies have confirmed that non-coding RNAs play a pivotal role in lipid metabolism and AS processes.Micro RNAs?mi RNAs?inhibit genes expression by binding to the 3' untranslated region?3'UTR?of the target genes and participate in multiple stages of AS development.Mi R-141-3p/200a-3p is involved in the lipid metabolism process,but the specific mechanism is unknown.Bioinformatics prediction showed that mi R-141-3p/200a-3p binds to the 3'UTR of CCDC80,suggesting that mi R-141-3p/200a-3p may be involved in the regulation of LPL expression by regulating CCDC80 expression.Long non-coding RNAs?lnc RNAs?can participate in the AS process by interacting with mi RNAs to regulate lipid metabolism.Long-chain non-coding RNA transfer-related lung adenocarcinoma transcription factor-1?lnc RNA metastasis-associated lung adenocarcinoma transcript-1,lnc RNA MALAT1?affects foam cell formation by regulating CDC36 expression,indicating that lnc RNA MALAT1 is involved in lipid metabolism,but it is involved in LPL The regulatory role has not yet been elucidated.Bioinformatics predicted that lnc RNA MALAT1 could bind to mi R-141-3p/200a-3p sequence,suggesting that MALAT1 may participate in the regulation of CCDC80 and LPL expression through mi R-141-3p/200a-3p.Based on this,we divided the study into the following four parts:?1? cultured VSMCs,overexpressed or silenced CCDC80,detected LPL expression levels,and clarified the regulation of CCDC80 on LPL;?2?overexpression or silencing CCDC80,TET2,detection ERK1/2 phosphorylation level,TET2 expression,LPL promoter DNA methylation level,LPL expression,elucidation of the molecular mechanism of CCDC80 promoting LPL promoter DNA methylation and inhibiting LPL expression through ERK1/2-TET2 pathway;?3?Apo E-/-mice were used as experimental subjects,transfected with CCDC80 or sh CCDC80,and their effects on APL expression in Apo E-/-mice,plasma lipid levels,aortic lipid accumulation and AS plaque area were observed.The overall level of clear CCDC80 promotes the mechanism of action of AS;?4?mi R-141-3p/200a-3p mimic/inhibitor or MALAT1/si MALAT1 transfected VSMCs,q RT-PCR and Western Blot examined the expression of CCDCC80 and LPL,clarify the regulation of MALAT1-mi R-141-3p/200a-3p on CCDC80 and LPL expression.Part I CCDC80 inhibits LPL expression in human aortic vascular smooth muscle cellsAim: To study the effect of CCDC80 on LPL expression.Methods: Human artery vascular smooth muscle cells?HA-VSMCs?were cultured in DMEM high glucose medium,and CCDC80 overexpressing and silencing adeno-associated virus vectors were constructed and transfected into cells.The transfective effects of CCDC80 overexpression and silencing was detected by Western Blot;q PCR and Western Blot methods were used to detect LPL m RNA and protein expression levels,respectively.Results: CCDC80 overexpressed or silenced with adeno-associated virus vectors were stably expressed in cells.q PCR and Western Blot assays showed that overexpression of CCDC80 significantly down-regulated LPL m RNA and protein levels in HA-VSMCs;CCDC80 sh RNA treatment significantly up-regulated LPL m RNA and protein expression in HA-VSMCs.Summaries:?1?CCDC80 overexpression down-regulates LPL m RNA and protein expression,while silencing CCDC80 up-regulates LPL m RNA and protein expression.?2?CCDC80 affects plasma TG metabolism may be achieved by regulating LPL expression.Part II The potential molecular mechanism of CCDC80 inhibiting LPL expressionAim: To determine the potential mechanisms of how CCDC80 inhibiting LPL expression.Methods: HA-VSMCs were cultured in DMEM high glucose medium,then transfected with CCDC80 or CCDC80 sh RNA adeno-associated virus vector.Overexpression and silencing effects of CCDC80 were detected by Western Blot.Total extracellular regulated protein kinase?ERK1/2?and phosphorylated ERK1/2 levels was detected by Western Blot.;then VSMCs cells were treated with 10 ?M ERK1/2 agonist Curcumin or ERK1/2 inhibitor PD98059,and q PCR and Western Blot methods were used to detect LPL m RNA and protein expression levels,respectively.To determined that CCDC80 regulates LPL expression through regulating the activation of ERK1/2.Secondly,over-expression or silencing of CCDC80,methylation-specific PCR method was used to detect the DNA methylation level of LPL promoter;the corresponding method was used to detect 5-methylcytosine?5-m C?and 5-hydroxymethylcytosine?5-hm C?level;q PCR and Western Blot methods were used to detect the m RNA and protein expression levels of ten-eleven translocation methyl cytosine dioxygenase 2?TET2?;Overexpression or silencing of TET2,Western Blot method to detect LPL expression;methylation-specific PCR method to detect LPL promoter DNA methylation level;corresponding method to detect cell 5-m C and 5-hm C levels;q PCR and Western Blot methods were used to detect the expression of LPL m RNA and protein,respectively.It was confirmed that CCDC80 affects LPL promoter DNA methylation modification and regulates its expression through TET2.Then,overexpress or silence CCDC80,while HA-VSMCs were treated with 10 ?M ERK1/2 agonist Curcumin or ERK1/2 inhibitor PD98059,Western Blot was used to detect the expression of TET2 and LPL;HA-VSMCs were transfected with CCDC80 sh RNA,Co-Immunoprecipitation?Co-IP?method was used to detect the interaction of TET2 and FOXO3 a.HA-VSMCs were transfected with FOXO3 a si RNA,silencing effect was detected by Western Blot,and LPL m RNA and protein expression were detected by q PCR and Western Blot.Results: CCDC80 overexpressed or silencing with adeno-associated virus vectors were stably expressed in cells.q PCR and Western Blot assay showed that overexpression of CCDC80 significantly down-regulates the m RNA and protein expression of LPL and TET2 in HA-VSMCs;decreasing the phosphorylation of ERK1/2,has no effect on the total ERK1/2 level;increasing 5-m C level,reducing the 5-hm C level.Methylation-specific PCR results showed that CCDC80 overexpression significantly increases LPL promoter DNA methylation levels.CCDC80 sh RNA treatment significantly up-regulates LPL and TET2 expression;increasing p-ERK1/2 and 5hm C levels;decreasing LPL promoter DNA methylation and 5m C levels.CCDC80 sh RNA co-treatment with ERK1/2 inhibitor PD98059 can down-regulate LPL and TET2 expression,increasing LPL promoter DNA methylation level and 5-m C level,and reducing 5-hm C level.Meanwhile,CCDC80 co-treating with ERK1/2 agonist Curcumin up-regulates the expression of LPL and TET2,decreasing the DNA methylation level and 5-m C level of LPL promoter,and increasing the level of 5-hm C.The results of Co-IP showed that CCDC80 sh RNA significantly reduced the interaction between TET2 and FOXO3a.FOXO3 a si RNA partially blocked the upregulation of LPL expression induced by CCDC80 sh RNA.Summaries:?1?CCDC80 inhibits LPL expression by increasing the level of DNA methylation of the LPL promoter.?2?CCDC80 inhibits LPL expression by decreasing ERK1/2 phosphorylation and down-regulating TET2 expression to promote DNA methylation of LPL promoter.?3?CCDC80 participates in the regulation of LPL expression by inhibiting the interaction of FOXO3 a with TET2.Part III CCDC80 accelerates atherosclerotic plaque formation in apoE^-/-miceAim: To investigate the effects of CCDC80 on LPL expression and activity,plasma lipid levels and AS plaque formation in apo E knockout(apoE^-/-)mice.Methods: Eight-week-old male apoE^-/-mice were randomly divided into control group?Control group?,CCDC80 overexpression group?CCDC80 group?and CCDC80 silent group?sh CCDC80 group?,with 10 mice in each group.Apo E-/-mice were transfected with recombinant adeno-associated virus?RAA?,r AA-CCDC80 and r AA-sh CCDC80,respectively.Apo E-/-mice were euthanized after 12 weeks of feeding on a high-fat/high-cholesterol diet.The apoE^-/-mice aorta was isolated and detected the protein expression of CCDC80 in aorta by Western Blot.The transfection effect was confirmed.Mice were weighed and recorded every two weeks during feeding.Before the sacrifice,apoE^-/-mice fasted for 12 hours and injected with 300 U/kg of heparin by tail vein injection.Ten minutes later,blood was taken by eyeball removal.The blood was collected into a tube with EDTA for centrifugation to obtain plasma.The levels of cereal third transaminase?ALT?,aspartate transaminase?AST?and alkaline phosphatase?ALP?in the blood samples were detected by chemiluminescence method;Determination of total cholesterol?TC?,non-high density lipoprotein cholesterol?non-HDL-C?,and high density lipoprotein cholesterol?HDL-C?and TG levels,in plasma using a commercial enzyme kit,the LPL activity assay kit detects LPL activity in plasma after heparin treatment.Apo E-/-mice aorta was isolated and detected the expression of LPL by q PCR and Western Blot.Aortic sinus was frozen and sectioned.Immunofluorescence staining was used to detect the expression of LPL.Immunofluorescence staining was used to detect LPL and vascular smooth muscle cell marker ?-Smooth muscle actin??-SMA?.The aortic arch of each group of mice was isolated,and the area of AS plaque at the aortic arch was observed by stereomicroscope.Longitudinal dissection of the aorta?from the aortic arch to the branch of the common iliac artery?,accumulation of lipids in the aorta was observed by Oil Red O staining.The area of aortic sinus AS,collagen content and lipid accumulation were detected by HE,Masson and oil red O staining,respectively.Results: The expression of aortic CCDC80 was detected by Western Blot.The results showed that CCDC80 and sh CCDC80 were stably transfected in apoE^-/-mice which fed with a high-fat and high-cholesterol diet.Overexpression or silencing of CCDC80 did not affect the body weight of the mice.Similarly,overexpression or silencing of CCDC80 had no effect on plasma levels of ALT,AST and ALP,indicating that overexpression or silencing of CCDC80 did not affect liver function in mice.Overexpression of CCDC80 significantly increased plasma TC,non-HDL-C and TG levels and had no effect on HDL-C levels.In addition,we also found that overexpression of CCDC80 significantly reduced plasma LPL activity and decreased aortic LPL expression.Immunofluorescence results showed that CCDC80 overexpression significantly decreased the fluorescence intensity of LPL at aortic valve plaque.And we also found that LPL at aortic valve plaques co-localization of ?-SMA;overexpression of CCDC80 increased plaque area at the aortic arch of apoE^-/-mice,promoted lipid accumulation in the aorta,increased AS plaque area and lipid accumulation in the aortic sinus,and accelerated AS occur.sh CCDC80 increased LPL expression and activity,decreased plasma TC,non-HDL-C and TG levels,decreased aortic lipid accumulation,inhibited AS plaque formation,and alleviated AS development in apoE^-/-mice.Summaries:?1?CCDC80 promotes aortic lipid accumulation in apoE^-/-mice and accelerates AS plaque formation.?2?CCDC80 increases plasma lipid levels by inhibiting LPL m RNA and protein expression,decreasing plasma LPL activity,thereby accelerating AS plaque formation in apoE^-/-mice.Part IV Expression regulation of CCDC80 by MALAT1-mi R-141-3p/200a-3p axisAim: To study the possible regulation molecular mechanism of CCDC80 expression.Methods: The mi RNAs which can interacte with the 3'UTR of CCDC80 were analyzed by the bioinformatics website,and the combined free energy was analyzed.The binding of mi RNA and CCDC80 was then detected by culturing HEK 293 T cells using the luciferase reporter gene.HA-VSMCs were transfected with 40 ?M mi RNA mimics or inhibitors for 24 hours,and then transfection efficiency was detected by q PCR;q PCR and Western Blot methods were used to detect the expression of CCDC80 and LPL m RNA and protein,respectively.The lnc RNAs interacting with mi R-141-3p and mi R-200a-3p were analyzed by the bioinformatics website.HEK 293 T cells were cultured and their binding was detected by a luciferase reporter gene.The cells were transfected with lnc RNA MALAT1 overexpression vector or si RNA,q PCR was used to detect overexpression and silencing effects,and examined the effects of overexpression or silencing of lnc RNA MALAT1 on mi R-141-3p and mi R-200a-3p expression by q PCR.lnc RNA MALAT1 was transfected into HA-VSMCs with mi R-141-3p mimic or mi R-200a-3p mimic,q PCR and Western Blot were used to detect the m RNA and protein expression levels of CCDC80 and LPL,respectively;lnc RNA MALAT1 si RNA and mi R-141-3p/200a-3p inhibitors were co-transfected into HA-VSMCs,q PCR and Western Blot were used to detect the m RNA and protein expression of CCDC80 and LPL,respectively.Results: Bioinformatics results showed that mi R-141-3p/200a-3p was bound to the 3'UTR of CCDC80,and the sequence of CCDC80 in different species was told to be conservative.In addition,the analysis revealed that the mi R-141-3p and mi R-200a-3p sequences are highly conserved,with only one base sequence being different.Mi R-141-3p/200a-3p-CCDC80 had lower free energy.The luciferase reporter gene showed that mi R-141-3p/200a-3p binds to the 3'UTR of CCDC80,and can significantly reduce its luciferase activity,but does not bind to the 3'UTR of the mutated CCDC80.The cells were transfected with mi R-141-3p mimic and mi R-200a-3p mimic,and the expression levels of mi R-141-3p and mi R-200a-3p were significantly increased,and the expression of CCDC80 m RNA and protein was significantly down-regulated,but LPL m RNA and protein were increased.Mi R-141-3p inhibitor and mi R-200a-3p inhibitor transfected cells significantly decreased the expression levels of mi R-141-3p and mi R-200a-3p, increased the expression of CCDC80,and down-regulated the expression of LPL.Bioinformatics analysis revealed that lnc RNA MALAT1 has a binding site with mi R-141-3p and mi R-200a-3p;the luciferase reporter gene confirmed that lnc RNA MALAT1 binds to mi R-141-3p and mi R-200a-3p.HA-VSMCs were transfected with lnc RNA MALAT1 overexpression plasmid or si RNA.q PCR assay confirmed that lnc RNA MAMLT overexpression and interference were successful.The results showed that lnc RNA MALAT1 overexpression reduced mi R-141-3p and mi R-200a-3p expression levels,while lnc RNA MALAT1 si RNA increased their expression levels;q PCR and western blot showed that mi R-141-3p/200a-3p mimics significantly inhibited lnc RNA MALAT1-mediated up-regulation of CCDC80 expression and down-regulation of LPL expression;mi R-141-3p/200a-3p inhibitors significantly inhibited lnc RNA MALAT1 si RNA The expression of CCDC80 was down-regulated and LPL expression was up-regulated.Summaries:?1?mi R-141-3p and mi R-200a-3p sequences are highly conserved,and both of them inhibit CCDC80 expression by binding to the 3'UTR sequence of CCDC80.?2?mi R-141-3p/200a-3p participates in the regulation of LPL expression by inhibiting CCDC80.?3?lnc RNA MALAT1 is able to bind to the mi R-141-3p and mi R-200a-3p sequences and inhibit their expression.?4?lnc RNA MALAT1 promotes the expression of CCDC80 and inhibits LPL expression by decreasing the level of mi R-141-3p/200a-3p.Conclusions?1?CCDC80 inhibits LPL m RNA and protein expression in human aortic vascular smooth muscle cells.?2?CCDC80 down-regulates TET2 expression by inhibiting the activation of ERK1/2,thereby increasing the DNA methylation level of LPL promoter and down-regulating LPL expression,FOXO3 a partly involved in the regulation of LPL by CCDC80.?3?CCDC80 inhibits LPL expression and activity,increases plasma lipid levels,and accelerates AS plaque formation in apoE^-/-mice.?4?CCDC80 expression is regulated by MALAT1-mi R-141-3p/200a-3p.