LncRNA HDA11-AS1 Regulating Adropin Histone Deacetylation Negatively And The Effect On Atherosclerosis

Atherosclerosis is the common pathological change in cardiovascular and cerebrovascular diseases which jeopardizes human health.Since hypertriglyceridemia is the independent risk factor in the development of atherosclerosis,it is very important to decrease the blood triglyceride in order to prevent and treat atherosclerosis.Lipoprotein lipase(LPL),the limited enzyme in the hydrolysis process of TG,is secreted from parenchymal cells such as vascular smooth muscle cells.After secretion,LPL gets into blood vessel and anchors on the vascular endothelial cells,then hydrolyses the TG of the chylomicrons(CM)and very low density lipoprotein(VLDL).In 2008,Kumar and his colleagues found a secreted protein named adropin that is an energy balance gene and encoded by Encho.Adropin is a secreted protein consisting of 76 amino acids,and the molecular weight of adropin is 4.5 k Da.Adropin locates on chromosome 9p13.3.Researchers have shown that adropin has the important effects on regulating lipidmetabolism,improving insulin resistance and increasing insulin sensitivity.A study reported that adropin can up-regulate the expression of LPL in Tilapia liver cells although the mechanism is still not unclear.All these studies suggest that adropin is involved in the TG metabolism through regulating LPL which then affects the development and progression of As.Therefore,adropin would possibly be a new target to develop effective therapy of atherosclerosis and other relevant cardiovascular diseases.Epigenetics has been emerging as a new strategy to regulate gene expression without changing the base sequence in genome.Our preliminary experiment have found that the level of adropin was reduced in mouse model of atherosclerosis while the deacetylation level in promoter region of adropin was increased although the underlying mechanism was not clear.Therefore,the regulation of adropin expreion through histone deacetylation is a new light on treating atherosclerosis.Previous studies have shown that HDAC11,the Ⅳ histone deacetylase(HDACs),is the key enzyme in catalyzing the deacetylation with H3K14 is the deacetylation site.Accordingly,we hypothesized that HDAC11 may regulate the expression of adropin through catalyzing the deacetylation of histone in adropin’s promoter region.Long non-coding RNAs(lnc RNAs)are defined as non-protein coding transcripts longer than 200 nucleotides.Lnc RNAs silence oractivate gene expression through the modification of histone,DNA methylation,chromatin remodeling,which might be involved in the development of cardiovascular disease(CVD).Natural antisense transcripts(NATs)is a kind of lnc RNAs transcribed from protein or nonprotein-coding genes opposite strand,they are complementation completely or partly with protein-coding genes.NATs adjust negatively the expression of sense gene through some ways,likewise,the specific secondary structure and restraining the process of translation.Bioinformatics analysis indicates that lnc RNAHDAC11-AS1 and HDAC11 locate on the same chromosome(3q25)with the opposite transcriptional direction,lnc RNAHDAC11-AS1 is the possible NATs of HDAC11.More importantly,we found there was a reduced expression of HDAC11 after transfection of the lentiviral vector over-expressing lnc RNAHDAC11-AS1 into HA-VSMCs suggesting lnc RNAHDAC11-AS1 negatively regulates HDAC11.Meanwhile,reduced expression of adropin(m RNA and protein)were observed after overexpression of HDAC11 while increased adropin expression were found after silence of HDAC11.According to the previous literatures,our bioinformatics analysis and preliminary data,we proposed our research hypotheses: HDAC11 promotes histone deacetylation of adropin;Lnc RNAHDAC11-AS1 prevents atherosclerosis by specifically silencing HDAC11 which thenresults in a reduced histone deacetylation of adropin,increased adropin expression followed by increased LPL expression and reduced level of TG.To demonstrate our hypotheses,we designed four experiments.Firstly,we detected histone deacetylation and expression of adropin after overexpression or silence of HDAC11 in HA-VSMCs.Secondly,we observed the expression of HDAC11 and adropin as well as the level of adropin histone deacetylation after overexpression or silence of lnc RNAHDAC11-AS1.Thirdly,we explored the LPL level after overexpression or silence of adropin and underlying mechanism.Lastly,apo E KO mice fed with high fat diet were injected with HDAC11 or lnc RNAHDAC11-AS1 virus vectors,several endpoints were measured including the adropin histone deacetylation in aorta,the expression levels of adropin and LPL,the level of serum lipid and atherosclerotic plaque.Our study revealed the deacetylation of adropin histone affects the expression level of adropin and lipid metabolism,we also identified the underlying mechanism by which Lnc RNAHDAC11-AS1 restrains atherosclerosis is through specifically silencing HDAC11 which then results in a reduced histone deacetylation of adropin,increased adropin expression followed by increased LPL expression and reduced level of TG.Our results will provide a new drug target to develop a novel therapy for atherosclerosis.Part I: Adropin histone deacetylation regulates the expression of adropinObjective: To observe the level of adropin histone deacetylation and the expression level of adropin after overexpression or silence of HDAC11 in HA-VSMCs;To explore the impact of HDAC11 on adropin histone deacetylation.Methods:1.HDAC11 and sh HDAC11 were transfected into HA-VSMCs,then the gene expression and protein expression of HDAC11 were detected with quantitative real-time PCR and Western blot,respectively;2.Quantitative real-time PCR and Western blot were conducted to detect the gene expression and protein expression of adropin after HDAC11 and sh HDAC11 transfection into HA-VSMCs;3.The deacetylation level of adropin histone H3K14 was detected using Western blot after HDAC11 and sh HDAC11 transfection into HA-VSMCs.Results:1.Transfection of HDAC11 could increase both gene expression and protein expression of HDAC11 whereas transfection of sh HDAC11 reduced HDAC11 expression in both gene level and protein level;2.Transfection of HDAC11 decreased the expression of adropin while transfection of sh HDAC11 upregulated adropin expression;3.As shown in the immunoblot,the deacetylation level of adropin histone H3K14 was reduced after HDAC11 transfection but dramatically increased after sh HDAC11 transfection.Summary: 1.HDAC11 regulates histone deacetylation of adropin in HA-VSMCs;2.HDAC11 negatively regulates the expression of adropin in HA-VSMCs.Part II: The impact of Lnc RNA HDAC11-AS1 on histonedeacetylation of adropin and the regulation mechanismObjective: To explore the impact of lnc RNA HDAC11-AS1 on HDAC11 expression,histone deacetylation of adropin,and adropin expression through overexpression or silence of lnc RNA HDAC11-AS1 in HA-VSMCs.Methods:1.Online bioinformatics analysis was conducted to identify the relationship between lnc RNA HDAC11-AS1 and HDAC11;2.Quantitative real-time PCR and Western blot were performed to check the expression levels of HDAC11 after overexpression or silence of lnc RNA HDAC11-AS1 in HA-VSMCs;3.The level of adropin histone H3K14 was examined by Western blot after overexpression or silence of lnc RNA HDAC11-AS1 in HA-VSMCs;4.m RNA and protein levels of adropin were determined by q RT-PCR and western blot respectively after overexpression or silence of lnc RNA HDAC11-AS1 in HA-VSMCs.Results:1.Bioinformatics analysis identified that lnc RNA HDAC11-AS1 and HDAC11 were located on chromosome 3(3q25),andtheir transcriptional directions are opposite,so lnc RNA HDAC11-AS1 is probably the natural antisense transcripts(NATs)of HDAC11;2.The m RNA and protein levels of HDAC11 were increased after silence of lnc RNA HDAC11-AS1,in contrast,the m RNA and protein levels of HDAC11 were reduced after overexpression of lnc RNA HDAC11-AS1;3.Immunoblot results showed that the deacetylation level of adropin histone H3K14 was increased after silence of lnc RNA HDAC11-AS1 in HA-VSMCs while overexpression of lnc RNA HDAC11-AS1 decreased the deacetylation level of adropin histone H3K14;4.As shown in the data of immunoblot and q RT-PCR,the m RNA and protein levels of adropin were decreased after silence of lnc RNA HDAC11-AS1 in HA-VSMCs,however,overexpression of lnc RNA HDAC11-AS1 increased the m RNA and protein levels of adropin;5.The deacetylation level of adropin histone H3K14 was increased by overexpression of HDAC11,this effect was reversed by co-transfection of lnc RNA HDAC11-AS1 and HDAC11 together.Summary: 1.Lnc RNA HDAC11-AS1 is the natural antisense transcripts(NATs)of HDAC11,and it suppresses the expression of HDAC11;2.Lnc RNA HDAC11-AS1 increases the expression of adropin by HDAC11 reducing the deacetylation level of adropin histone H3K14.Part III: Adropin regulates LPL and the mechanismObjective: To reveal the effect and the mechanism of adropinregulating LPL by examining the expression level of LPL after overexpressed or silenced adropin,and to explore the signal pathway.Methods:1.Using quantitative real-time PCR and Western blot to test the expression level of LPL by overexpressed or silenced adropin;2.Quantitative real-time PCR and Western blot method were used to analyze the m RNA and protein expression of LPL using the specific AMPK inhibitor,WZ4003,which treated HA-VSMCs with adropin together;3.The level of AMPK expression and phosphorylation were tested by Western blot analysis.Results:1.Quantitative real-time PCR and Western blot results showed the expression of LPL was increased by overexpressed adropin,otherwise,silenced adropin could reduce the expression of LPL;2.After overexpressing adropin,the expression of LPL was raised,however,the promotion would be reversed by WZ4003 which was the specific AMPK inhibitor,all these data were came from Real-time quantitative PCR and Western blot;3.The results of Western blot showed that the level of AMPK phosphorylation was raised by overexpressed adropin,while phosphorylation of AMPK was decreased in response to silenced adropin,all these pointed out adropin impacted LPL through activated AMPK.Summary:1.Adropin could up-regulate the expression of LPL;2.Adropin impacted LPL through AMPK pathway.Part Ⅳ: The impact of Lnc RNA HDAC11-AS1 on AorticAtherosclerosis in Apo E Knockout MiceObjective: To investigate the aortic sinus pathology,lipid accumulation in lesion,the expression of adropin and LPL in aorta,the change in blood lipid,and then to investigate the effects of lnc RNA HDAC11-AS1 on the development of atherosclerosis in apo E KO mice.Methods:1.Eight-weeks-old male apo E KO mice were fed with high-fat diet,and then were given a tail vein injection with lentivirus vector of lnc RNA HDAC11-AS1 or HDAC11,respectively;2.10 weeks later,the apo E KO mice were sacrificed using overdose anesthetics,blood were collected,and then heart perfusion with 4% paraformaldehyde were performed and the formation of lesion in aortic arch and three branch vessels were observed;3.The whole aorta was split from the aortic root to the common iliac artery,then the aorta was opened longitudinally to observe the lipid accumulation in arterial wall after staining with oil red O;4.The aortic sinus of apo E KO mice were sectioned by rapid frozen section technology to investigate the lesion area of aortic sinus after hematoxylin-eosin staining,the Image Pro Plus technology was used to analyze the lesion area;5.The aortic sinus of apo E KO mice were investigated to examine the lipid accumulation in aortic sinus after oil red O staining,the Image Pro Plus technology was used to detect the area ratio between lesion and lumen;6.Collagen contents in lesion wereexamined with masson staining of the section of aortic sinus from apo E KO mice;7.The serum levels of adropin and LPL in apo E KO mice were measured after lnc RNA HDAC11-AS1 transfection with ELISA;8.Serum triglyceride(TG),total cholesterol(TC)and free acid(FFA)were measured with the automatic biochemistry analyzer.Results:1.The stereomicroscope showed that the apo E KO mice injected with HDAC11 had the most widespread atherosclerosis lesion in aortic arch and branch vessels while the mice injected with lnc RNA HDAC11-AS1 had the least atherosclerosis lesion area;2.Oil red O staining showed that the group injected with HDAC11 had the highest ratio of positive staining area over intravascular surface area while the group injected with lnc RNA HDAC11-AS1 had the lowest positive staining area on the aortic tunica intima as well as the reduced percentage of intravascular surface area;compared to the group injected with HDAC11 alone,injection of both lnc RNA HDAC11-AS1 and HDAC11 decreased the positive staining area and the percentage of intravascular surface area suggesting that HDAC11 could promote atherosclerotic lesion formation by increasing the lipid accumulation in vascular wall of apo E KO mice whereas lnc RNA HDAC11-AS1 reverses the effect and reduces the atherosclerotic lesion formation;3.The hematoxylin-eosin staining found that the group injected with HDAC11 had the highest ratio of atherosclerotic lesions area to lumen of aortic sinus while the groupwith lnc RNA HDAC11-AS1 injection had the lowest ratio,injection of both lnc RNA HDAC11-AS1 and HDAC11 reduced the ratio compared to injection of HDAC11 alone;4.The results from oil red O staining showed that the group injected HDAC11 had the most percentage of aortic sinus red-stained area to lumen,however,the percentage of aortic sinus red-stained area to lumen was least in the group injected lnc RNA HDAC11-AS1 compared with HDAC11;The group injected lnc RNA HDAC11-AS1 and HDAC11 also had the less percentage of aortic sinus red-stained area to lumen.All these data reminded us that HDAC11 could increase the lipid accumulation in aortic sinus atherosclerosis lesion,but lnc RNA HDAC11-AS1 could change this result by reducing the lipid accumulation;5.The Masson staining showed that the group injected with HDAC11 had the highest amount of collagen fiber in the atherosclerosis lesion of aortic sinus while the group with lnc RNA HDAC11-AS1 injection had the least collagen accumulation in the atherosclerosis lesion,injection of both lnc RNA HDAC11-AS1 and HDAC11 reduced the collagen accumulation compared to injection of HDAC11 alone;6.Compared to the group with HDAC11,the levels of adropin and LPL increased dramatically in the group injected with lnc RNA HDAC11-AS1 or sh HDAC11;The levels of adropin and LPL increased slightly in the group injected with both lnc RNA HDAC11-AS1 and HDAC11 suggesting lnc RNA HDAC11-AS1 promoting theexpression of adropin and LPL in apo E KO mice;7.The automatic biochemistry analyzer found that the serum levels of TG,TC in the group injected with HDAC11 were the highest,the other groups had reduced levels of TG and TC as well as increased level of FFA.Summary:1.Lnc RNA HDAC11-AS1 inhibits the atherosclerosis in apo E KO mice;2.Lnc RNA HDAC11-AS1 restrains the lipid accumulation in the atherosclerotic plaque and decreases the level of plasma lipids in apo E KO mice.Conclusion:1.HDAC11 regulates histone deacetylation of adropin in HA-VSM Cs then inhibits the expression of adropin.2.Lnc RNA HDAC11-AS1 increases the expression of adropin by HDAC11 reducing the deacetylation level of adropin histone.3.Adropin impacted LPL through AMPK pathway.4.Lnc RNA HDAC11-AS1 restrains the lipid accumulation in the at herosclerotic plaque and decreases the level of plasma lipids in apo E KO mice.