| Background:Atherosclerosis(AS)is the pathological basis of coronary heart disease.Developing strategies to effectively prevent and manage the initiation and progression of AS is a major long-term medical issue that causes concerns and needs solving worldwide.Dysfunction of vascular endothelial cells(VEC)is characteristic by metabolic disturbance of low-density lipoprotein(LDL)and is one of risk factors in AS.When enter into VEC,LDL is modified as oxidized LDL(ox-LDL).Ox-LDL induces oxidative stress of VEC and subsequent apoptosis of endothelial cells.Besides,ox-LDL is the trigger of VEC inflammation.Thus,it is meaningful to investigate the pathogenesis of ox-LDL-induced AS and corresponding targets to prevent and manage coronary disease.In recent studies,non-coding RNAs(nc RNAs)are reported to regulate pathophysiological functions of cells including cell apoptosis,metastasis,invasion,migration,proliferation and inflammation,and to be involved in initiation of multiple diseases,becoming important issues for pathogenesis of many diseases and disorders.Among them,circular RNA(circ RNA)is a circular endogenous non-coding RNA formed by back-splicing.Compared with other nc RNAs,circ RNA is more resistant to exonuclease and more stable,attracting more and more attentions.Current mounting evidence shows that circ RNA is also involved in onset and progression of many cardiovascular diseases and disorders including AS.Main functions of circ RNA consist of transcriptional regulation of host genes,sponging of micro RNA(mi RNA),regulation of cellular functions as protein scaffold when binds to functional proteins and encoding proteins.mi RNA mainly modulates functions of host genes in nucleus and mainly acts as mi RNA sponge in cytoplasm.circ RNA acts as a competing endogenous RNA and impacts on mi RNA as a competing endogenous RNA(ce RNA).According to previous findings,has?circ?0000345(circ RSF1)originates from remodeling and spacing factor1(RSF1),and its expression in ox-LDL stimulated human umbilical vein endothelial cells(HUVECs)is significantly decreased.It is also reported in other studies that circ RSF1 regulateshepatic cell inflammation,suggesting that circ RSF1 is involved in VEC injury induced by ox-LDL treatment.Nevertheless,the role and mechanism of circ RSF1 in VEC injury still remain unclear and need investigations.Sponging mi RNA to regulate expression of target genes is the main regulatory mechanism of circ RNA.circ RSF1 demonstrates regulatory function by binding to mi RNA,indicating that it is located in cytoplasm,and that may regulate associated mi RNA to affect expression of target proteins and be involved in the initiation of AS.mi RNAs are short non-coding RNAs containing approximately 21 nucleotides,and are structurally conserved and widely present in eukaryotes.They bind to 3'untranslated region(3'UTR)of target m RNA at post-transcriptional level,depress or degrade target m RNA.Therefore,screening circ RSF1-associated mi RNAs-m RNAs contributes to revealing regulatory mechanism of AS.By screening circ RSF1-associated mi RNAs,we observed that mi R-135b-5p,target gene of circ RSF1,was mi RNA widely involved in cell proliferation,apoptosis and migration.In addition,clinical data in other study also show that the expression of mi R-135b-5p in AS is significantly enhanced,and that mi R-135b-5p promotes proliferation and migration of endothelial cells,suggesting that as a vital regulator,mi R-135b-5p may be involved in the regulation of AS.Furthermore,among mi R-135b-5p-targeted m RNA,histone deacetylase 1(HDAC1)modulate the progression of AS via acetylating non-histone targets including transcription factors.For example,tumor suppressor p53 is the essential transcription factor for cell cycle and initiating cell apoptosis,and p53 is inactivated by deacetylation by HDAC1 and deprived of activation of the promoter Bax,resulting in lower percentage of cellular Bax/Bcl-2and subsequent inhibition of cellular apoptosis.Besides,nuclear factor-?B(NF-?B)is a classical nuclear factor in regulating host inflammation,immune response and cell apoptosis and its p65 subunit is easily affected by dynamic acetylation of lysine.Additionally,previous studies demonstrate that HDAC1 negatively modules NF-?B-mediated gene transcription and down-regulates associated inflammatory factors by binding to p65.At present,a large number of studies have shown that HDAC1 can regulate cell proliferation,apoptosis,and inflammation,and participate in the occurrence and development of AS.Therefore,we speculate that circ RSF1 may regulate the progression of AS via affecting mi R-135b-5p/HDAC1 axis.Objective:Ox-LDL-induced HUVECs is applied as in vitro cell model,and mi R-135b-5p/HAC1 axis is the key start point in the current thesis to investigate the role and regulatory mechanism of circ RSF1 in ox-LDL-induced HUVECs injury.Method:1.To evaluate the stability of circ RSF1 and confirm its intracellular location:The expression of circ RSF1 and RSF1 after Actinomycin D and RNase R treatment was measured by q PCR to evaluate the stability.The intracellular location of circ RSF1 was analyzed by q PCR according to circ RSF1 expression after nucleocytoplasmic separation.2.To evaluate the impact of circ RSF1 over-expression by constructing ox-LDL-induced HUVECs(OLH)injury model: ox-LDL was used to construct HUVECs injury model and circ RSF1 expression after ox-LDL treatment by q PCR.After transfection of plasmid of circ RSF1 over-expression into OLH,the impact of circ RSF1 over-expression on viability of OLH was measured by MTT.Then cell apoptosis rate and protein expression of apoptosis pathway protein(Bcl-2/Bax/cleaved caspase-3)were assessed by flow cytometry and Western blot,respectively,to evaluate the impact of circ RSF1 over-expression on apoptosis of OLH.The expression of inflammatory factors(IL-1?,IL-6,TNF-? and IL-8)in cell medium supernatant was further measured by ELISA,and the expression of cell-adhesion-associated proteins(VCAM1/ICAM1/E-selectin)was assessed by Western blot to observe the impact of circ RSF1 over-expression on OLH inflammation.3.To predict and screen target mi RNA of circ RSF1: Firstly,circbank database was used to predict target mi RNA(mi R-135b-5p)of circ RSF1.Then circ RSF1 enrichment was measured by pull-down experiment,and the activity of luciferase in HUVECs by dual-luciferase-reporter assay was assessed to observe the interaction of circ RSF1 and mi R-135b-5p.Lastly the expression of mi R-135b-5p after circ RSF1 silencing,over-expression and ox-LDL treatment was assessed by q RT-PCR to verify that circ RSF1 directly targets mi R-135b-5p in HUVECs.4.To verify the impact of circ RSF1 on OLH viability,apoptosis and inflammation via the target mi R-135b-5p: the impact of the combination of over-expression of circ RSF1 and mi R-135b-5p over-expression on cell viability was measured by MTT.Then cell apoptosis rate and protein expression of apoptosis pathway protein(Bcl-2/Bax/cleaved caspase-3)were assessed by flow cytometry and Western blot,respectively.The expression of inflammatory factors(IL-1?,IL-6,TNF-? and IL-8)in cell medium supernatant,and the expression of cell-adhesion-associated proteins(VCAM1/ICAM1/E-selectin)was assessed by ELISA and Western blot,respectively,to observe the impact of circ RSF1over-expression on OLH inflammation.5.To predict and screen target m RNA of mi R-135b-5p: Firstly,circbank database was used to predict target m RNA and binding sites of mi R-135b-5p.Then the activity of luciferase in HUVECs by dual-luciferase-reporter assay was assessed to observe the interaction of mi R-135b-5p and HDAC1.Lastly the gene and protein expression of HDAC1 after anti-mi R-135b-5p transfection,mi R-135b-5p over-expression,ox-LDL treatment and the combination of circ RSF1 and mi R-135b-5p over-expression was assessed by q RT-PCR and Western blot to verify that mi R-135b-5p directly targets HDAC1 in HUVECs.6.To verify the impact of mi R-135b-5p on OLH viability,apoptosis and inflammation via the target HDAC1: after the treatment of the combination of anti-mi R-135b-5p transfection and HDAC1 gene silencing,cell viability was measured by MTT and then cell apoptosis rate and protein expression of apoptosis pathway protein(Bcl-2/Bax/cleaved caspase-3)were assessed by flow cytometry and Western blot,respectively,to observe the impact on cell apoptosis.The expression of inflammatory factors(IL-1?,IL-6,TNF-? and IL-8)in cell medium supernatantand the expression of cell-adhesion-associated proteins(VCAM1/ICAM1/E-selectin)was assessed by ELISA and Western blot,respectively,to observe the impact on OLH.Results:1.Circ RSF1 was more stable than circ RSF1 after Actinomycin D and RNase R treatment.Nucleocytoplasmic separation showed that Circ RSF1 was mainly located in cytoplasm of HUVECs.2.After ox-LDL treatment in HUVECs for 48 h,circ RSF1 expression was decreased,with lower cell viability and higher cell apoptosis rate,and in addition down-regulation of anti-apoptosis protein Bcland up-regulation of apoptosis pathway protein(Bax/cleaved caspase 3),inflammatory factors(IL-1?,IL-6,TNF-? and IL-8),and cell-adhesion-associated protein(VCAM1/ICAM1/E-selectin)were also observed,indicating endothelial injury model was successfully constructed by ox-LDL treatment in HUVECs.Circ RSF1over-expression promotes cell viability,inhibited cell apoptosis of ox-LDL HUVECs.Besides,up-regulation of anti-apoptosis protein Bclanddown-regulation of apoptosis pathway protein(Bax/cleaved caspase 3),inflammatory factors(IL-1?,IL-6,TNF-? and IL-8),and cell-adhesion-associated protein(VCAM1/ICAM1/E-selectin)were observed,suggesting that circ RSF1 promoted cell viability,inhibited ox-LDL induced cell apoptosis and inflammation.3.Binding site of mi R-135b-5p and circ RSF1 was predicted by circbank database.The activity of luciferase in HUVECs was significantly reduced when mi R-135-5p bound to circ RSF1 WT,and the binding site was Ago2 protein.The expression of mi R-135b-5p in HUVECs was enhanced by circ RSF1 silencing,and reversely circ RSF1 higher expression inhibited mi R-135b-5p expression in HUVECs,suggesting that mi R-135b-5p is the target mi RNA of circ RSF1.4.MTT assay demonstrated that over-expression of mi R-135b-5p reversed the promotion of circ RSF1 over-expression in cell viability.Besides,lower apoptosis rate,up-regulation of anti-apoptosis protein Bcland down-regulation of apoptosis pathway protein(Bax/cleaved caspase 3)were observed after circ RSF1 over-expression,which was reversed by mi R-135b-5p over-expression.Circ RSF1 over-expression significantly suppressed extra-and intra-cellular inflammation,which was reversed by mi R-135b-5p over-expression,indicating that circ RSF1 affected ox-LDL-induced cell viability,apoptosis and inflammation via mi R-135b-5p.5.Circbank database showed that HDAC1 was potential target of mi R-135b-5p.Dual-luciferase reporter assay demonstrated that mi R-135b-5p directly targeted HDAC1 in HUVECs.The expression of mi R-135b-5p in HUVECs was enhanced by circ RSF1 silencing,and reversely highercirc RSF1 expression inhibited mi R-135b-5p expression in HUVECs,suggesting that mi R-135b-5p is the target mi RNA of circ RSF1.Anti-mi R-135b-5p transfection induced HDAC1 expression,and reversely mi R-135b-5p inhibited HDAC1 expression in HUVECs.Meanwhile,circ RSF1 regulated m RNA and protein expression of HDAC1 via inhibiting mi R-135b-5p.In addition,ox-LDL suppressed HDAC1 expression in HUVECs,indicating that HDAC1 was the target m RNA of mi R-135b-5p.6.MTT assay showed that the promotion of anti-mi R-135b-5p treatment in cell viability was reversed by HDAC1 silencing.Lower apoptosis rate,up-regulation of anti-apoptosis protein Bcland down-regulation of apoptosis pathway protein(Bax/cleaved caspase 3)were observed after anti-mi R-135b-5p treatment,which was reversed by HDAC1 silencing.In addition,anti-mi R-135b-5p treatment significantly inhibited extra-and intra-cellular inflammation,which was also reversed by HDAC1 silencing,indicating that mi R-135b-5p affected ox-LDL-induced cell viability,apoptosis and inflammation via HDAC1.Conclusion:1.The expression of circ RSF1 decreased in ox-LDL-treated HUVECs,but its overexpression can inhibit the apoptosis and inflammation as well as enhance cell viability of ox-LDL-treated HUVECs;2.The regulatory effect of circ RSF1 on the function of ox-LDL-treated HUVECs is achieved by directly targeting mi R-135b-5p,while high expression of mi R-135b-5p can partially reverse the effect of circ RSF1,suggesting that circ RSF1 acts as a molecular sponge of mi R-135b-5p to play a regulatory role;3.circ RSF1/mi R-135b-5p can regulate HDAC1,silencing HDAC1 gene can reverse the effect of anti-mi R-135b-5p on apoptosis and inflammation of ox-LDL-treated HUVECs,suggesting circ RSF1 plays a regulatory role by regulating the mi R-135b-5p/HDAC1 axis.Innovation and significanceThis study confirmed that circ RSF1 promotes cell viability of ox-LDL-induced HUVECs and inhibited cell apoptosis and inflammation,indicating the role of circ RSF1 in prevention and management of AS.At the same time,the regulation mechanism of circ RSF1 regulates cell viability,apoptosis and inflammation in ox-LDL-induced endothelial cell injury model via acting as mi R-135b-5p sponge to module HDAC1-mediated acetylation of key proteins.High expression of circ RSF1 or inhibition of mi R-135b-5p expression can reduce endothelial cell damage induced by ox-LDL,which is expected to become a new strategy for the prevention or treatment of atherosclerosis. |
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