| BackgroundCoronary heart disease(CHD)is one of the most common heart diseases in clinical medicine.Its current morbidity and mortality is on the top of diseases in China.Atherosclerosis(AS)is the common pathological basis of cardiovascular diseases such as CHD which pathogenesis is very complex.The disorder of lipid metabolism is an independent risk factor for CHD.High density lipoprotein(HDL)is a kind of heterogeneous protein with roughly equal content of lipid and protein,and it has important cardiovascular protective function.In recent years,the study of blood lipid metabolism has found that simply raising the level of high density lipoprotein cholesterol(HDL-C)can not reduce the incidence of CHD,either can reduce the incidence of cardiovascular events.This suggests that the change of HDL function is more important than the amount of HDL-C.Reverse cholesterol transport(RCT)means transporting cholesterol from peripheral tissues(including atherosclerotic plaque)into the liver for recycling or being excreted in bile,which includes the cholesterol removal in liver and the cholesterol efflux in peripheral tissue.Cholesterol efflux transport proteins in peripheral tissue are expressed in macrophages and cholesterol efflux from macrophages is the first step in RCT.The RCT process can reduce cholesterol deposition in the blood vessel wall,thereby preventing the occurrence of atherosclerosis.Serum amyloid P component(SAP)was first found in amyloidosis,but also stable in serum.SAP can be deposited in atherosclerotic plaque lesions,and has a high affinity with HDL and very low density lipoprotein(VLDL).SAP is not only synthesized by the liver cells in normal condition,but also by macrophages in the plaque.Its ability is increasing cholesterol efflux,thereby could removing cholesterol in the cells.But the concrete mechanism still needs further investigation.Proline/serine-rich coiled-coil protein 1(Psrc1),also known as DDA3,is a protein coding gene.Psrcl protein is a necessary substance in the process of mitosis in normal eukaryotic cells.Psrc1 can participate in the p53/TP53 regulation of growth process.Studies have found that the level of Psrcl gene expression is closely related to the total serum cholesterol concentration.The high expression of Psrcl gene can cause the level of HDL-C increased,the level of LDL-C decreased,and reduce the risk of coronary heart disease.These data suggest that Psrcl may play an important role in the change of HDL function and atherosclerosis(AS).But its mechanism has not been clarified.ObjectivesMouse RAW264.7 macrophages were used as the research objects to identigy the effect of SAP on lipid metabolism.We sequentially elucidate the related molecular mechanisms by preforming modern molecular biology,such as radioactive isotope labeling technique,Western blot,QRT-PCR,ELISA.These results may provide theoretical basis for understanding effect of SAP on cholesterol transport process and further clarifying process of AS.MethodsPart I Effect of SAP on Ox-LDL induced cholesterol efflux and the related molecular mechanisms in macrophages.1.Effect of SAP on Ox-LDL induced cholesterol efflux rate in macrophageConcentration effect:Cells were divided into 5 groups:1)control group,2)SAP(1 ?M)group,3)SAP(2.5 ?M)group,4)SAP(5 ?M)group,5)SAP(10 ?M)group,and cells were treated for 24 h;Time effect:Cells were divided into various indicated period groups(6,24,48,72 h).Cells were treated by SAP in two concentrations(1 ?M and 10 ?M).The radioactive isotope labeling method was performed to detect the cholesterol efflux rate of RAW264.7 macrophages.2.Effect of SAP on cholesterol transport related proteins in RAW264.7 macrophagesCells were divided into 5 groups:1)control group,2)SAP(1 ?M)group,3)SAP(2.5 ?M)group,4)SAP(5 ?M)group,5)SAP(10 ?M)group,and cells were treated for 24 h;The expression of lipid transfer proteins were detected by Western blot.3.Effect of SAP on cholesterol transport related proteins mRNA in RAW264.7 macrophagesCells were divided into 2 groups:1)control group,2)SAP(5 ?M)group,and cells were treated for 24 h;The totle RNA was isolated from cells in both groups and reverse transcription for cDNA.The expression of lipid transfer proteins mRNA were detected by QRT-PCR.4.Effect of SAP on mRNA of ICAM-land FcyR II b in RAW264.7 macrophagesCells were divided into 2 groups:1)control group,2)SAP(5 ?M)group,and cells were treated for 24 h;The totle RNA was isolated from cells in both groups and reverse transcription for cDNA.The expression on mRNA of ICAM-1and Fc?R? were detected by QRT-PCR.5.Effect of SAP on IL-6 and IL-10 cytokines in cell supernatantConcentration effect:Cells were divided into 5 groups:1)control group,2)SAP(1 ?M)group,3)SAP(2.5 ?M)group,4)SAP(5 ?M)group,5)SAP(10 ?M)group,and cells were treated for 24 h;Time effect:Cells were divided into various indicated period groups(6,24,48,72 h).Cells were treated by SAP in four concentrations(1,2.5,5 and 10 ?M).ELISA was performed to determine the IL-6 and IL-10 concertrations in cell supernatants.Part ? Effect of SAP on gene expression profile in RAW264.7 macrophages.1.Effect of SAP on RAW264.7 cell gene expressionCells were divided into 2 groups:1)control group,2)SAP(5 ?M)group,and cells were treated for 24 h;RNA-sequencing was the method to detect the differentially expressed genes in each group.2.Effect of SAP on RAW264.7 cell functional clustering of differentially expressed genesCells were divided into 2 groups:1)control group,2)SAP(5 ?M)group,and cells were treated for 24 h;GO enrichment analysis was performed by using BiNGO 2.3 with the GOslim dataset.3.Effect of SAP on RAW264.7 cell identification of atherosclerosis-related genesCells were divided into 2 groups:1)control group,2)SAP(5 ?M)group,and cells were treated for 24 h;PubMed database was conducted a systematic and comprehensive literature search of all differentially expressed genes.4.Effect of SAP on RAW264.7 cell altered expression of non-coding RNA genesCells were divided into 2 groups:1)control group,2)SAP(5 ?M)group,and cells were treated for 24 h;PubMed database was conducted a systematic and comprehensive literature search of non-coding RNA genes.5.Network analysis of SAP-regulated genesCells were divided into 2 groups:1)control group,2)SAP(5 ?M)group,and cells were treated for 24 h;The web-based network tool STRING was used to investigate functional interaction among SAP-regulated genes.Part ? Effect of SAP on Ox-LDL induced cholesterol efflux and the related molecular mechanisms in Psrcl-siRNA macrophages.1.Transfection efficiency and silencing effect of Psrcl-siRNA on RAW264.7 macrophagesPsrcl-siRNA sequence verification:Cells were divided into 5 groups:1)negative control group(NC group),2)Psrcl-siRNA-1 group,3)Psrc1-siRNA-2 group,4)Psrcl-siRNA-3 group,5)Psrcl-siRNA-4 group;Psrcl-siRNA transfection time verification:Cells were divided into 4 groups:1)24 h Psrcl-siRNA sequence verification groups,2)48 h Psrcl-siRNA sequence verification groups,3)72 h Psrcl-siRNA sequence verification groups,4)96 h Psrc1-siRNA sequence verification groups;Psrcl-siRNA concentration verification:Cells were divided into 3 groups:Psrc1-siRNA-1(100 nM)group,2)Psrcl-siRNA-1(50 nM)group,3)Psrcl-siRNA-1(20 nM)group;The totle RNA was isolated from cells in both groups and reverse transcription for cDNA.The expression on mRNA of Psrcl was detected by QRT-PCR.2.Effect of SAP on Ox-LDL induced cholesterol efflux rate in Psrcl-siRNA macrophageConcentration effect:Cells were divided into 6 groups:1)negative control group(NC group),2)NC+ SAP(5 ?M)group,3)Psrcl-siRNA group,4)Psrcl-siRNA+SAP(5 ?M)group,5)normal control group 6)normal + SAP(5 ?M)group;The radioactive isotope labeling method was performed to detect the cholesterol efflux rate of Psrcl-siRNA RAW264.7 macrophages.3.Effect of SAP on cholesterol transport related proteins mRNA in Psrcl-siRNA RAW264.7 macrophagesCells were divided into 4 groups:1)negative control group(NC group),2)NC+ SAP(5 ?M)group,3)Psrcl-siRNA group,4)Psrcl-siRNA+SAP(5 ?M)group,and cells were treated for 24 h for both siRNA and SAP;The totle RNA was isolated from cells in all groups and reverse transcription for cDNA.The expression of lipid transfer protein mRNA was detected by QRT-PCR.4.Effect of SAP on cholesterol transport related proteins in RAW264.7 macrophagesCells were divided into 4 groups:1)negative control group(NC group),2)NC+ SAP(5 ?M)group,3)Psrcl-siRNA group,4)Psrcl-siRNA+SAP(5 ?M)group,and cells were treated for 24 h for both siRNA and SAP;The expression of lipid transfer protein was detected by Western blot.5.Effect of SAP on mRNA of ICAM-land Fc?R ? b in RAW264.7 macrophages1)negative control group(NC group),2)NC+ SAP(5 ?M)group,3)Psrc1-siRNA group,4)Psrcl-siRNA+SAP(5 ?M)group,and cells were treated for 24 h for both siRNA and SAP;The totle RNA was isolated from cells in both groups and reverse transcription for cDNA.The expression on mRNA of ICAM-1and Fc?R? was detected by QRT-PCR.ResultsPart ? Effect of SAP on Ox-LDL induced cholesterol efflux and the related molecular mechanisms in macrophages.1.SAP enhanced Ox-LDL induced cholesterol efflux rate in macrophageRAW264.7 cells were treated by different concentrations(0-10 ?M)SAP.Compared with control group,cholesterol dfflux rate was significantly increased in SAP(1,2.5,5,10 ?M)group.(P<0.05)The most statistical group is SAP(5 ?M)group.(P<0.05)The effect was in a dose-dependent manner in the experimental concentration range.RAW264.7 cells were treated by SAP(1 ?M)and SAP(10 ?M)for indicated times(6,24,48,72 h).Compared with control group,cholesterol dfflux rate was significantly increased in 24 h groups obviously.The effect was in a time-dependent manner in the experimental concentration range.2.Effect of SAP on cholesterol transport related proteins in RAW264.7 macrophagesRAW264.7 cells were treated by different concentrations(0-10 ?M)SAP.Compared with control group,SAP(1,2.5,5,10 ?M)increased the expression level of ABCA1,ABCG1,SR-BI,PPARy and LXRa proteins,and effect was in a dose-dependent manner.These results revealed that SAP up-regulated expression of cholesterol transport proteins to enhance reverse cholesterol transport function.3.Effect of SAP on cholesterol transport related proteins mRNA in RAW264.7 macrophagesRAW264.7 cells were treated by SAP(5 ?M).Compared with control group,SAP increased the expression mRNA level of ABCA1,ABCG1,SR-BI,PPAR? and LXRa proteins.These results further revealed that SAP up-regulated expression of cholesterol transport proteins to enhance reverse cholesterol transport function.4.Effect of SAP on mRNA of ICAM-land Fc?R ? b in RAW264.7 macrophagesRAW264.7 cells were treated by SAP(5 ?M).Compared with control group,SAP increased the expression mRNA level of ICAM-1and Fc?RII b proteins.The results indicate that SAP up-regulated expression of ICAM-1and Fc?R?b may enhance inflammatory response.5.Effect of SAP on IL-6 and IL-10 cytokines in cell supernatantRAW264.7 cells were treated by different concentrations(0-10 ?M)SAP.Compared with control group,IL-6 and IL-10 were increased in SAP(15 2.5,5,10?M)group.(P<0.05)The effect was in a dose-dependent manner in the experimental concentration range.RAW264.7 cells were treated by different concentrations(0-10 ?M)SAP for indicated times(24,48,72 h).Compared with control group,IL-6 and IL-10 were significantly increased in 24 h groups obviously.The effect was in a time-dependent manner in the experimental concentration range.These results further indicate that SAP up-regulated secretion of IL-6 and IL-10 may enhance inflammatory response.Part II Effect of SAP on gene expression profile in RAW264.7 macrophages.1.Effect of SAP on RAW264.7 cell gene expressionTo capture the global gene changes associated with SAP,RAW264.7 cells were collected on 24h of SAP(5 ?M)treatment.Through RNA sequencing,we obtained 12,050,636 reads from SAP treated cells and 11,651,186 reads from control cells,respectively.Compared to control,134 genes were down-regulated and 41 genes were up-regulated after SAP treatment.The range of fold changes was 41.01-2.00 and 54.14-2.01 for down-regulated and up-regulated genes,respectively.2.Effect of SAP on RAW264.7 cell functional clustering of differentially expressed genesEnrichment analysis revealed that a total of 9 GO terms exhibited significance as overrepresented terms(P<0.05).In the biological process category,5 GO terms,namely biosynthetic process,macromolecule metabolic process,nucleic acid metabolic process,response to stimulus and cell death,were found to be significantly enriched.GO terms related to cell chromosome and nucleus were significantly enriched under the cellular component category.Enriched GO terms in the molecular function category were binding and nucleic acid binding.A total of 25 differentially expressed genes fell into the response to stimulus category.Included were 18 down-regulated genes(Atr,Fancm,Tnfsfl5,Thbd,Tlr13,F3,Acot11,Tlr3,Ahrr,Mtl,Ednl,Rifl,Mt2,Rrm2b,1110,Lig4,Mlh3 and Kin)and 7 up-regulated genes(Tnfsfl0,H2-Abl,Bcl,Ltb,Hspala,Gadd45g and Hspalb).3.Effect of SAP on RAW264.7 cell identification of atherosclerosis-related genesWe conducted a systematic and comprehensive literature search of all 175 differentially expressed genes in the PubMed database.By the use of Boolean operators,individual gene search was combined with key-words:atherogenesis OR atherosis OR sclerosis OR "coronary heart disease" OR "coronary artery disease".The search tag[Title/Abstract]was added after each key word.In this way,we identified 10 protein coding genes associated with AS.According to RNA sequencing data,7 genes were down-regulated and 3 were up-regulated upon SAP treatment.To validate RNA sequencing data,we used qRT,PCR to assess mRNA levels of 14 genes,including 10 AS related protein genes.RNA samples isolated from an independent set of biological replicates were used.In general,the expression trend of these genes measured by qRT-PCR was consistent with RNA sequencing data.4.Effect of SAP on RAW264.7 cell altered expression of non-coding RNA genesWe examined all the 175 differentially expressed genes and discovered 19 non-coding RNA genes,including 11 long non-coding RNAs,4 pseudogenes,3 snRNA precursor/hosting RNAs and 1 miRNA precursor/hosting RNA.The expression trend of these genes measured by qRT-PCR was consistent with RNA sequencing data.In the SAP group,the Mirl7hg is significantly down-regulated which is resided on the plus strand of chromosome 14.5.Network analysis of SAP-regulated genesWe investigated functional interaction among SAP-regulated genes.The highly connected nodes,also known as hub genes,represent functionally important genes in the network.Connectivity analysis showed that Atr,Hspala,Hspalb,Mblac2 and Vrkl were hubs of the network.Additionally,5 genes(Ednl Egrl,Ppplr3b,1110,Nlrp3 and Lepr),which have known roles in atherogenesis,were also present in the network.Part III Effect of SAP on Ox-LDL induced cholesterol efflux and the related molecular mechanisms in Psrcl-siRNA macrophages.1.Transfection efficiency and silencing effect of Psrcl-siRNA on RAW264.7 macrophagesAccording to results of part ?,we selected an AS related gene,Psrc1.The transfection method was used in the experiment.SiRNA is transferred into cells by liposome,and it is a kind of transient transfection.Therefore,in process of optimization of transfection conditions,fluorescence microscopy was used to assess transfection efficiency roughly.After transfection of siRNA 1 h,under the fluorescence inverted microscope,we can see that there are about 30%of the total cells in the field of view with green fluorescence.After 3 h and 6 h transfected with no obvious quenching.Quantitative RT-PCR method was used to test transfection effect.Compared with the negative control group,RNA levels of the 4 different segments of the Psrcl were transfected with a low expression state in 24 h,48 h,72 h and 96 h.The effect of Psrcl-siRNA-1(sequence:ggaagaagacauaacaguatt;uacuguuaugucuucuucctt)transfection effect was the best.Macrophages were transfected Psrcl-siRNA-1 with different concentrations of 100 nM,50 nM,and 20 nM to optimize the transfection conditions in 24 h.Psrc1 decreased in all groups and the best concerntration is 50 nM.Determining the subsequent interference experiment condition is Psrcl-siRNA-1 50 nM in 24 h.2.Effect of SAP on Ox-LDL induced cholesterol efflux rate in Psrcl-siRNA macrophageAccording to the experimental results of the optimized transfection conditions,the expression of Psrcl in macrophages was disturbed by optimized transfection conditions.Cells were divided into 6 groups:1)negative control group(NC group),2)NC+SAP(5 ?M)group,3)Psrc1-siRNA group,4)Psrcl-siRNA-+SAP(5 ?M)group,5)normal control group 6)normal + SAP(5 ?M)group.The results showed that there was no significant difference in cholesterol efflux rate between NC+ SAP(5 ?M)group and normal + SAP(5 p,M)group.And cholesterol efflux rate decreased significantly in Psrcl-siRNA group.The cholesterol efflux rate was increased after SAP treatment.3.Effect of SAP on cholesterol transport related proteins mRNA in Psrcl-siRNA RAW264.7 macrophagesAccording to the experimental results of the optimized transfection conditions,cells were divided into 4 groups:1)negative control group(NC group),2)NC+ SAP(5 ?M)group,3)Psrcl-siRNA group,4)Psrcl-siRNA+SAP(5 ?M)group,and cells were treated for 24 h for both siRNA and SAP.Detection of reverse cholesterol transport related protein mRNA levels of ABCA1,ABCG1,SR-B I?PPARy and LXRa.Before the official start of the experiment,Psrc1 gene expression was detected by QRT-PCR in the extracted samples to verify the effect of transfection.The totle RNA was isolated from cells in all groups and reverse transcription for cDNA.The expression of lipid transfer protein mRNA was detected by QRT-PCR.NC+ SAP(5 ?M)group compared with Psrcl-siRNA+SAP(5 ?M)group,the latter group mRNA levels of Psrcl,ABCA1,ABCG1,SR-B I,PPARy and LXRa were down-regulated.In Psrcl-siRNA group,above mentioned 6 genes mRNA level was quite decreased.After SAP treatment,those genes mRNA expression was elevated.4.Effect of SAP on cholesterol transport related proteins in RAW264.7 macrophagesAccording to the experimental results of the optimized transfection conditions,cells were divided into 4 groups:1)negative control group(NC group),2)NC+ SAP(5 ?M)group,3)Psrcl-siRNA group,4)Psrcl-siRNA+SAP(5 ?M)group,and cells were treated for 24 h for both siRNA and SAP.Detection of reverse cholesterol transport related protein expression of ABCA1,ABCG1,SR-B I,PPARy and LXRa.The results showed that protein expression levels of ABCA1,ABCG1,SR-B I,PPAR? and LXRa were down-regulated in Psrcl-siRNA and Psrc 1-siRNA+SAP(5?M)group.And cholesterol transport related proteins decreased significantly in Psrc1-siRNA group.The cholesterol transport related proteins expression was increased after SAP treatment.5.Effect of SAP on mRNA of ICAM-land Fc??R b in RAW264.7 macrophagesAccording to the experimental results of the optimized transfection conditions,cells were divided into 4 groups:1)negative control group(NC group),2)NC+ SAP(5 ?M)group,3)Psrcl-siRNA group,4)Psrcl-siRN A+S AP(5 ?M)group,and cells were treated for 24 h for both siRNA and SAP.Detection mRNA levels of ICAM-1and Fc?R ? b.The totle RNA was isolated from cells in all groups and reverse transcription for cDNA.The expression of mRNA of ICAM-1and Fc?R ? b was detected by QRT-PCR.NC+ SAP(5 ?M)group compared with Psrcl-siRNA+SAP(5 ?M)group,the latter group mRNA levels of ICAM-land Fc?R ? b were down-regulated.In Psrcl-siRNA group,above mentioned genes mRNA level was quite decreased.After SAP treatment,both gene mRNA expression was elevated.Conclusions1.SAP in the concentration range(1?10 ?M)can increase the cholesterol efflux rate.The expression of cholesterol transport related protein and mRNA levels of ABCA1?ABCGl?SRBI?PPARy?LXRa were increased.2.SAP in the concentration range(1?10 ?M)can increase expression of mRNA level of ICAM-land Fc?R? b.SAP also promote the secretion of IL-6 and IL-10 on macrophage.3.After SAP treated RAW264.7 cells,there are 175 differentially expressed genes,including 134 genes were down-regulated and 41 genes were up-regulated and 19 1ncRNA genes as well.4.In functional clustering of 175 differentially expressed genes,there are 9 GO terms were found to be significantly enriched.They are namely biosynthetic process,macromolecule metabolic process,nucleic acid metabolic process,response to stimulus and cell death,cell chromosome,nucleus,binding and nucleic acid binding.5.In the PubMed database,10 protein coding genes were identified associated with AS.According to RNA sequencing data,7 genes were down-regulated(Ednl?Egrl?Ppplr3b?Unc5b?Eef2k?Nlrp3?IL-10)and 3 were up-regulated(Lepr?Cx3 cr1?Psrc 1)upon SAP treatment.6.Silencing the Psrcl expression in RAW264.7 cells,cholesterol efflux rate decreased and the mRNA expression and protein level of cholesterol transport related proteins decreased as well.7.SAP treated Psrcl-siRNA RAW264.7 cells,cholesterol efflux rate increased and the mRNA expression and protein level of cholesterol transport related proteins increased as well.8.Silencing the Psrcl expression in RAW264.7 cells,mRNA expression level of ICAM-land Fc?R?b decreased.SAP treated Psrcl-siRNA RAW264.7 cells,mRNA expression level of ICAM-1and Fc?R?b increased.9.We draw a conclusion through this research and published literatures:SAP regulates the reverse cholesterol transport function of RAW264.7 macrophages by partial Psrcl expression. |
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