| Purpose:The aim of this study was to investigate the relationship between APOE promoter-219G/T,-427T/C,-491A/T polymorphism,plasma lipoprotein and apolipoprotein E levels and the risk of coronary heart disease(CHD)in population of Tianjin,China.The effect of APOE gene transcription at the promoter site of-427T/C was investigated by fluorescence reporter gene method.After statins,the regulatory effect of APOE-219G/T,-427T/C,-491A/T on plasma lipoprotein in CHD patients with different gene polymorphisms provides theoretical basis for the genetic basis of CHD in China,and further provides theoretical basis for more comprehensive and accurate personalized treatment of CHD.Method:1.The relationship between APOE promoter-219G/T,-427T/C,-491A/T gene mutation and plasma lipoprotein level and coronary heart disease: A case-control study was conducted in Tianjin Han population from October 2015 to September 2016 in the Department of Cardiology,Tianjin Thoracic Hospital.The patients were divided into two groups,coronary heart disease group and normal control group.Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)was used to analyze APOE-219G/T,-427T/C,-491A/T genotypes.The serum APOE levels were determined by ELISA.The relationship between APOE-219G/T,-427T/C,-491A/T genetic polymorphism,apolipoprotein E levels in serum,plasma lipoprotein metabolism and risk of coronary heart disease was analyzed.2.The effect of APOE promoter-427T/C gene mutation on transcriptional activity of downstream gene expression in cell DNA except promoter site: According to the results,carriers of C allele at-427T/C locus were risk factors for coronary heart disease,and the experimental results showed that apolipoprotein E content in carriers of C allele tended to decrease compared with that in carriers of T allele,considering that this was the influence of coronary heart disease and T allele.Because of the possible pathway of lipid,we only selected homozygote genes-427 TT and-427 CC to amplify the promoter region-342 bp ?-580 bp gene fragment by PCR.After purification and sequencing,the recombinant plasmids pGL2-APOE/-427 TT and pGL2-APOE/-427 CC were constructed by restriction enzyme digestion and ligase.After sequencing,the recombinant plasmids were transfected into 293 T cells by cationic liposome mediated transfection.Cells were collected,luciferase reporter gene intensity was determined,and carrier was calculated.The relative activity of luciferase of recombinant plasmids with different genotypes was analyzed by statistical analysis.Finally,the different effects of APOE genotypes on transcriptional activity were obtained.3.All patients with coronary heart disease were followed up after detection of gene polymorphisms at APOE-219G/T,-427T/C,-491A/T promoter sites.All patients were given atorvastatin calcium 20 mg per night continuously.After 12 weeks,the blood lipids were examined in the outpatient clinic,and the relationship between the different APOE promoter genotypes-219G/T,-427T/C,-491A/T and the effect of atorvastatin calcium on regulating blood lipids was observed.Result:1.Compared with the general data of the coronary heart disease group and the control group,the age of the two groups had significant difference,the age of the coronary heart disease group was significantly higher than that of the control group;the male ratio of the two groups had significant difference,with statistical significance.In CHD group,the levels of serum TG and LP(a)were significantly higher than those in control group,and the levels of serum HDL and APOAI were significantly lower than those in control group.2.Comparing the differences of blood lipid and apolipoprotein between different genotypes of all subjects(-219GG/-219GT+TT,-427TT/-427TC+CC,-491AA/-491AT+TT),we can see that in the control group.Compared with GG genotype,the levels of TC,LDL-C,APOA and HDL-C in 219 locus GT+TT genotype were significantly higher than those in GG genotype.It can be seen that the concentration of APOB in-427 locus TC+CC genotype was significantly higher than that in TT genotype in CHD group,and that in control group-427 locus TC+CC genotype was significantly higher than that in TT genotype.+ The apoA/B ratio of TT genotype was significantly different from that of AA genotype.There were no significant differences in TC,TG,HDL-C and other blood lipids.There were no significant differences in blood lipids and apolipoproteins in the control group.3.The frequency of-219 T allele was 0.268 in CHD group and 0.288 in control group;the frequency of-427 C allele was 0.211 in CHD group and 0.098 in control group;the frequency of-491 T allele was 0.053 in CHD group and 0.074 in control group.4.The frequency of-427 C allele in CHD group was significantly higher than that in control group,with statistical difference.Multivariate logistic regression analysis showed that-427 C was a risk factor for coronary heart disease.5.Comparing the effects of APOE promoter-219G/T and-491A/T loci on APOE concentration in coronary heart disease and control group,it was found that there was no significant difference in APOE concentration between gene polymorphisms of the two loci.Compared with TT genotype,APOE concentration of TC + CC genotype at -427T/C locus tended to increase,but there was no statistical difference.6.The relative activity of luciferase in 293 T cells transfected with pGL2-APOE/-427 TT was significantly higher than that of pGL2-APOE/-427 C C(P < 0.05),which indicated that the apolipoprotein E transcriptional activity of T allele carriers was higher than that of C allele carriers,and the difference was significant.7.After taking atorvastatin calcium,plasma lipid level was correlated with APOE gene polymorphism in patients with coronary heart disease.There was no statistical difference between GT+TT genotype group at-219 locus and GG genotype group in the change rate of blood lipid level before and after lipid-lowering treatment.Compared with TT genotype group,TC+CC genotype group at-427 locus had more significant decrease in TC and LDL levels before and after lipid-lowering treatment.Statistical significance;-491 AT + TT genotype group compared with AA genotype group before and after lipid-lowering treatment,there was no significant difference in the change rate of blood lipid levels.Conclusion:1.APOE promoter-427 C allele carriers are risk factors for coronary heart disease.2.Reporter gene experiment results showed that in vitro,the transcriptional activity of variant C allele was significantly lower than that of wild T allele carriers.APOE promoter-427T/C site was a functional site regulating APOE promoter transcriptional activity and influencing serum APOE concentration.3.APOE-427 C allele carriers were more sensitive to atorvastatin calcium lipid-lowering effect. |
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