Identification Of Prognostic Value Of Paraoxonase 1(PON-1) In Chronic Obstructive Pulmonary Disease Patients Who Underwent Coronary Artery Bypass Grafting(CABG)

Coronary artery heart disease(CHD)is caused by abnormal lipid metabolism,by formation of atheromatous plaque in the coronary arteries intimal,by vascular lumen occlusion.Coronary artery bypass grafting(CABG)is the main surgical procedure for CHD.CHD is often accompanyed with concomitant disease,including hypertension,heart dysfunction,diabetes,and chronic obstructive pulmonary disease(COPD).Compared with the patients who have diabetes or hypertension,the patients with COPD are more likely to undergo accidents during the perioperative period,undergo increasing the incidence and mortality of postoperative accidents.At present,the medical community attach more and more importance to patients with CHD and COPD.CHD and COPD have some common risk factors.During the clinical treatment process,in addition to relying on patient history,signs and relative inspection to know risk factors that may impact on the prognosis of patients having CHD and COPD,the relevant molecular markers are used to predict prognosis of CHD.Early in the disease,Proper treatment is of great significance.At the same time,paying attention to patients with CHD and COPD can ultimately improve the prognosis of such patients and prolong their survival time。Paraoxonase 1(PON1)is synthesized by human liver.Because of its antioxidant properties,PON1 has become a focus of current researchs.Studies have found that PON1 has a protective effect on low density lipoprotein(LDL),so it has the function of anti-atherosclerosis.PON1 has anti-inflammatory effects.The results of genotype study and enzymatic PON1 phenotype test showed the big difference of PON1 activity in a population.Changes in PON1 activity are closely related to the occurrence and development of a variety of diseases.PON1 activity of patients with diabetes,familial hypercholesterolemia,myocardial infarction and other diseases has been decreased to varying degrees.Studies on PON1 activity in recent years have shown that gene polymorphism is not enough to be a risk factor for coronary heart disease,because in patients with coronary atherosclerosis,decreased PON1 activity causes a significant down-regulation of HDL antioxidant effect.Gene polymorphism refers to the existence of two or more discontinuous variant genes or alleles in a population at the same time.Mutations in regulatory functional regions and adjacent sites can affect protein function by affecting protein gene expression and activity.Single nucleotide polymorphism(SNP),that is,the difference of scattered single base.SNP is usually a diallelic or dimorphic variation.At present,a large number of research show that PON1 gene polymorphism are related to the occurrence and development of a variety of cardiovascular and cerebrovascular events,including coronary heart disease,ischemic stroke,cerebral infarction,etc.So it is used as a risk factor to predict the occurrence of the above diseases.In recent years,a large number of studies have been done on the relationship between PON1 gene polymorphism and coronary heart disease,but no studies have been conducted on the relationship between PON1 gene polymorphism and postoperative clinicopathological parameters of patients with CHD and COPD.For this reason,this study is the first time to discuss the above issues.Since researchers discovered miRNA,more and more evidence has been shown that miRNA is closely related to the occurrence and development of a variety of diseases.In the cells at the site of disease,miRNA expression is usually significantly different from that of normal tissue cells in the body.The cardiovascular system has kinds of miRNA,which plays a variety of roles in cardiovascular diseases.The high expression of miRNA in the cardiovascular system and the abnormal expression of miRNA during myocardial injury be found,that have brought new way to the treatment of myocardial ischemia diseases,and researchers expect to reduce myocardial ischemia and hypoxia injury by regulating the expression of miRNA.The research on the role of miRNA in cardiovascular diseases is still in its infancy.In this study,three phenotypes of rs3735590 polymorphic locus of biallelic genes of PON1 gene sequence were studied respectively(CC: 212;TC: 75;TT: 5).The relationship between the clinical pathological parameters and genes phenotypes of PON1 in patients with CHD and COPD;the correlation between miRNA-616 and PON1 was studied by using the method of in vitro cell experiment,so as to provide theoretical basis for the early prevention and assessment of diagnosis after CABG of patients with CHD and COPD.ObjectiveIn order to study the correlation between PON1 gene polymorphism and postoperative prognosis of patients with CHD and COPD,we researched the relationship between three PON1 gene type(CC: 212,TC: 75 and TT: 5)and clinical pathology parameters in patients with CHD and COPD.The correlation between mir-616 and genotype of PON1 was studied by in vitro cell experiment,the mechanism of PON1 expression and activity change in patients with CHD and COPD was explored,so as to provide theoretical basis for the early diagnosis of complications related to CABG.Method1.A total of 292 patients with CHD and COPD who underwent CABG at the China-japan uion hospital of jilin university from 2010 to 2017 were selected.All patients were examined by coronary angiography and confirmed as coronary heart disease,peripheral blood samples of patients were collected.The DNA samples of each blood sample were extracted and the genotypes of PON1 gene were determined by restriction enzyme digestion.According to the different genotypes of PON1,all the selected patients were divided into three groups:CC: 212 group,TC: 75 group and TT:5 group,and then the three genotypes were compared with the age,sex,low density lipoprotein(LDL)and high density lipoprotein(HDL).Kaplan-Meier and Cox regression analysis were used to investigate whether PON1 rs3735590 was associated with event-free survival in patients with CHD after CABG.2.The primary human hepatotypes were isolated and cultured,and their PON1 genotypes were identified.The TC and TT groups were merged in one group with T carriers for statistical analyses.The target gene of miR-616 was predicted by biological software.The 3 ’UTR amplification primers were designed according to the PON1 gene sequence information.The 3’ UTR sequence of PON1 gene was amplified by PCR,and then cloned into the gene containing luciferase reporter gene,to construct a the wild type plasmid PON13’UTR WT.and PON13’UTR MUT.Then Different concentrations of miR-616 mimic and inhibitor(25 nM,50 nM,75 nM)were transfected into PON1 CC-type human hepatotypes with PON13’UTR-WT and PON13’UTR-Mut plasmids containing the luciferase reporter gene,respectively.To Detect luciferase expression in cells.MiR-NC and different concentrations of miR-616 mimic / inhibitor(25 nM,50 nM,75 nM)were transfected into PON1 CC-type hepatocytes.After 48 h,fluorogenic quantitative PCR was used to detect miR-616,PON1 mRNA and protein expression levels were measured by western blot,the PON1 activity measured by ELISA.MiR-NC and different concentrations of miR-616 mimic / inhibitor(25 nM,50 nM,75 nM)were transfected into PON1 T hepatotypes.After 48 h,the expression of miR-616 was detected by fluorescence quantitative PCR and western blot was detected PON1 mRNA and protein expression levels,the activity of PON1 were measured by ELISA..Results1.T test results show that,there was no significant difference in age,sex,overall level and LDL of the three PON1 genotypes(P> 0.05),but the HDL of thePON1 CC group was significantly lower than that of the PON1 TC and TT group(P <0.05).The results showed that the survival time of patients with CC genotype was significantly lower than that of TC genotype and TT genotype group(P <0.05),which indicated that the survival time of CABG patients was related to the polymorphism T allele of PON1 rs3735590.2.PON1 is predicted to be the target gene for miR-616,and the position of miR-616 bound to PON1 is located in its 3’UTR region.With the increase of miR-616 mimic transfection concentration,the expression of luciferase in PON1 WT group was decreased in a dose-dependent manner,while the expression level of luciferase in PON1 Mut group did not change significantly.With the increase of miR-616 mimic transfection concentration,the expression level of miR-616 in PON1 CC hepatotypes was gradually increased in a dose-dependent manner.The expression of PON1 mRNA and protein was decreased in a dose-dependent manner.With the increase of miR-616 inhibitor,the expression level of miR-616 was decreased in a dose-dependent manner.The expression of PON1 mRNA and protein was gradually increased in a dose-dependent manner.The activity of PON1 increased gradually increases.With the increase of miR-616 mimic transfection concentration,the expression level of miR-616 in PON1 T hepatotypes was gradually increased in a dose-dependent manner.With the increase of miR-616 inhibitor,the expression of miR-616 in PON1 T hepatotypes was decreased,but the expression of PON1 mRNA and protein did not change significantly(P> 0.05),the activity of PON1 did not significantly change too.ConclusionMain PON1 gene phenotype in patients with coronary artery heart disease complicated with chronic obstructive pulmonary disease is CC type,and patients with PON1 CC have a shorter postoperative event-free survival and a worse prognosis than patients with PON1 CT and PON1TT;miR-616 attenuates expression and activity of PON1 in PON1 CC liver cell,but not in PON1 T liver cells.We speculated that may be the reasons that lead to lower PON1 activity in patient with PON1 CC,that lead to higher inflammatory reaction than patients with TC and TT genotype,and survival time is shorter in patient with PON1 CC.PON1 CC can be used as an independent risk factor for prognosis of patients with CHD and COPD.