| ObjectivesEssential hypertension has become a global epidemic disease,an important risk factor for cardiovascular and cerebrovascular diseases,and one of the main causes of chronic kidney disease.The pathological mechanism of hypertension is complicated,and the genes of the natriuretic peptide family and its receptor play an important role in the pathological mechanism of hypertension.Previous studies have found that the NPR3 gene rs 1173747 polymorphism in hypertensive patients affects the transcriptional expression of NPR3 and is associated with elevated blood pressure.Studies have found that miR143 targets the negative regulation of NPR3 in human left ventricular cardiomyocytes,but does the rs1173747 polymorphism affect the regulatory effect of miR143?Does the NPR3 gene rs1173747 site polymorphism and miR143 target negatively regulate NPR3 affect NPR3 receptor ligand binding and its downstream effector molecular levels?Regarding such issues,it is currently unclear.To this end,this study selected hypertensive patients and healthy people as the research object,to explore the role of NPR3 gene rs1173747 polymorphism and miRNA143 in the pathological mechanism of hypertension,trying to verify the above problems.Methods1.Select research objects and collect samplesFrom June to September 2019,100 patients with essential hypertension and 100 healthy people who underwent a health check-up at the medical examination center of our hospital were selected as the case group and the control group.Case group:The three generations of paternal and maternal lineages are all Han people who live in Yunnan Province,are between 40-80 years old,and are diagnosed with primary hypertension.Patients with secondary hypertension were excluded,and those with incomplete information and those with failure to detect observation indicators were excluded.Control group:The three generations of paternal and maternal lineages are all Han people living in Yunnan Province,with systolic blood pressure<140 mmHg,diastolic blood pressure<90 mmHg,no other cardiovascular diseases,and are between 40-80 years old.Conduct a questionnaire survey on eligible people,improve personal information and biochemical index testing,sign informed consent,and collect serum and whole blood specimens.2.AS-PCR detection of rs1173747 polymorphism of NPR3 gene in two groupsConsult the NPR3 gene rs1173747 gene sequence to design and synthesize AS-PCR specific primers.Optimize PCR reaction system and amplification conditions of NPR3 gene rs1173747 loci polymorphism detection.The optimized AS-PCR method was used to detect the polymorphism at the rs1173747 site of the NPR3 gene of the research subject,that is,the DNA of the whole blood sample of the research subject was extracted by the centrifugal column method,and the template DNA was added to the PCR reaction solution containing specific primers to set up the PCR.The reaction procedure is to amplify the target fragment.The amplified product is added to the spotting well of agarose gel,electrophoresis is performed,and the electrophoresis result is analyzed to determine the genotype.3.Detection of serum miR143Based on the NPR3 gene rs1173747 polymorphism and clinical basic information,50 equal specimens were selected from each of the two groups of subjects,using centrifugal column method to extract the serum sample miRNA,and the purity of the miRNA was detected in the spectrophotometer And concentration,add a certain volume of miRNA to the reverse transcription reaction solution,set the reverse transcription program,and perform the reverse transcription reaction.Add a certain volume of reverse transcription product cDNA to the RT-PCR reaction solution,and add specific miR143 primers,set up the RT-PCR reaction program,and perform amplification of the target fragment.After the amplification,view and analyze the CT value to U6 Simultaneous amplification as an internal control was used to calculate the relative expression level of miR143 in serum samples.4.Detect serum ANP,NO,VEGF,ET-1 and cAMP levelsAccording to the polymorphism of the rs 1173747 locus of NPR3 gene,44 specimens with the relative expression of miR143 were selected from the two groups of subjects,using imported ELISA kits,according to the instructions for serum ANP,NO,VEGF,ET-1 and cAMP concentration detection.5.Statistical methodsUse SPSS Statistics 17.0 statistical software package for statistical analysis.The frequency of population distribution of NPR3 gene SNPs genotypes and alleles was counted directly,and chi-square test was used to analyze the statistical significance of the difference between the two groups.The interquartile range was used to express the levels of serum ANP,NO,cAMP,VEGF,and ET-1 in the study subjects,and the statistical significance of the differences between the two groups was analyzed using rank sum test.The mean±standard deviation was used to express the relative expression level of serum miR143,and the t-test of two independent samples was used to analyze the statistical significance of the difference between the two groups.Pearson correlation analysis was used to study the correlation between blood pressure and serum ANP,NO,cAMP,VEGF,ET-1,and miR143.The subjects' serum ANP,NO,ET-1,cAMP and VEGF were analyzed by single factor regression analysis with miRNA143,and the blood pressure was analyzed by multifactorial regression with ANP,NO,ET-1,cAMP,VEGF and miRNA143.Results1.A total of 83 AA genotypes,100 AC genotypes,and 17 CC genotypes were detected in 200 subjects;the frequency of AC genotypes and C alleles in the case group was higher than that in the control group(P<0.05)).2.Serum miR143 expression levels were tested in 50 case groups and 50 control groups(AA genotype carriers,25 AC genotype carriers each),and the results showed that the relative expression level of serum miR143 in the case group was 0.97±0.04.It was 1.07±0.09 lower than the control group,and the difference was statistically significant(t=-7.286,p=0.000).3.Serum ANP,NO,ET-1,cAMP,and VEGF levels were tested in 44 cases in the case group and the control group respectively.The results showed that the serum ANP and cAMP concentration levels in the case group were lower than those in the control group,ET-1 and The VEGF level was higher than that of the control group,and the difference was statistically significant(p<0.05).4.Analysis of the effect of NPR3 gene rs1173747 polymorphism on serum ANP,NO,VEGF,ET-1,cAMP concentration levels,the results showed that the serum ANP concentration level of AC genotype carriers was higher than that of AA genotype,the difference was statistically significant Significance,p<0.05.Among them,the control group AC genotype carrier serum ANP concentration level was greater than AA genotype carrier;VEGF water was less than AA genotype carrier,p<0.05.5.Analysis of the effect of NPR3 gene rs1173747 polymorphism on the expression level of serum miRNA143.The results showed that the expression level of miRNA143 in AC genotype carriers was higher than that in AA carriers,the difference was statistically significant,p<0.05.6.Analyze the effect of miRNA143 expression level on serum ANP,cAMP,NO,ET-1,VEGF concentration levels.The results showed that the serum ANP concentration level in 88 subjects was positively correlated with miRNA 143 expression.Among them,44 cases of AC genotype carrier ANP were positively correlated with miRNA143;VEGF and miRNA143 were negatively correlated;the results of regression analysis were consistent.7.Analysis of the effect of serum miRNA143 on blood pressure,the results showed that 88 subjects SBP and DBP were negatively correlated with miR143,of which,only ABP genotype carriers DBP and miR143 were negatively correlated;AC genotype carriers SBP and DBP and miR143 Both were negatively correlated.The regression analysis results are consistent.8.Taking the occurrence of hypertension as the dependent variable,logistic regression analysis was performed using the genotype of rs 1173747 locus of NPR3 gene,serum miRNA143,ANP,cAMP,NO,ET-1,VEGF as independent variables.The results showed that the genotype of rs1173747 locus,ANP and miR143 have statistical significance with partial regression coefficients of hypertension.Conclusions1.NPR3 gene rs1173747 polymorphism and miR143 may be involved in blood pressure regulation.2.miR143 may affect the NPR3/ANP receptor ligand effect through the regulation of the NPR3 gene transcript.3.The rs1173747 polymorphism of NPR3 gene may affect the regulatory effect of miR143 on NPR3 gene transcript.4.The above-mentioned effects may change in the pathological mechanism of hypertension. |
PDF Full Text Request