Effect Of MiR-4787-5p And MiR-4306 On The Pathogenesis Of Acute Aortic Dissection

Background and objective Acute Aortic Dissection(AAD)is a kind of cardiovascular emergency and it is characterized by sudden,progress rapidly,high mortality and morbidity.According to research reported,AAD incidence is about 3/100000 per year,and it about 50% died within 48 h.The hospital mortality of AAD is about 20%-30% and 10-year mortality rate can reach 37%-53%.With the improvement of medical technology,mortality of AAD has declined obviously,but compared with coronary heart disease and other cardioascular diseases,the treatment level of AAD is still low.The misdiagnosis rate,missed diagnosis and mortality is still high.Therefore,further explore pathogenetic process of AAD is of great significance to guide the research about early diagnosis,timely and effective treatment and the prevention of complications.At present,the research on the pathogenesis of AAD is very much,but the molecular pathogenesis has not been fully elucidated.Micro RNA(miRNA)is a kind of endogenous non-coding small RNA molecules.It is long about 18 to 22 nucleotides and it has the characteristics of highly conservative.Mi RNA mainly combines with the target gene m RNA 3'-untranslated region to control target gene transcription and translation level,and then participates in related biological functions which include cell proliferation,cell differentiation and cell apoptosis.A study shows the miRNA can exist widely and steady in the peripheral blood plasma or serum and it can detected by the method of fluorescence quantitative reverse transcription-polymerase Chain Reacto(q RT-PCR).The miRNA expression level can vary with the pathophysiology of the disease.Therefore,miRNA widely participate in regulating the process of various diseases.The studies have found that miRNA plays a main role in the pathogenesis of AD.Hence,in order to further explore the miRNA expression in the AAD,the lab early has passed Agilengt micrornas chip to finish miRNA expression profile analysis and it preliminary screen different expression of miRNA in plasma between AAD and normal people.The lab choosed two expression obviously differences of the miRNAs which are miR-4787-5p and miR-4306.The previous research result in the lab showed miR-4787-5p in AAD expression was statistically significant,but miR-4306 was not statistical significance,which are not consistent with the result of miRNAs expression profile chip.In order to be more accurate to verify differentially expression level of miRNAs in AAD and reduce the experiment error,this research will expand sample size and use q RT-PCR method to validate the difference of expression level of miR-4787-5p and miR-4306 between AAD patients and healthy control group.In addition,this research will also be a preliminary explore about the target genes of miR-4787-5p and miR-4306 in AAD and discusse its role in the pathogenesis of AAD.Research methods: The experiment was divided into two groups: patients with acute aortic dissection(AAD group)and normal volunteers in the control group(group Control).With the approval of the hospital ethics committee,we collected plasma from AAD patients and healthy volunteers in the first affiliated hospital of zhengzhou university from January 2015 to January 2017.All into the group signed the informed consent.AAD patients and healthy volunteers were with the same age,sex ratio.The research detailly recorded the groups basic information,including blood pressure on admission to hospital and within 24 hours of D-dimer,C-reactive protein(CRP),brain natriuretic peptide(BNP),White blood cell(WBC),red blood cell(RBC),hemoglobin(Hb),platelet(PLT),Width of the ascending aorta,and so on.First,using independent sample t test and chi-square test analysed two groups of basic clinical data and selecting the statistically significant indicators and whether AAD correlation to analysis.Second,the study through q RT-PCR method expanded sample size and used q RT-PCR method to validate the difference of expression level of miR-4787-5p and miR-4306 between AAD patients and healthy control group.Receiver operating characteristic(ROC)and area under the receiver operating characteristic curve(AUC)were used to test the specificity and efficiency of selected miRNAs for early diagnosis of AAD.At last,bioinformatic analysis were used to predict target genes of miR-4787-5p and miR-4306.At last,Luciferase reporter assays were performed to certify the correctness of the target genes.Results: 1.In the present study,we enrolled 102 patients with AAD and 58 controlsl.AAD patients included 81 men and 21 females,aged 28 to 78 years,an average age of 52.3 plus or minus 10.4 years.The control group included 48 men and 10 women,30 to 77 years of age,and an average age of 53.3 plus or minus 11.6 years.There were no statistically significant differences but hypertension,whether elevated blood pressure on admission,width of the ascending aorta,D-dimer,CRP,BNP,WBC,RBC,Hb,PLT in clinical characteristics between the AAD and control groups(P<0.05 was regarded as significant difference).2.Using bivariate correlation analysis the indexes which were statistically difference between AAD groups and the control groups and whether was AAD.The results showed that RBC and Hg were weak negative relationship with AAD,P<0.05,the correlation coefficient was 0.286,0.291.Platelets was generally negative correlation,P<0.05,the correlation coefficient is 0.411;High blood pressure is generally positive correlation with AAD,P<0.05,the correlation coefficient is 0.383;Blood pressure on admission and brain natriuretic peptide was significantly positive correlation with AAD,P<0.05,the correlation coefficient were 0.614,0.644;Width of the ascending aorta,white blood cells,D-dimer and c-reactive proteinare highly positive correlation with AAD,P<0.05,the correlation coefficient were respectively 0.761,0.772,0.820,0.832.3.The research further checked the expression changes of miR-4787-5p and miR-4306 between two groups with q RT-PCR method by expanding the sample and compared with independent t test(P<0.05 was regarded as significant difference).We found that the expression of miR-4787-5p and miR-4306 in the plasma of AAD patients were upregulated greatly(P=0.000<0.01<0.05)and differences were statistically significant,which are consistent with the microarray results.ROC analysis showed that the sensitivity and specificity of has-miRNA-4787-5P were 78% and 80% for the diagnosis of AAD and AUC result of has-miRNA-4787-5P was 0.834(95%CI,0.769-0.900).For has-miRNA-4306,the sensitivity and specificity were 86% and 88% separately and AUC result of has-miRNA-4306 was 0.921(95%CI,0.876-0.966).4.Bioinformatic analysis predicted that the 3'-UTR of polycystin1(PKD1)and transforming growth factor-?1(TGF-?1)respectively contain binding sites for miR-4787-5p and miR-4306.5.Dual luciferase report experimental verification results were as follows.Restructuring carrier group of cells which transfection miR-4787-5p agomir and contain PKD1 wild type 3'UTR region restructuring carrier group and transfection miR-4306 agomir and contains TGF-beta 1 wild type 3' UTR region in the luciferase activity appeared significantly lowered(P<0.05).Mi R-4787-5p and miR-4306 respectively with PKD1 3'UTR region and TGF beta1 3'UTR region existed interaction.Mi R-4787-5p and miR-4306 may be respectively negative regulation the expression of PKD1 and TGF-beta1.Therefore,the study indicated that PKD1 and TGF-?1 are respectively direct target of miR-4787-5p and miR-4306 by dual luciferase assay.Conclusions: Plasma miR-4787-5p and miR-4306 might be potential diagnostic biomarkers for AAD.Furthermore,miR-4787-5p and miR-4306 may be involved in pathogenesis of AAD.