Verification And Clinical Application Of CircRNA As The Biomarker Of Rheumatoid Arthritis Concurrent With Coronary Heart Disease

BackgroundRheumatoid arthritis(RA)is a systemic autoimmune disease,which is characterized by chronic destructive joint disease.This disease tends to begin slowly with minor symptoms,and the onset of clinical and imaging findings often happens in late stage when irreversible damage has occurred,so early diagnosis,treatment and individualized treatment of RA needs to be emphasized.Studies have shown that the mortality rate of RA patients is three times higher than that of normal people,among which cardiovascular disease accounts for over half of the deaths,make it the main cause of death.However,traditional cardiovascular risk factors such as gender,obesity,hyperlipidemia,hypertension and family history can not fully explain the high incidence of coronary heart disease(CHD)in RA.Basic studies have shown that CHD and RA share the same inflammatory,mechanisIm,and RA itself is an independent risk factor for early atherosclerosis and CHD.In 2015,the European League against Rheumatism,EULAR,has clearly put forward the recommendations for cardiovascular disease risk management in patients with RA,which shows how harmful it is for RA patients to have CHD.Thus it has clinical significance to find the potential risk factors for cardiovascular disease in RA patients,and to find the biomarkers to detect and prevent CHD in RA patients.In recent years,the diagnostic of Circular RNAs(circRNAs)represents a new research hotspot.And circRNA has a distinctive closed ring structure and plays an important role in recgulating gene expression,which can both directly and indirectly affect the expression of microRNAs(miRNA),resulting in the imbalance of expression of miRNAs and proteins.It matters a lot both in the development of organisms and the diagnosis and treatment of diseases.Studies have found that circRNAs play an important role in many diseases,including neurological and cardiovascular and cerebrovascular diseases such as Alzheimer’s disease(AD)and atherosclerotic vascular disease(ASCVD),as well as autoimmune diseases,like diabetes and various tumors.The expression profiles of circRNA from different tissues and cells can be studied by using gene chip technology.Furthermore,the tissue-specificity,the disease-specificity and high stability make circRNAs promising biomarkers.And circRNA’s unique ceRNA can provide a broader idea for the development of drugs.Undoubtedly,the study of circRNA can provide new ideas for the study of life.In the near future,circRNA will play an important role in the prevention.diagnosis,treatment and prognosis of various diseases.In the previous study of research group,researchers set up RA concurrent with CHD group,RA group,CHD group and healthy control group(HCG).and used circRNA chip technology to analyze the differential expression of circRNA in plasma samples and screened out those circRNAs whose diversity were more than 1.5 times and had statistical significance:232 in RA concurrent with CHD group and HCG,95 in RA concurrent with CHD group and RA group,473 in RA concurrent with CHD group and CHD group.Five circRNA genes were preliminarily identified to be significantly different in plasma expression between RA concurrent with CHD group and the other three groups,including:circR-0007850,circR-0403658,circR-0100332,circR-0092388 and circR-0007260.This reaserch intends to evaluate their diagnostic efficacy by using q-PCR for further verification and screening and taking their clinical indicators into consideration,which is a more in-depth study based on the previous one.ObjectiveTo explore the feasibility and clinical significance of differentially expressed circRNAs as the biomarkers for the diagnosis of RA concurrent with CHD.Research methodsPart1 Preliminary validation and screening of 5 plasma circRNAs by q-PCR1.1 Research objectsWe selected those who were hospitalized or got the physical examination in Yuxi People’s Hospital(the Sixth Affiliated Hospital of Kunming Medical University)from June 2016 to April 2017 and divided them into four groups:(I)20 cases of RA concurrent with CHD;(2)20 cases of RA group;(3)20 cases of CHD group;(4)20 cases of HCG Based on the RA concurrent with CHD group,we took gender and age as the matching conditions,matched individuals as much as possible;CHD was diagnosed by coronary angiography,and typical RA was diagnosed according to the 1987 classification criteria of the American College of Rheumatology(ACR)and for atypical and early RA;diagnosis was made according to the 2010 classification criteria of the ACR or EULAR to avoid misdiagnosis or missed diagnosis.1.2 MethodsBased on the results of previous gene chip analysis,the five circRNA genes(circR-0007850,circR-0403658,circR-0100332,circR-0092388,circR-0007260)were tested for further validation and screening.1.3 Statistical methodsFluorescence quantitative results were used to calculate the gene expression between different tissues by 2(-Delta Ct)method.Statistical analysis was performed by mean plus or minus standard deviation.One-way ANOVA was used to compare the samples of the four groups,and p<0.05 was considered to be statistically significant.Part2 Evaluation of the diagnostic efficacy of three circRNAs by taking relevant clinical indicators into consideration2.1 Research objectsSelect 30 RA patients with CHD,who were hospitalized in Yuxi People’s Hospital,as the study group,meanwhile,the age,sex and number of cases in the same period of hospitalization were matched.Then patients were divided into 30 RA group,CHD group and HCG,with 30 cases in each group.Take the RA concurrent with CHD group as the benchmark,gender and age as the matching conditions,and the individuals were matched as much as possible.Exclusion and inclusion criteria of the objects were the same as that of the 1.1 above.2.2 MethodIn the independent sample,these three circRNA which were screened out in part one were tested by q-PCR,and the serum lipid,myocardial enzyme,coagulation,blood routine,RF,ACCP,ESR and inflammation indexes were also detected,which were satistically analyzed considering the clinical routine indexes.The clinical value of these three circRNA as the diagnostic markers of RA with CHD was evaluated by using ROC curve.2.3 Statistical methodsFluorescence quantitative results were used to calculate the changes in gene expression between different groups by usin the 2(-Delta Ct)method.Statistical analysis was performed using mean plus or minus the standard deviation.Statistical analysis was performed using spss22.0 and DPS data analysis system(V14.10 Advanced Edition),and the non-parametric Mann-Whitney test was used to compare the differential expression of circRNA between RA concurrent with CHD and other three groups.The difference multiples of q-PCR results among groups were analyzed by Tukey method of DPS data analysis system(V14.10 Advanced Edition),and P<0.05 was considered to of statistical significance.The area under the ROC curve was used to evaluate the accuracy of diagnostic indicators.Pearson single factor correlation analysis was used to analyze the correlation between circRNA expression level and some clinical indicators.It is to evaluate the feasibility and application value of single and combined circRNA,which is typically used to diagnose cardiovascular disease and differential expression,in the diagnosis of RA concurrent w th CHD.ResultsPart 1 Preliminary validation and screening of 5 plasma circRNAs by q-PCRIt was preliminarily verified that circR-0007850 was up-regulated by 4.41 times compared with CHD group.circR-0403658 was down-regulated by 0.07 time and 0.05 time respectively compared with RA group and HC,which was 2.61 times higher than that of CHD group;circR-0100332 was up-regulated by 10.57 times and 6.8 times respectively compared with CHD group and HCG;and circR-0092388 was up-regulated by 2.5 times and 2.52 times respectively compared with CHD group and HCG.The results of the four circRNAs were basically consistent with the results of gene chip screening,however,there was no difference among the expression of circR-0007260 and those of the other three groups.After statistical analysis.circR-0007850.circR-0100332 and circR-0092388 were screened out for further diagnostic efficacy evaluation.Part 2 Evaluation of the diagnostic efficacy of three circRNAs by combining clinically relevant indicators1.The expression of circR-0007850 was of statistical significance compared with CHD group(P<0.05,Fold chang was 3.31);the expression of circR-0100332 had statistical significance compared with HCCQ RA group and CHD group(P<0.05,Fold chang were 4.01,0.62 and 2.88,respectively),the expression of circR-0092388 had statistical significance compared with HCG,RA group and CHD group too(P<0.05,Fold chang were 1.60,0.62 and 1.57,respectively).2.Correlation analysis with clinical traditional indicators showed that circR-0007850 was positively correlated with coagulation indexes PT and TT(r=0.409,0.384,P=0.037,0.047),negatively correlated with CK-MB and PCT(r=-0.471,-0.456,P=0,018,0.022);and circR-0100332 was positively correlated with TT(r=0.611,P=0.002),negatively correlated with CK-MB and PCT(r=-0.409.-0.382,P=0.037.0.048);circR-0092388 was positively correlated with APTT(r = 0.592,P=0.003),negatively correlated with blood lipid index LDL-C(r=-0.382,P=0.048)and WBC(r =-0.418,P = 0.033),but the three circRNA genes not correlated with inflammatory markers PCT.CRP,IL-6 and rheumatoid related markers ACCP,RF;3.The ROC curve was used to evaluate the diagnostic performance of circR-0007850,circR-0100332 and circR-0092388.The area under the RA concurrent with CHD ROC curve was 0.190(95%CI,0.059-0.321)and 0.788(95%CI,0.650-0.927)and 0.215(95%CI,0.078-0.352)respectively.The diagnostic performance of circR-0007850,circR-0100332,and circR-0092388 for diagnosis of RA concurrent with CHD was evaluated,and their diagnostic sensitivities were 53.3%,76.7%,and 56.6%,respectively;corresponding specificities were 93.3%,96.7%,and 90.0%;and coincidence rates were 56.6%,86.7%and 73.3%.Conclusions1.CircR-0007850,circR-0403658,circR-0100332 and circR-0092388 were proved to be significantly up-regulated in RA patients with CHD,which suggested that they may be related to the onset and development of RA concurrent with CHD.2.CircR-0007850 is correlated with clinical indicatorsCK-MB.PT,TT and PCT;circR-0100332 is correlated withCK-MB,TT,PCT;and circR-0092388 is correlated with LDL-C,APTT and WBC.3.As a marker,circR-0100332 has important reference value in diagnosing RA concurrent with CHD.