Study On Screening,verification And Clinical Application Of Plasma CircRNA As A Biomarker Of Rheumatoid Arthritis With Coronary Heart Disease

BackgroundRheumatoid arthritis(RA),mainly in the form of non-suppurative inflammation in the joints and surrounding tissues,is a chronic autoimmune disease and characterized by joint synovitis,With the progressive development,it can not only lead to the deformity and dysfunction with patient’s damaged joints,but also cause various complications,which affect the patients’ physical and mental health and the quality of life seriously.According to recent years’ epidemiological data,the incidence of cardiovascular disease,especially coronary heart disease(CHD)increases significantly among patients with RA,increased by nearly 50%～60%,compared with the general population.The incidence of myocardial infarction among patients with RA history of more than 10 years increases by 3.1 times,which suggests that CHD may be one of the serious complications of RA.RA is an independent risk factor for cardiovascular disease,however,RA with CHD has not yet attracted enough clinical attention for a long time and the life expectancy of patients with RA is shortened significantly.The exact pathogenesis between RA and cardiovascular disease is still unclear and more medical researchs are needed to improve our understanding of the problem.Enhancing the vigilance of RA patients complicated with cardiovascular disease,screening for cardiovascular disease among RA patients as early as possible,reducing the incidence of cardiovascular disease and improving the survival cycle of RA,have become an important subject which needs to be explored.What’s more,seeking a marker which can accurately and effectively predict and detect the risk of CHD among RA patients early,will have an important clinical value and practical significance in preventing the occurrence of RA complicated with CHD.The latest researches had showed that circRNA played an important role in the course of the occurrence and development of the disease.CircRNA can be used as a new potential medium and marker in human diseases,can lead to mRNA and protein expression disorders by directly or indirectly affecting miRNA expression,can also affect protein expression directly.Some studies had shown that circRNA played a very important part in the development of diseases such as cancer,diabetes,neurological diseases,autoimmune diseases and atherosclerosis.According to the cluster analysis of circ RNA expression profile data from different tissues and cell lines by gene chip technology,circRNA can be specifically identified in specific tissues and cells.So far,scientists have used circRNA chip technology in brain tissue,lung tissue,colon cancer,pancreatic cancer,gastric cancer,liver cancer,cells and other studies,the specific expression of corresponding circRNA is found.In addition,circRNA has made some results in monitoring the recurrence after cancer surgery.Therefore,the change of circRNA level is closely related to the development of many diseases and may be a highly specific and sensitive marker for noninvasive diagnosis of these diseases.Objectives1.The differential expression of circRNA in plasma among RA patients with CHD was analyzed by circRNA gene chip technique.2.Explore the significance of plasma circRNA differential expression as the noninvasive diagnostic marker of RA with CHD.Materials and MethodsPart I Screening the differential expression of plasma circRNA chip between RA with CHD and other groups1.1 Research groupsThree RA patients with CHD are selected as the study group from June 2016 to August 2016 in Yuxi City People’s Hospital,during the same period of hospitalization,matching with age and gender,each 3 cases with simple RA patients,simple CHD and healthy people are regarded as the control group.The diagnosis of RA is based on the 2010 American Rheumat Association RA diagnostic criteria.While the diagnosis of CHD is based on the reporting standards of 1979 international societies and associations of cardiology and World Health Organization Clinical Nomenclature Standardization Joint Professional Group.In the morning,10 ml of fasting venous blood of the subjects is collected in the EDTA-K3 anticoagulant tube,centrifuged at 3500r/min for 15 minutes,the plasma is dispensed into a 2 ml freezer and stored in a refrigerator at-70℃.1.2 MethodsApply high-throughput circRNA chip technology to screen the differences of expression profile of circRNA in the plasma among RA patients with CHD,simple RA patients,simple CHD patients and healthy people,each group for 3 cases,after preprocessing the raw data,we screen the circRNA of the difference expression and have a cluster analysis.1.3 Chip analysisScan each specimen Cy3 fluorescence intensity by Axon gene chip 4000B chip scanner,then use the Agilent Scanner G2505C to scan the image into Agilent Feature Extraction 11.0 Software for image data analysis.The data was normalized treated by R software package.Hierarchical clustering was used to distinguish circRNAs expression patterns among samples.The circRNA of the difference expression between the two groups is screened by the change multiple and the P value take the change multiple>1.5 times,t test with P value<0.05 as a statistically significant result-According to screening the difference expression of the circRNA by the chip to plot the scatter diagram and the volcano plot,we had the supervised hierarchical clustering analysis of the obtained data.As for each circRNA,the first five miRNAs with the greatest possibly relative with circRNA is predicted by the miRNA binding site software from Arraystar,USA.Part Ⅱ Verify differential expression of plasma circRNAs between RA with CHD and control group2.1 Research groups10 RA patients with CHD are selected as the study group from June 2016 to April 2017 in Yuxi City People’s Hospital,during the same period of hospitalization,matching with age and gender,each 10 cases with simple RA patients,simple CHD and healthy people are regarded as the control group.The selection and exclusion criteria of the subjects,collection and treatment of specimens are the same as 1.1.2.2 MethodFive up-regulated and down-regulated genes(circR-0089762,circR-0077462 circR-0081545,circR-0004121 and circR-0044837)are selected for fluorescence quantitative PCR detection.2.3 Statistical methodsFluorescence quantitative results use 2(-ΔCt)method to calculate changes in gene expression among different organizations.Statistical analysis is performed by mean± standard deviation.The comparisons of four groups are based on single factor analysis of variance in Oneway-ANOVA,P<0.05 is considered to be statistically significant.Part III Further validation and clinical application research of differential expression of plasma circRNA between RA with CHD and control group3.1 Research objects30 RA patients with CHD are selected as the study group from June 2016 to June 2017 in Yuxi City People’s Hospital,during the same period of hospitalization,matching with age and gender,each 30 cases with simple RA patients,simple CHD and healthy people are regarded as the control group.The selection and exclusion criteria of the subjects,collection and treatment of specimens are the same as 1.1.3.2 MethodsTwo up-regulated and down-regulated genes(circr-0089762 and circr-0004121)are selected for further fluorescence quantitative PCR detection.3.3 Statistical methodsFluorescence quantitative results 2(-ΔCt)method is used to calculate changes in gene expression among different organizations.Statistical analysis used mean plus or m:inus standard deviation method.The comparisons of four groups are based on single factor analysis of variance in Oneway-ANOVA,P<0.05 is considered to be statistically significant.SPSS 22.0 and software are used for statistical analysis and the non-parametric mann-whitney test is used to compare the difference expression of circRNA between the RA concurrent CHD and the control group.While Kruskal-wallis test is used to compare the differences among the three or more groups.The area under the ROC curve is used to evaluate the accuracy of diagnostic indicators.The correlation between the expression level of circRNA and some clinical indicators is analyzed byPearson single-factor correlation analysis.ResultsPart I Screening for differential expression of circRNA chip between RA complicated with CHD and each control groupBy analyzing chip,the differential expression of circRNA in plasma of RA patients with CHD,the healthy population,the simple RA and the simple CHD are mainly analyzed.Thoes numbers of circRNA that have greater than 1.5 times of different expression levels and statistically significant are:232 in RA with CHD and healthy people,95 in RA with CHD and pure RA,473 in RA with CHD and simple CHD.Part II Verify differential expression of plasma circRNAs of RA complicated with CHD and control group5 circRNAs(CircR-0089762,circR-0077426,circR-0081545,circR-0004121 and circR-0044837)are verified by qPCR in four groups of 40 plasma samples by genomic circRNA expression assay gene microarray.The results of three circRNAs are the same as those of the chip,which is statistically significant.Among them the circr-0089762 and circr-0004121 are significantly up-regulated,and the two are selected for further verification.Part III Further validation and clinical application research of differential expression of plasma circRNA of RA complicated with CHD and control groupAs for circR-0089762 and circR-0004121.The expression of circR-0089762 between the groug of RA with CHD groug and simple RA group were statistically significant(P<0.021).Compared with the other three groups,the expression of circR-0004121 in RAgroup with CHD had significance(P<0.001).The area under the ROC curve of diagnose RA with CHD is 0.269(95%CI,0.147-0.390)and 0.816(95%CI,0.689-0.926)with autoantibodies(ACCP and RF),then inflammatory factors(PCT,IL-6 and CRP),correlation analysis of blood lipid index(TC,TG,HDL-c and LDL-c),circR-0089762 is closely related to CRP,RF and RF(r=0.639,0.508,P=0.002,0.022).While circR-0004121 is closely related to PCT(r=0.775 P = 0.000).Conclusion1.Compared with simple RA patients,simple CHD and healthy people,RA patients’ with CHD plasma circRNA expression profile is significantly changed,which suggests that circrnas expressed in these differences may be closely related to the occurrence of RA and CHD.2.By validating the circr-0089762 and circr-0004121,the two genes are significantly down-regulated and up-regulated among RA patients with CHD,which suggests that there may be a relationship between RA and CHD during the occurrence and development.3.Circr-0089762 and circr-0004121 are correlated with clinical indicators CRP,RF and PCT.4.CircR-0004121 has a certain diagnostic value for RA combined with CHD.