A Study On The Relationship Between TCM Syndrome Characteristics And CircRNA Expression Profiles In Patients With Type Ⅱ Cardio-renal Syndrome

ObjectTo study the general laboratory test data and the distribution of major TCM syndromes in patients with type Ⅱ cardio-renal syndrome and differences between the laboratory tests among different syndromes of type Ⅱ cardio-renal syndrome,and to detect the expression of circRNA in patients with type Ⅱ cardio-renal syndrome and the differences among the different syndromes of the expression of circRNA,to screen the symbols of each TCM syndrome of type Ⅱ cardio-renal syndrome,to obtain the pathological mechanisms of type Ⅱ cardio-renal syndrome and different syndromes of type Ⅱ cardio-renal syndrome,and to verify the symbols of type Ⅱ cardio-renal syndrome and each TCM syndrome of type Ⅱ cardio-renal syndrome,in order to provide objective evidence for the diagnose of TCM syndrome of type Ⅱ cardio-renal syndrome.The results showed that the biomarkers of typeⅡ cardio-renal syndrome patients could be used as a biomarker for the diagnosis of type Ⅱ cardio-renal syndrome.Method1.The laboratory test data collection of healthy people and type Ⅱcardio-renal syndrome patientsA total of 267 patients with type Ⅱ cardio-renal syndrome were enrolled in the Department of Cardiology,Guang’anmen Hospital,China Academy of Chinese Medical Sciences from 2009 to 2016.We used Epidata3.1 database to establish the clinical data database of patients with type Ⅱ cardio-renal syndrome.Meanwhile,we collected 241 healthy people.To compare the clinical features between them.2.The laboratory test data collection of patients with different stages of type Ⅱcardio-renal syndromeA total of 267 patients with type Ⅱ heart and kidney syndrome were enrolled in the Department of Cardiology,Guang’anmen Hospital,China Academy of Chinese Medical Sciences from 2009 to 2016.According to the level of eGFR,the patients were divided into three groups（CKD3-5）.To compare the clinical features of the results of the different stages of type Ⅱ cardio-renal syndrome.3.Analysis of TCM Syndromes in Patients with type Ⅱ cardio-renal syndromeTo summary the TCM syndrome of 267 cases of type Ⅱ cardio-renal syndrome,we removed below 10%of the symptom frequency,and got a total of 48 variables,and applying the clustering and factor analysis statistical methods,and then sum up the common factors,we obtained the main TCM syndromes of type Ⅱ cardio-renal syndrome,and according to the TCM syndromes,we compared laboratory test among the different TCM syndromes of type Ⅱ cardio-renal syndrome.4.Detection circRNA expression profiles in patients with type Ⅱ cardio-renal syndromeTwenty patients with type Ⅱ cardio-renal syndrome and five healthy subjects were enrolled,we applied circRNA chip technique produced by Agilent Company to detect the distinguished circRNA expression profiles among the type Ⅱ cardio-renal syndrome patients and healthy subjects,and then according to the distinguished mRNA expression profiles,we analyse the different mRNA function pathways among them.5.Detection circRNA expression profiles among the patients with different TCM syndromes of type Ⅱ cardio-renal syndromeTwenty patients with different TCM syndromes of type Ⅱ cardio-renal syndrome were enrolled,and 5 patients in each TCM syndrome,meanwhile 5 healthy subjects were selected,we filtered the distinguished circRNA expression profiles among the different TCM syndromes of type Ⅱ cardio-renal syndrome patients and healthy subjects,and pairwise comparison among different TCM syndromes of type Ⅱcardio-renal syndrome.And then we analyse the different mRNA function pathways among them.6.Analysis of symbolic circRNA in patients with different TCM syndromes of type Ⅱ cardio-renal syndromeBy analyzing different expression of circRNA in patients with different TCM syndromes of type Ⅱ cardio-renal syndrome to obtain symbolic circRNAs in different TCM syndromes of type Ⅱ cardio-renal syndrome,and explore function pathways in different TCM syndromes using biological information method.7.Validation of circRNA in patients with type Ⅱ cardio-renal syndrome andTCM syndromes of type Ⅱ cardio-renal syndromeUsing RT-PCR technique,we enrolled 15 patients with type Ⅱ cardio-renal syndrome and 12 healthy subjects,8 patients with non-interaction between heart and kidney,blood stasis with water retention syndrome of type Ⅱ cardio-renal syndrome,and another negatively with non-interaction between heart and kidney,blood stasis with water retention syndrome to validate symbolic circRNA in patients with type Ⅱ cardio-renal syndrome in order to obtain molecular biological basis of type Ⅱ cardio-renal syndrome and heart and kidney,blood stasis with water retention syndrome.Results1.Comparison of laboratory test results in patients among type Ⅱcardio-renal syndrome and healthy subjectsIndependent samples t-test showed that there were some differences in erythrocyte parameters,white blood cell parameters,platelet parameters,serum enzymes,liver and kidney function,blood lipid,plasma protein,thyroid hormone and HCY in CRS group.Spearman correlation analysis showed that there are certain correlations among erythrocyte parameters,leukocyte parameters,platelet parameters,serum enzymes,liver and kidney function,blood lipid,plasma protein,thyroid hormone and occurrence of type Ⅱ cardio-renal syndrome.Logistic stepwise regression analysis showed that age,serum HCY level were an important factor in the pathogenesis of type Ⅱ cardio-renal syndrome.2.Comparison of laboratory test results in patients among different stages of type Ⅱ cardio-renal syndromeAnalysis of variance showed that significant differences existed in erythrocyte parameters,serum enzymes,serum lipids,renal function,plasma protein,thyroid hormone,homocysteine,B-type natriuretic peptide,urinary protein and cardiac ultrasound in CKD3-5 patients.3.TCM syndromes in patients with heart and kidney syndromeApplying cluster and factor analysis,we got the following four syndromes:spleen-kidney yang deficiency,qi deficiency with blood stasis syndrome;heart and kidney yang deficiency,detriment to yang affects yin syndrome;non-interaction between heart and kidney,blood stasis with water retention syndrome;yin and yang deficiency,blood stasis with phlegm syndrome.4.Detection results of circRNA in patients with heart and kidney syndromeCompared with healthy subjects,the results showed that there were 1989 circRNA upregulated and 895 circRNA downregulated.The differentially expressed mRNA were analyzed by GO Method,the differentially expressed mRNA were enriched in cell periphery,plasma membrane,plasma membrane part,intergral component of plasma membrane,intrinsic component of plasma membrane and so on in cellular component,and receptor activity、molecular transducer activity、signaling receptor activity、transmembrane signaling receptor activity、signal transducer activity and so on in molecular function,signaling、response to chemical、single organism signaling、cell communication、cell surface receptor signaling pathway and so on in biological process.KEGG analysis revealed that differentiated expression of mRNA in patients with type Ⅱ cardio-renal syndrome and healthy control group was enriched in Hematopoietic cell lineage and Cell adhesion molecules（CAMs）.5.The results of circular RNA of TCM syndromes in patients with type heart and kidney syndromeCompared with healthy subjects,2614 circRNA were upregulated and 516 circRNA were downregulated in patients with spleen-kidney yang deficiency,qi deficiency with blood stasis syndrome of type Ⅱ cardio-renal syndrome.847 circRNA were upregulated and 1947 circRNA were downregulated in patients with heart and kidney yang deficiency,detriment to yang affects yin syndrome of type Ⅱcardio-renal syndrome,3272 circRNA were upregulated and 2381 circRNA were downregulated in patients with non-interaction between heart and kidney,blood stasis with water retention syndrome of type Ⅱ cardio-renal syndrome,3262 circRNA were upregulated and 1590 circRNA were downregulated in patients with yin and yang deficiency,blood stasis with phlegm syndrome of type Ⅱ cardio-renal syndrome.Compared with spleen-kidney yang deficiency,qi deficiency with blood stasis syndrome,there were 120 circRNA upregulated and 75 circRNA downregulated in patients with yin and yang deficiency,blood stasis with phlegm syndrome.Compared with heart and kidney yang deficiency,detriment to yang affects yin syndrome,there were 1760 circRNA upregulated and 80 circRNA downregulated in patients with spleen-kidney yang deficiency,qi deficiency with blood stasis syndrome,620 circRNA upregulated and 139 circRNA downregulated in patients with non-interaction between heart and kidney,blood stasis with water retention syndrome,1887 circRNA upregulated and 224 circRNA downregulated in patients with yin and yang deficiency,blood stasis with phlegm syndrome.Compared with non-interaction between heart and kidney,blood stasis with water retention syndrome,there were 329 circRNA upregulated and 103 circRNA downregulated in patients with spleen-kidney yang deficiency,qi deficiency with blood stasis syndrome,202 circRNA upregulated and 27 circRNA downregulated in patients with spleen-kidney yang deficiency,qi deficiency with blood stasis syndrome.Compared with healthy group,the differentially expressed mRNA were enriched in multiple biological pathways,such as locomotion,single organism signaling,signaling,cell communication,anatomical structure morphogenesis and so on in different TCM syndromes,and differentially expressed mRNA were enriched in a number of biological pathways,such as DNA repair,embryo development ending in birth or egg hatching,chordate embryonic development,cellular component organization,single-organism cellular process and so on in TCM syndromes.6.RealTime PCR of circRNA in whole blood in patients with type Ⅱ cardio renal syndromeCompared to health group,ΔCTlevelof hsa_circ₀001763 in the whole blood of patients with type Ⅱ cardio-renal syndromewas significantly down-regulated andΔ CT level of hsa_circ₀079598was significantly up-regulated.Compared to negatively with non-interaction between heart and kidney,blood stasis with water retention syndrome,ΔCTlevel of hsa_circ₀025640 in the whole blood of patients with non-interaction between heart and kidney,blood stasis with water retention syndrome of type Ⅱ cardio-renal syndromewas significantly down-regulated,and all the results were consistent with the results of circRNA expression pofiles.The ROC curve showed that the area under the curve of hsa_cir₀001763 was 0.933,the diagnostic sensitivity was 86.67%,the specificity was 83.33%,the correct rate of the overall prediction of type Ⅱ cardio-renal syndrome was 85.18%,the area under the curve of hsa_cir₀079598 was 0.911,the diagnostic sensitivity was 73.33%,the specificity was 91.67%,the correct rate of the overall prediction of type Ⅱcardio-renal syndrome was 81.48%,the area under the curve of hsa_cir₀025640 was 0.875,the diagnostic sensitivity was 87.50%,the specificity was 71.43%,the correct rate of the overall prediction of type II cardio-renal syndrome was 80.00%,so the higher level of hsa_cir₀001763,and lower level of hsa_cir₀079598,the greater possibility of suffering from type Ⅱ cardio-renal syndrome,and the higher level of hsa_circ₀025640,the greater possibility of suffering from non-interaction between heart and kidney,blood stasis with water retention syndrome of type Ⅱ cardio-renal syndrome.Conclusion1.There is complex condition in patients with type Ⅱ cardio-renal syndrome,with the renal function damage increasing,hematopoietic system,coagulation system,immune system,circulatory system,urinary system are reducing.2.The nature of type Ⅱ cardio-renal syndrome is deficiency root and excess tip.There are four types of main syndrome,such as spleen-kidney yang deficiency,qi deficiency with blood stasis syndrome,heart and kidney yang deficiency,detriment to yang affects yin syndrome,non-interaction between heart and kidney,blood stasis with water retention syndrome,yin and yang deficiency,blood stasis with phlegm syndrome.3.The pathologic mechanism of type Ⅱ cardio-renal syndrome is related to hematopoietic cell lineage,andthe mechanism of immune inflammation,such as Toll receptor signaling pathway,MAPK signal pathway,Wnt signaling pathway and so on.4.The key raised circRNA are hsa-circRNA 14362-14,hsa_circ₀127021,hsa_circ₀117337,hsa_circ₀060535,hsa_circ₀138971,key down circRNA are hsa-circRNA9066-1,hsa_circ₀136708 in Type Ⅱ cardio-renal syndrome with spleen-kidney yang deficiency,qi deficiency with blood stasis syndrome;the key down circRNA are hsa_circ₀103678,hsa-circRNA6809-9,hsa-circRNA11277-1,hsa_circ₀122389 in Type Ⅱ cardio-renal syndrome with heart and kidney yang deficiency,detriment to yang affects yin syndrome;The key raised circRNA are hsa_circ₀012956,hsa_circ₀100172,hsa_circ₀025640,hsa_circ₀025641,hsa_circ₀117312,has_circ₀133589,the key down circRNA are has_circ₀103164,hsa_circ₀098592,hsa-circRNA11756-1 in Type Ⅱ cardio-renal syndrome with non-interaction between heart and kidney,blood stasis with water retention syndrome;the key raised circRNA are hsa_circ₀113754,hsa-circRNA7413-12,the key down circRNA are hsa_circ₀106982,hsa circ 0000777,Hsa-circRNA3410-17 in TypeⅡ cardio-renal syndrome with yin and yang deficiency,blood stasis with phlegm syndrome.