Bioinformatics Analysis Of RNA Differential Expression Profiles Of Qi Stagnation And Blood Stasis Syndrome And Potential Diagnostic Biomarker Studies

Background:Syndrome is the basis and target of clinical diagnosis and treatment in Traditional Chinese Medicine?TCM?.It is generally believed that the Syndrome has biological basis.Exploring the biological basis of Syndrome is beneficial to clarify the scientific essence of TCM.For decades of years,researchers have explored the biological basis of Syndrome in many aspects,such as organ,cell and molecule,physiology,biochemistry and ultrastructure.Academician Chen Keji and Shen Ziyin have made significant progress in blood stasis syndrome and kidney yang deficiency syndrome respectively.With the rapid development of modern omics technology,the study of the biological basis of Syndrome has entered into the level of gene,transcription and proteomics.Exploring biomarkers has become the main research direction at present.Biomarkers are widely used in many aspects such as disease prevention,diagnosis,individualized treatment and prognosis judgement in modern medicine.In the field of Syndrome research,it is mainly used for syndrome diagnosis,syndrome typing and study on the target of TCM medicine.The formation and evolution of Syndrome are essentially the result of interaction between genes and environment.The process of gene expression into protein playing biological effects is regulated by many links and multiple factors.RNA is a key link on gene regulation and also a hot spot in biomarker research.Qi stagnation and blood stasis syndrome?QSBSS?is one of the common clinical syndromes in Chinese medicine.It involves 76 diseases,including cardiovascular disease,digestive system disease and gynecological diseases.At present,it has been found that it is related to the pathological changes of microcirculation disorders,hemorheological changes,inflammation,coagulation and fibrinolysis system disorders,endothelial dysfunction,dyslipidemia,immune dysfunction and other pathological changes.In addition,the study of QSBSS also has some progress in the omics research.However,the pathogenesis of QSBSS syndrome is not fully elucidated.There is still lack of biomarkers for clinical diagnosis of QSBSS.Objective:To analyze the RNA differential expression profiles of QSBSS using the bioinformatics methods and screen the target RNA which might be a potential biomarker for QSBSS.Through quantitative real-time fluorescence polymerase chain reaction?qRT-PCR?and diagnostic efficacy analysis,to confirm the potential diagnostic biomarkers of QSBSS which provide an experimental basis for the objective diagnosis of QSBSS,and also provide ideas and methods for the study of the biomarkers of TCM syndrome.Methods:1.Bioinformatic Analysis of RNA Differential Expression Profiles Related to QSBSS Based on LiteratureRetrieving the relevant literature on the study of QSBSS in CNKI and PubMed database,and the results of RNA differential expression were extracted and summarized.GeneCards???:The Human Gene Database was used to annotate the gene function,and The Database for Annotation,Visualization and Integrated Discovery was used for functional enrichment and signal transduction analysis of differentially expressed RNA.The miRNA-target interactions module,miRNA-lncRNA interactions module,miRNA-circRNA interactions module of starBase v2.0and the multiple proteins module in of STRING Version 10.5 were carried out to establish the ceRNA-mRNA-protein interaction network of QSBSS and screen the target RNA which might be a potential biomarker for QSBSS.Then,the results combined with the differentially expressed RNAs of QSBSS which were found in our previous study were prepared to the next experimental verification.2.The Study of Potential Biomarkers on Transcriptome of QSBSS24 cases of QSSBSS patients were enrolled to be divided into 5 groups,5 cases of hypertension,5 cases of coronary heart disease,5 cases of chronic gastritis,5 cases of remission period of cerebral infarction,4 cases of health.The peripheral blood samples of the patients were collected,and the white blood cells were separated and purificated to extract the total RNA by Trizol method.The concentration and integrity of RNA were detected by spectrophotometer and agarose gel electrophoresis.RNA was transcribed to cDNA,and Roche LCPDS2 was used to design specific primers of different RNA for qRT-PCR reaction to calculate the expression level.The statistical significance of differential expression between groups was tested by the independent samples t test and one-way ANOVA analysis.Finally,the receiver operating characteristic?ROC?curve was used to analyze the diagnostic efficacy,sensitivity and specificity of the biomarker.Results:1.Bioinformatic Analysis of RNA Differential Expression Profiles Related to QSBSS Based on Literature?The literature analysis showed that 41 studies reported the results of QSBSS on omics,in which the number of protein expression studies was the most,followed by mRNA,SNP,miRNA,DNA methylation,lncRNA,circRNA.?GO function enrichment showed that?a?in biological process,QSBSS related genes are mainly relevant to cell response to organic cyclic compounds,negative regulation of transcription,negative regulation of neuronal apoptosis gene expression,and inflammatory response,?b?in cellular component,QSBSS related genes are mainly relevant to protein complex,nucleoplasm and cell surface,?c?in molecular function,QSBSS related genes are mainly relevant to identical protein binding,enzyme binding,and transcription regulatory region DNA binding.Among of them,the inflammatory response is the most relevant to QSBSS.?KEGG signal pathway analysis showed that the signaling pathway related to QSBSS related genes included proteoglycans in cancer,TNF signaling pathway,pathways in cancer,human T lymphocyte virus infection.Among of them,the TNF signaling pathway is the most relevant to QSBSS.?According to all the bioinformatics analysis results,TGF-betal,HSP70,CD62p,DES,HLA-DQA1,UCHL5,circRNA09849,circRNA11523,circRNA18046,and circRNA24450 were chosen as the target RNA to be verified in the next study.2.The Study of Potential Biomarkers on Transcriptome of QSBSS?Compared with the two groups of target RNA expression,there was a significant difference in the expression of circRNA₀9849 and UCHL5 between QSBSS group and the healthy control group,p<0.05,and there was no significant difference in the expression of other target RNA between the two groups,p>0.05.?Compared with the target RNA expression among muti-groups,the expression of UCHL5 was significantly different among different groups,p=0.010,and there was no significant difference in the expression of other target RNA among groups,but there was a trend of higher or lower than that of the healthy control group.The results of pairwise comparison showed that there was a significant difference in the expression of UCHL5 between QSBSS groups with different diseases and healthy groups,but there was no significant difference among QSBSS groups with different diseases.It suggested the non-disease specificity of UCHL5.?The expression of UCHL5 decreased by 1.53 folds in the QSBSS patients,which wasthe biological significance.?The analysis of diagnostic efficacy of UCHL5 showed that the area under the ROC curve was 0.900,p=0.01322,suggesting the high diagnostic accuracy,the Jordan index is 0.75,and the corresponding sensitivity is 0.75,the specificity is 1,suggesting the high sensitivity and specificity.Conclusion:The RNAs related to QSBSS connect to multi-functions and multi-pathways.Among of them,the inflammatory response and the tumor necrosis factor signal pathway are the most relevant.UCHL5 is a potential diagnostic biomarker of QSBSS,which has a high diagnostic accuracy,sensitivity and specificity.