The Association Of CircRNA And LncRNA In Peripheral Blood With Qi Deficiency And Blood Stasis Syndrome In Ischemic Stroke

Objective Traditional Chinese medicine（TCM）syndrome differentiation plays an important role in the diagnosis and treatment of Ischemic Stroke（IS）,which severely affects the disease burden.However,there is still a lack of objective basis for syndrome differentiation in traditional Chinese medicine.The exploration of objective diagnostic biomarkers is conducive to the objectification and standardization of syndrome differentiation in traditional Chinese medicine and the promotion of accurate syndrome differentiation and treatment in traditional Chinese medicine.This study aims to identify circular RNA（circRNA）and long non-coding RNA（lncRNA）that may affect the occurrence of qi deficiency and blood stasis syndrome in ischemic stroke,and build a non-coding RNA diagnostic model of qi deficiency and blood stasis syndrome in IS through machine learning method.And explore the relationship between these circRNAs,lncRNAs and the clinicopathological parameters of IS syndrome of Qi deficiency and blood stasis,so as to provide potential biomarkers for TCM syndrome differentiation of ischemic stroke,and also provide basis for further elucidate the potential pathological mechanism of TCM syndrome type of ischemic stroke.Methods 1.Non-coding RNA identification related to the occurrence of qi deficiency and blood stasis syndrome in ischemic stroke;Firstly,in 30 cases of IS with wind phlegm and blood stasis syndrome,30 cases of IS with qi-deficiency and blood-stasis syndrome,and 30 healthy controls,5 circRNAs（hsa＿circRNA＿101693,hsa＿circRNA＿102334,hsa＿circRNA＿065793,hsa＿circRNA＿000535,hsa＿circRNA＿000581）and 5 lncRNAs（LINC00928,RP11-524O1.4,ENST00000467369,RP11-386M24.6,XLOC＿013852）were determined by real-time quantitative PCR（qRT-PCR）,which were used to identify non-coding RNA molecules that were related to the occurrence of IS syndrome of qi deficiency and blood stasis,but not related to IS syndrome of wind phlegm and blood stasis.Then,the sample size was expanded,and the expression levels of non-coding RNA molecules related to the syndrome of qi deficiency and blood stasis in IS identified in the previous study were measured by qRT-PCR in180 cases of IS and 180 cases of healthy control population.2.Correlation analysis between non-coding RNA and clinicopathological parameters of Qi-deficiency and blood-stasis syndrome in ischemic stroke:The correlation between differentially expressed circRNAs and lncRNAs and clinicopathological parameters such as blood pressure,blood glucose,blood lipid and coagulation function in IS patients with Qi-deficiency and blood-stasis syndrome was analyzed.3.Construction of non-coding RNA diagnostic model for the syndrome of qi deficiency and blood stasis in ischemic stroke:Five machine learning methods,including artificial neural network model,decision tree model,support vector machine model,XGBoost model and random forest model,were used to construct the non-coding RNA diagnostic model of IS qi deficiency and blood stasis syndrome.150 cases of IS syndrome of qi deficiency and blood stasis and150 healthy control samples were taken as the training set,and 30 cases of IS syndrome of qi deficiency and blood stasis and 30 healthy control samples were taken as the validation set to determine the best diagnostic model.Results 1.Non-coding RNA identification of the occurrence of qi deficiency and blood stasis syndrome in ischemic stroke（1）Among 30 IS patients with qi deficiency and blood stasis syndrome and30 healthy controls,the expression level of hsa＿circRNA＿000535（P=0.046）,hsa＿circRNA＿000581（P=0.002）,RP11-386M22.4（P=0.003）and XLOC＿013852(P=4.084×10^-5)in peripheral blood was statistically significant between the patients with Qi-deficiency and blood-stasis syndrome and the control group.However,the expression levels of hsa＿circRNA＿101693,hsa＿circRNA＿102334,hsa＿circRNA＿065793,LINC00928,RP11-524O1.4 and ENST00000467369 in peripheral blood showed no statistically significant difference between the IS patients with qi deficiency and blood stasis syndrome and the healthy controls（P>0.05）.In addition,there was no significant difference in the expression levels of 10 non-coding RNAs in peripheral blood between ischemic stroke patients with wind phlegm and blood stasis syndrome and healthy controls（P>0.05 for all）.（2）Large sample verification of non-coding RNA related to qi deficiency and blood stasis syndrome in ischemic stroke:The expression levels of hsa＿circRNA＿000535（P=0.004）,hsa＿circRNA＿000581（P=3.556×10-7）,lncRNA RP11-386M24.6（P=0.032）and lncRNA XLOC＿013852(P=7.691×10^-5)in peripheral blood showed statistically significant differences between the case group and the control group.2.Correlation analysis of non-coding RNA and clinicopathological parameters of ischemic stroke with Qi deficiency and blood stasis syndrome（1）Correlation analysis of non-coding RNA and coagulation indexes of patients with Qi deficiency and blood stasis syndrome in ischemic strokeThe expression levels of hsa＿circRNA＿000581（P=0.016）and lncRNA XLOC＿013852（P=0.039）in peripheral blood of IS patients with abnormal platelet（PLA）syndrome of Qi deficiency and blood stasis were significantly lower than those of IS patients with normal PLA.The expression level of Hsa＿circRNA＿000581 in peripheral blood was significantly negatively correlated with the level of fibrinogen（FIB）in patients with IS Qi deficiency and blood stasis syndrome（rs=-0.162,P=0.043）.Peripheral blood expression level of lncRNA RP11-386M24.6 to International normalized ratio（INR）（rs=0.227,P=0.004）,prothrombin time（PT）（rs=0.213,P=0.007）were significantly positive correlation.Moreover,the prothrombin time activity（PTA）of IS patients with Qi deficiency and blood stasis syndrome in the RP11-386M24.6 low expression group was significantly higher than that of IS patients with RP11-386M24.6 high expression of Qi deficiency and blood stasis syndrome（t=3.15,P=0.002）.（2）Correlation analysis of non-coding RNA and blood lipid levels in patients with Qi deficiency and blood stasis syndrome in ischemic strokeThe expression level of lncRNA RP11-386M24.6 in peripheral blood of patients with Qi deficiency and blood stasis syndrome in high triglyceride（Triglyceride,TG）was significantly lower than that of patients with Qi deficiency and blood stasis syndrome in normal triglycerides（P=0.011）.（3）Correlation analysis of non-coding RNA and blood uric acid level in patients with Qi deficiency and blood stasis syndrome in ischemic strokeThe expression level of lncRNA XLOC＿013852 in peripheral blood was significantly negatively correlated with the blood uric acid（UA）level of patients with IS Qi deficiency and blood stasis syndrome（r_s=-0.169,P=0.037）.The blood uric acid level of IS patients with Qi deficiency and blood stasis syndrome with low expression of XLOC＿013852 was significantly higher than that of XLOC＿013852 high expression group（P=0.043）.（4）Correlation analysis of non-coding RNA and the degree of neurological deficit in patients with ischemic stroke with Qi deficiency and blood stasis syndromeIn patients with Neurological deficits within 3 days,the expression level of peripheral blood lncRNA RP11-386M24.6 in patients with moderate to severe IS Qi deficiency and blood stasis syndrome was significantly lower than that of patients with mild neurological deficits,and the difference was statistically significant（P=0.041）.3.Construction of a non-coding RNA diagnostic model for ischemic stroke with Qi deficiency and blood stasis syndrome（1）The diagnostic value of a single non-coding RNA in the diagnosis of ischemic stroke syndrome of Qi deficiency and blood stasis:In the training sample,the AUC（95%CI）,sensitivity and specificity of hsa＿circRNA＿000535 are 0.578（51.30%～64.30%）and 0.793,respectively And 0.380;the AUC（95%CI）,sensitivity and specificity of hsa＿circRNA＿000581 are 0.637（57.30%～70.00%）,0.627 and 0.673,respectively;the AUC（95%CI）,sensitivity and specificity of lncRNA RP11-386M24.6 The degrees were 0.504（43.80%～57.00%）,0.400 and0.687;the AUC（95%CI）,sensitivity and specificity of lncRNA XLOC＿013852were 0.583（51.80%～64.80%）,0.533 and 0.633,respectively.In the verification sample,the AUC value（95%CI）of hsa＿circRNA＿000535 is 0.650（50.9%-79.1%）,the sensitivity and specificity are 0.467 and 0.867,respectively;the AUC value（95%CI）of hsa＿circRNA＿000581 is 0.736（60.7%-86.4）%),sensitivity and specificity are 0.700 and 0.767 respectively;the AUC value（95%CI）of lncRNA RP11-386M24.6 is 0.720（57.6%-86.4%）,the sensitivity and specificity are 1.000 and 0.667 respectively;lncRNA The AUC value（95%CI）of XLOC＿013852 is 0.809（70.3%-91.5%）,the sensitivity and specificity are 0.800and 0.667,respectively.（2）Construction of a non-coding RNA multi-factor diagnostic model for ischemic stroke with Qi deficiency and blood stasis syndrome①Construct a diagnostic model with training samples:In the training samples,the best diagnostic model fitted by the artificial neural network model is the combination"hsa＿circRNA＿000581+lncRNA XLOC＿013852",with AUC（95%CI）,sensitivity and specificity of 0.647（53.53%）.～75.92%),0.627 and0.735.The best diagnostic model fitted by the decision tree model is the combination"hsa＿circRNA＿000581+lncRNAXLOC＿013852+hsa＿circRNA＿000535",with AUC（95%CI）,sensitivity and specificity of 0.714（61.46%～81.41%）,0.641 and 0.672,respectively.The best diagnostic model fitted by the support vector machine model is the combination"hsa＿circRNA＿000581+lncRNA XLOC＿013852",with AUC（95%CI）,sensitivity and specificity of0.701（59.40%～80.72%）,0.741 and 0.674,respectively.The best diagnostic model fitted by the XGBoost model is the combination"hsa＿circRNA＿000581+lncRNA XLOC＿013852",with AUC（95%CI）,sensitivity and specificity of0.717（60.94%～82.51%）,0.721 and 0.702,respectively.The best diagnostic model fitted by the random forest model is the combination"hsa＿circRNA＿000581+lncRNA XLOC＿013852+hsa＿circRNA＿000535+lncRNA RP11-386M24.6",AUC（95%CI）,sensitivity and specificity are 0.597（50.06%～69.33%）,0.527 and 0.667.②Verify the diagnostic model with the verification sample:In the verification sample,the AUC（95%CI）,sensitivity and specificity of the combination of"hsa＿circRNA＿000581+lncRNA XLOC＿013852"under the support vector machine model are 0.927（81.75%～100%）and 0.750,respectively And 1.000.The AUC（95%CI）,sensitivity and specificity of the combination of"hsa＿circRNA＿000581+lncRNA XLOC＿013852"under the XGBoost model are0.641（37.92%～90.21%）,0.375 and 0.833,respectively.Conclusion 1.Identify 4 non-coding RNAs related to the occurrence of Qi deficiency and blood stasis syndrome in ischemic stroke:hsa＿circRNA＿000535,hsa＿circRNA＿000581,lncRNA XLOC＿013852 and lncRNA RP11-386M24.6.2.Hsa＿circRNA＿000581 may be involved in the coagulation process of ischemic stroke with Qi deficiency and blood stasis syndrome;lncRNA XLOC＿013852 may be related to the uric acid metabolism of ischemic stroke with Qi deficiency and blood stasis syndrome;lncRNA RP11-386M24.6 may be related to ischemic stroke with Qi deficiency and blood The blood coagulation process,blood lipid metabolism and the degree of neurological deficit of blood stasis syndrome are related.3.Clarify that the"has＿circRNA＿000581+lncRNA XLOC＿013852"in support vector machine model has the potential to be used as a diagnostic model for ischemic stroke with Qi deficiency and blood stasis syndrome.