A Study On Serum MicroRNAs As Biomarkers Of Post-stroke Cognitive Impairment

Objective:In this study,significantly differentially expressed Serum microRNAs(miRNAs)were collected from three groups of subjects-Post-stroke Cognitive Impairment(PSCI),Post-stroke Cognitive Normality(PSCN),Age-matched Control(AMC)-by using Solexa sequencing and Quantitative Real-time PCR(RT-qPCR).Meanwhile,the cognitive function of the above subjects was evaluated via Montreal Cognitive Assessment(MoCA)in order to explore the correlation between the significantly differentially expressed miRNAs and the risk of cognitive impairment after stroke,and then to determine whether the significantly differentially expressed miRNAs can be an objective basis for early diagnosis and intervention of cognitive impairment after stroke.Methods:In the present study,40 patients were enrolled as subjects in each of the following three groups:PSCI group,PSCN group and AMC group.Subjects from all those three groups were matched in terms of their age,gender,years of education and underlying diseases.And subjects from the PSCI group and the PSCN group were matched in terms of the types of stroke,the focus,the course of disease and the degree of motor dysfunction.In the first stage,five serum samples were selected from the PSCI group,PSCN group and AMC group respectively.All these samples were matched in terms of the subjects' age,gender,years of education and underlying diseases.Meanwhile,samples from the PSCI group and the PSCN group were matched in terms of the subjects' stroke type,focus,course of disease and degree of motor dysfunction.All the selected samples were sent to Guangzhou Qidiao Biological Co.,Ltd.for high-throughput Solexa sequencing,and significantly differentially expressed miRNAs were screened out from the PSCI group,the PSCN group and the AMC group respectively.Second stage:Large-scale and second-stage accurate quantitative verification was carried out based on the results of the high-throughput Solexa sequencing.Quantitative Real-time PCR(qRT-PCR)was used to detect serum miRNAs significantly differentially expressed in the primary screening stage,and the 2-ACt Method was used to measure the expression level of those serum miRNAs.In order to investigate the correlation between significantly differentially expressed microRNAs and cognitive dysfunction after stroke,a correlation analysis was made between the expression level of significantly differentially expressed serum miRNAs and the score that the subjects achieved in the Montreal Cognitive Assessment(MoCA)by using Spearman correlation.And in order to test the predictive value of differentially expressed microRNAs in cognitive dysfunction after stroke,further analyses based on the receiver operating characteristic curve(ROC curve)and the area under the curve(AUC)were carried out in the present study.Results:Through high-throughput Solexa sequencing and qRT-PCR validation,6 serum miRNAs were detected in the PSCI group that were significantly differentially expressed when compared with those in the PSCN group and the AMC group.They were:hsa-miR-191-5p,hsa-miR-20a-5p,hsa-miR-20b-5p,miR-5991-y,hsa-miR-193b-5p and hsa-miR-93-5p.Among them,hsa-miR-191-5p,hsa-miR-20a-5p,hsa-miR-20b-5p,miR-5991-y and hsa,miR-93-5p were significantly up-regulated in the serum of PSCI patients(P<0.05),and hsa-miR-193b-5p was significantly down-regulated in the serum of PSCI patients(P<0.05),which was consistent with the high-throughput Solexa sequencing.Spearman correlation analysis showed that there was a relatively strong positive correlation between the MoCA score and the expression level of hsa-miR-191-5p,hsa-miR-20a-5p,hsa-miR-20b-5p,miR-5991-y and hsa-miR-93-5p(P<0.05),but a relatively strong negative correlation between the MoCA score and the expression level of hsa-miR-193b-5p(P<0.05).Furthermore,the ROC curve was constructed and AUC(95%CI)was obtained.The AUCs of hsa-miR-193b-5p,miR-5991-y,hsa-miR-20a-5p,hsa-miR-93-5p,hsa-miR-20b-5p and hsa-miR-191-5p were 0.905,0.817,0.751,0.741,0.650 and 0.642 respectively(P<0.01).The Sensitivity and Specificity were hsa-miR-193b-5p(100%,85.7%),miR-5991-y(77.5%,80.0%),hsa-miR-20a-5p(63.2%,66,7%),hsa-miR-93-5p(64.1%,62.2%),hsa-miR-20b-5 p(65.0%,57.0%),hsa-miR-191-5p(58.5%,60.5%).Conclusions:1.Through high-throughput Solexa sequencing and qRT-PCR validation,6 significantly differentially expressed serum miRNAs were detected.Among them,hsa-miR-191-5p,hsa-miR-20a-5p,hsa-miR-20b-5p,miR-5991-y and hsa-miR93-5p were significantly up-regulated in the serum of PSCI patients,and hsa-miR-193b-5p was significantly down-regulated in the serum of PSCI patients.2?When the expression level of hsa-miR-191-5p,hsa-miR-20a-5p,hsa-miR-20b-5p,miR-5991-y and hsa-miR-93-5p got higher,or when the expression level of hsa-miR-193b-5p got lower,the riskiness of post-stroke cognitive impairment showed an increase.3?Because it's convenient and noninvasive to detect the serum miRNAs,they have good potential as biomarkers of post-stroke cognitive impairment in its early diagnosis as well as prognosis.