| Part I Dynamic changes of peripheral blood mi R-124-3p expression in patients with acute cerebral infarction before and after intravenous thrombolysis of rt-PAObjective:Mi R-124-3p is the most abundant and specifically mi RNA in brain tissue,which is involved in the pathophysiological process of the occurrence and development of acute cerebral infarction(ACI).It was reported that the expression profile of mi RNA in peripheral blood of patients with CI changed after intravenous thrombolysis with recombinant tissue plasminogen activator(rt-PA).However,the expression of peripheral blood mi R-124-3p before and after intravenous thrombolysis of rt-PA in ACI patients and its clinical significance are unclear.The purpose of this study was to observe the dynamical change of mi R-124-3p in of ACI patients before and after intravenous thrombolysis with rt-PA,to explore the relationship between the expression of mi R-124-3p and the severity of neurological deficit and prognosis,and to analyze the potential value of mi R-124-3p as a biomarker of prognosis after intravenous thrombolysis.Methods:From January 2019 to January 2020,ACI patients who were admitted to the department of neurology and received intravenous thrombolysis of rt-PA and completed follow-up were included and served as CI group(CI group).Healthy subjects who were matched with age,sex and cerebrovascular risk factors in the physical examination center in the same period were selected as the control group.The expression of mi R-124-3p in of ACI patients before intravenous thrombolysis,6 hours after intravenous thrombolysis,24 hours after intravenous thrombolysis and 7 days or before discharge after intravenous thrombolysis was detect by real-time reverse transcription polymerase chain reaction(q RT-PCR).The expression level of mi R-124-3p in control group was only needed to detect once.The NIHSS scores of ACI patients were evaluated before blood sampling.The infarct volume of ACI patients at 24 hours after thrombolysis was calculated,and the clinical data of patients and m RS score at 90 days after onset were collected.Repeated measurement variance test was used to analyze the changes of mi R-124-3p expression level and NIHSS score by time in CI group.The correlation between mi R-124-3p and severity of neurological impairment was used to analyze by spearman correlation analysis.The relative operating characteristic curve(ROC)was used to evaluate the prognostic value of mi R-124-3p expression level in patients with CI.Result:In this study,35 patients with CI were included,including 25 males and10 females.There was no significant difference in gender,cerebrovascular risk factors and age between the CI group and control group(P>0.05).Compared with NIHSS score on admission,the NIHSS score of CI patients gradually decreased at 6 hours,24 hours and 7 days after intravenous thrombolysis[9(5,13)vs 4(3,9)vs 4(2,7)vs 4(2,9),P<0.05].Compared with control group,the expression of mi R-124-3p in CI group increased significantly before intravenous thrombolysis,and decreased gradually at 6 hours,24 hours,and 7 days after intravenous thrombolysis,but still significantly higher than that in the control group(P<0.001).Compared with before thrombolysis,the expression of mi R-124-3p decreased significantly 7 days after thrombolysis(P=0.0002).24 hours after thrombolysis,the level of peripheral blood mi R-124-3p in patients with infarct volume>10cm~3(n=8)was significantly higher than that in patients with infarct volume≤10cm~3(n=27).Correlation analysis showed that 24 hours after thrombolysis,the level of mi R-124-3p in peripheral blood was positively correlated with infarction volume(r=0.574,P<0.05).Most of the patients with CI had a good prognosis after thrombolysis(m RS score≤2 on 90 days).ROC analysis showed that increased mi R-124-3p levels before thrombolysis and at three time points after thrombolysis showed good diagnostic accuracy in identifying the presence of poor prognosis,and the areas under the curve were0.708,0.719,0.746,and 0.714,respectively(P<0.05).Conclusion:The expression of mi R-124-3 in peripheral blood of ACI patients increased before thrombolysis and then decreased gradually after thrombolysis.The level of mi R-124-3p in peripheral blood at 24 hours after thrombolysis was positively correlated with the volume infarct,which could reflect the severity of neurological impairment in ACI to some extent.The increased level of mi R-124-3p in peripheral blood may be a biomarker of poor prognosis of intravenous thrombolysis in patients with ACI.Part II Prediction of mi R-124-3p target gene and verification of its expression changes in patients with acute cerebral infarction before and after intravenous thrombolysis in rt-PAObjective: In this study,bioinformatics method was used to predict the possible target genes of hs-mi R-124-3p.q RT-PCR was used to verify the dynamic changes of the target genes before and after intravenous thrombolysis in patients with ACI.Combined with functional enrichment analysis,the possible mechanism of the target genes screened in the occurrence and development of CI was preliminarily explored.Methods: Firstly,12 authoritative mi RNA prediction platforms were used to predict the target genes of has-mi R-124-3p.The target genes intersected in at least four mi RNA prediction platforms were selected as potential target genes.The differentially expressed genes were screened from data set of CI in GEO database.Intersection analysis was conducted between target genes predicted by authoritative mi RNA prediction software and differentially expressed genes in GEO database.Protein interaction network analysis and functional enrichment analysis using overlapping genes,and to identify the hug target gene of mi R-124-3p.The hug target genes were then input into mi RDB,mi RNAs were predicted backwards,and the predicted m RNAs were screened.Secondly,q RT-PCR was used to detect the expression of target m RNA in different time periods of thrombolysis in patients with ACI to further determine the targeted relationship between mi R-124-3p and predicted target gene.Results: Using 12 authoritative miRNA prediction software and GSE58294 data sets,combined with Target Scan and Pub Med,it was predicted that the target m RNA of mi R-124-3p was silent information regulator1(SIRT1).Input SIRT1 into mi RDB software for reverse verification,and SIRT1 was identified as one of the targets of mi R-124-3p.The q RT-PCR results showed that the relative expression levels of SIRT1 m RNA in peripheral blood of patients with ACI before thrombolysis,6 hours,24 hours and 7 days after thrombolysis were 1.34(1.13,1.60),2.48(1.60,2.76),1.82(1.39,2.82),0.07(0.03,0.20).Repeated ANOVA analysis showed that the change of SIRT1 m RNA expression was opposite to the trend of mi R-124-3p expression during the corresponding period of time,showing a negative regulatory relationship.Functional enrichment analysis showed that the target genes predicted by mi R-124-3p were enriched in signal pathways such as "cytokine receptor interaction","stimulation of nerve tissue interaction" and "regulation of oxidation",among which FOXO signal pathway SIRT1 is one of the important molecular pathways.Conclusion: In this part,bioinformatics analysis and qRT-PCR results show that SIRT1 is one of the target genes of mi R-124-3p,and the role of mi R-124-3p targeted regulation of SIRT1 in the occurrence and development of ACI and its specific molecular mechanism need to be further studied. |
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