MicroRNAs Expression Profile In Peripheral Blood Of Children With Drug-resistant Epilepsy

objective:This study airns to use high-throughput sequencing and quantitative reverse transcriptase polymerase chain reaction(qRT-PCR)assays to analyze microRNAs(miRNAs)expression profile in the peripheral whole blood of children with drug-resistant epilepsy,and to screen the specifically differential expression miRNAs,in the hope of finding potential diagnostic biomarkers for early identification of children with drug-resistant epilepsy.By using bioinformatics analysis methods to predict the target genes of differentially expressed miRNAs and perform the gene classification,molecular functions and involvement pathways of candidate target genes,further to analyze the possible role of miRNAs in the mechanism of drug resistance in children with epilepsy,hoping to provide clues for the exploration of new and effective therapeutic targets for children with drug-resistant epilepsy.Methods:1.Children with epilepsy who had definite diagnosis and accepted the antiepileptic drugs therapy for at least 1 years,from January 2018 to June 2019 in the outpatient department of Pediatric neurology of the First Affiliated Hospital of Zhengzhou University were chose as case group.According to the definition of drug-resistant epilepsy defined by the International League Against Epilepsy in 2010 and epileptic symptom control situation,children with epilepsy were divided into drug-resistant epilepsy group(R group),drug-reactive epilepsy group(F group).The healthy children undergoing physical examination in the same hospital in the same time were chose as control group(J group).2.2.5 ml empty stomach peripheral whole lood early in the morning was collected from children in case group and control group,next total RNA of each sample was extracted and reverse-transcribed to cDNA.After PCR amplification,Illumina NovaSeq6000 platform was used for high-throughput sequencing and analysis of miRNAs expression profiles in peripheral whole blood of children with drug-reactive epilepsy and drug-refractory epilepsy.3.Analysis of the significantly differential expression of miRNAs among groups was performed by using the DESeq software,then the candidate miRNAs were screened out.The expression levels of candidate miRNAs in whole blood samples was verified by qRT-PCR amplification,so as to further obtain the target miRNAs.4.The target genes of target miRNAs were predicted and screened by using online databases such as miRWalk?TargetScan?miRDB?miRTarBase.Gene Ontogy(GO)categories and functional association analysis and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis of Candidate target genes were performed by using DAVID online database.Results:1.During the high-throughput sequencing,5 cases were included in each of the 3 groups.During the PCR amplification,the drug-resistant epilepsy group,drug-reactive epilepsy group and control group have respectively 5 cases,7 cases and 6 cases.There were no statistically significant differences of age and sex between children with epilepsy and healthy children in the two study stages.2.Analysis of differential miRNAs in peripheral whole blood of children with drug-resistant epilepsy:Compared with the control group,there were 95 differentially expressed miRNAs in drug-resistant epilepsy group,of which 70 miRNAs upregulated and 25 miRNAs downregulated.Compared with the drug-reactive epilepsy,there were 68 differentially expressed miRNAs in drug-resistant epilepsy group,of which 22 miRNAs upregulated and 46 miRNAs downregulated.3.Analysis of differential miRNAs in peripheral whole blood of children with drug-reactive epilepsy:Compared with the control group,there were 82 differentially expressed miRNAs in drug-reactive epilepsy group,of which 71 miRNAs upregulated and 11 miRNAs downregulated.4.qRT-PCR amplification Validation:11 candidate miRNAs,with down-regulated expression:Let-7a?let-7f?miR-15a-5p?miR-1299,and up-regulated expression:miR-19b-5p?miR-99a-5p?miR-99b-5p?miR-125a-5p?miR-125b-5p?miR-142-5p?miR-100,were verified by qRT-PCR amplification,and the results showed that the variation trends of expression levels of let-7f?miR-99a-5p?miR-99b-5p?miR-125a-5p?miR-125b-5p?miR-142-5p?miR-100 were consistent with the results of high-throughput sequencing.5.ROC curve analysis:Results showed that when the cut off value of miR-99a-5p?miR-99b-5p?miR-125b-5p?miR-142-5p?miR-100 was greater than 0.56,0.62,5.53,2.24,2.09 and 0.59,respectively,these miRNAs were expected to be biological biomarkers for the diagnosis of drug-resistant epilepsy with high AUC(?0.871),sensitivity(?80%)and specificity(?85.7%).Among them,miR-99a-5p(AUC=0.971)and miR-125b-5p(AUC=0.971)were the best for the diagnosis of drug-resistant epilepsy.6.Prediction and KEGG and GO analysis of candidate target genes:The number of candidate target genes for seven target miRNAs was respectively 532,73,45,590,681,67 and 93,and number of intersection target genes predicted by using four online databases was respectively 81,8,3,58,81,4 and 2.KEGG analysis of the candidate target genes showed that the candidate target genes may be involved in pathways including:AMPK signaling pathways,FoxO signaling pathways,HIF-1 signaling pathways,MAPK signaling pathway,Sphingolipid signaling pathway,p53 signaling pathway,Neurotrophin signaling pathway,mTOR signaling pathways,Jak-STAT signaling pathway,PI3K-Akt signaling pathway,Glioma,TGF-beta signaling pathways.GO analysis showed that the candidate target genes were mainly enriched in post-embryonic development,regulation of transcription and protein autophosphorylation in biological process;mainly enriched in nucleoplasm,cytosol,membrane,cytoplasm and nucleus in cellular components and mainly enriched in protein binding,mRNA 3'-UTR binding,RNA polymerase ? core promoter proximal region sequence-specific DNA binding,protein serine/threonine kinase activity,protein kinase activity in molecular function.Conclusions:1.This study shows that significant differences of miRNAs in peripheral whole blood between children with epilepsy and healthy children,and between children with drug-reactive epilepsy and children with drug-resistant epilepsy,suggests that seizures and responses to treatment could affect the expression of miRNAs in peripheral whole blood in children with epilepsy.2.This study screens 6 differentially expressed miRNAs from the drug-resistant epilepsy group:miR-99a-5p?miR-99b-5p?miR-125a-5p?miR-125b-5p?miR-142-5p?miR-100.Among them,miR-99a-5p?miR-125b-5p may be expected to be one of potential biomarkers for the diagnosis of drug-resistant epilepsy.3.This study suggests that mir-99a and mir-125b may be one of therapeutic targets for drug-resistant epilepsy,and five pathways regulated by candidate target genes of mir-125b:HIF-1 signaling pathways,MAPK signaling pathway,Sphingolipid signaling pathway,Neurotrophin signaling pathway,mTOR signaling pathways,may be related to the mechanism of drug resistance in drug-resistant epilepsy.