Validation And Bioinformatic Analysis Of Propofol-induced Differentially Expressed MicroRNAs In Primary Cultured Neural Stem Cells

Every year,thousands of preterm and newborn infants experienced general anaesthesia.Long-term effect of general anesthesia on the developing brain is a widely discussed and controversial issue,it is also one of the hot topics in anesthesiology,neurobiology and pharmacology.Almost 30 years ago,the American Academy of Pediatrics Committee on Fetus and Newborn publicated a joint policy statement strongly advocating for the use of anaesthesia in all neonates.While the US Food and Drug Administration(FDA)alert in December 2016 on the safety of general anesthesia and sedations in patients less than 3 years of age and pregnant women,this statement caused many family members panic.The National Association of anesthesia keep a neutral attitude towards the warning of FDA,until now there is no exact answer to this question.Propofol,an ideal intravenous anesthetic,is widely used in general anesthesia induction and maintenance,intensive care unit(ICU)sedation,and endoscopic procedures,which is mainly due to its highly favorable pharmacokinetics,rapid-onset,short half-life,less accumulation,etc.However,the beneficial or detrimental effect of propofol on central nervous system is still on debate.There were some reviews demonstrating that propofol had neuroprotective activities for its anti-inflammatory effect.In recent years,however,it has been reported that propofol can induce apoptosis of neural stem cells in rat embryos through multiple signaling pathways,reduce the number of neurons and cause abnormal synapse development,thereby affecting learning and memory ability and cognitive function.Therefore,to further explore the mechanism of propofol in the process of neurodevelopment is an important scientific issue.Method1.Drug AssayThe primary culture of rat embryonic neural stem cells(NSCs)was divided into 5 groups.The Null group was without any treatment.The Vehicle group was given DMSO at a constant concentration.Propofol was divided into three groups:25 ?mol/L group,50 ?mol/L group and 100 ?mol/L group.2.Cell Viability Assaypropofol(dissolved in DMSO)of different final concentrations(25,50,100 ?M)was added.The Vehicle and Null group were treated with same volume vehicle(DMSO)or DMEM/F12 respectively.After treated different time(6h,12h,24h),cells were washed by PBS(3x5min)and CCK8 was added into each well.Four hours later,absorbance was measured using microplate reader at 450 nm.Cell viability rate equals to(ODtreatment/ODcontrol)×100%.3.Next Generation SequencingAfter the cell viability assay,the optimum time and concentration of propofol on neural stem cells were screened out.Total RNA of the cells was extracted after drug treatment.cDNA gene bank was obtained by PCR amplified and gel purified.Finally,the libraries were sequenced on an Illumina HiSeqTM 2000 system.qRT-PCR was used to detect differentially expressed microRNAs.4.Prediction of microRNA target geneThe target gene prediction of the verified differentially expressed microRNAs was performed using the miRWalk 2.0 database.we selected four continuously updated microRNA target prediction databases(miRWalk,miRanda,miRDB,TargetScan)to predict the target genes of differentially expressed microRNAs.5.Predicted target genes GO and KEGG enrichment analysisThe GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes,and pathway enrichment analysis of microRNA clusters' predicted target genes were analyzed by DAVID.P value of GO terms and pathways less than 0.05 were considered significantly enriched.6.Quantitative Real-time PCR and Western BlotThe mRNA expression levels of differentially expressed microRNAs and target genes Gabbrl,Cancalb and Cabbr2 were detected by real-time fluorescence quantitative PCR.The protein expression of Gabbrl,Cancalb and Cabbr2 was detected by Western Blot.7.Statistical analysisAll statistical analysis was performed using SPSS 20.0 Statistics,Data were expressed as mean ? SD.edgeR package was used to analyze the differences of microRNA expression,P<0.05 was considered statistical significance.Results1.Effect of Propofol on Survival of NSCsThe results showed that propofol decreased NSCs viability in a dose-dependent manner(compared with Null group and Vehicle group,P<0.05),but not in an obvious exposed time-dependent manner.No statistical difference between Null group and Vehicle group2.Propofol Regulate microRNAs Expression in Rats' NSCsTo identify the propofol induced microRNAs expression change in NSCs,two cDNA libraries were constructed from Vehicle and Propofol group.High-throughput sequencing was then performed on the cDNA libraries.27 microRNAs(10 upregulated and 17 downregulated)with the discrepancy of |log2(foldchange)|?1 were found in the NSCs after 6 h exposed to propofol at 50 ?M/L.27 microRNAs were examined by qRT-PCR.The expression of rno-miR-410-5p,mo-miR-194-3p,mo-miR-3583-3p,rno-miR-466b-5p,rno-miR-143-5p,and rno-miR-377-5p were differently expressed significantly.We classified the up-regulating microRNAs(miR-194-3p,miR-143-5p,miR-377-5p)as cluster A while down-regulating microRNAs(miR-410-5p,miR-466b-5p,miR-3583-3p)as cluster B.3.Prediction of Target GenesThen all the predicted target genes of the two microRNAs' cluster from four databases were intersected to build a set for further analysis.The number of intersected gene set of cluster one is 365,and cluster two is 209,respectively4.GO and KEGG pathway Analysis of Predicted Target GenesUsing the predicted target genes for GO and KEGG enrichment analysis,it was found that Gabbrl,Cancalb and Gabbr2 are highly enriched in the GABAergic synapse pathway.5.Propofol modulate the expression of Gabbrl,Cacnalb and Gabbr2Compared with vehicle group,the expressions of Gabbrl and Cancalb in propofol group were significantly down-regulated and Cabbr2 expression was up-regulated(P<0.05),which is consistent with the regulation of miRNA target genes.ConclusionIn this study,miR sequencing technology was used to analyze and verify the differential expression of six microRNAs in rat embryonic neural stem cells after propofol exposure.The results of the bioinformatics analysis and subsequent verification indicated that the up-regulated miR-377-5p,miR-194-3p,miR-143-5p and down-regulated miR-3583-3p,miR-466b-5p and miR-410-5p could regulate Gabbr1,Cancalb and Gabbr2 in GABAergic signaling pathway respectively.It is suggested that propofol may regulate the apoptosis of NSCs through GABAergic signaling pathway,thereby affecting the neurodevelopment and the ability of learning and memory.Our findings may help elucidate the molecular mechanisms of propofol neurotoxicity and may provide potential therapeutic targets for the neurotoxicity of propofol.