Differential Expression Analysis Of Brain Striatum In Manganism Rats

?Objective?To observe the changes in gene expression profile of rat striatum after manganese exposure,explore the molecular mechanism of manganese-induced dopaminergic neuronal damage,further analyze the gene targets of manganism and seek biological treatment of it.?Methods?1.This study used 25mg/kg MnCl₂·?4H₂O,0.2ml/100g for intraperitoneal injection,once every 48h,after 1 month of manganese in SD rats,the rat striatum was taken on ice and immediately poured into liquid nitrogen.The striatum RNA was extracted and total RNA concentration,RNA integrity number?RIN?28S/18S value and fragment size were detected to establish a gene library.2.The gene library was detected by Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System.The range of inserts of the library was detected by Agilent 2100Bioanalyzer,and the concentration of the library was quantified by ABI StepOnePlus Real-Time PCR System.At the same time,the data is filtered to remove joint contamination,unknown base too high and low quality readings to obtain clean reads.3.Use HISAT to compare clean reads to the reference genome sequence.After alignment with the reference genome,each sample was transcribed using StringTie,and then the reconstructed transcript was compared to the reference annotation information using Cuffcompare,the new transcript was selected,and the new transcript was encoded with CPC software.It is predicted that a new transcript with predicted protein coding potential will be finally added to the reference gene sequence to obtain a complete reference sequence information,which is subsequently analyzed based on this reference sequence.4.Using the two algorithms of DEseq2 and PossionDis for differential gene detection,screening for genes with FDR?0.001 and multiple differences of more than 2 times,defined as differentially expressed genes.Functional analysis of differentially expressed genes was further carried out using GO function and KEGG database,and the interaction gene of differentially expressed genes was screened by String database and correlated analysis was performed.5.RT-PCR was performed on the selected differentially expressed genes and their interaction genes.The differences between whole genome analysis and PCR validation were compared,and the possible molecular regulation mechanisms of differentially expressed genes were analyzed.?Results?1.Quality testing of each sample before sequencing.The concentration of each sample in this study was 50 ng/?l,RIN value was greater than 8.0,28S/18S was greater than 1.3,total RNA was sufficient,no DNA,protein/salt Ion and other pollution,the sample is colorless,transparent and non-viscous,indicating that the sample meets the sequencing quality detection standards,and a gene library can be established.2.Detected using the Illumina HiSeq platform,generating an average of 6.72 Gb of data per sample.The average alignment ratio of the sample to the genome was 88.12%,the value of Clean Reads Q20?%?of each sample in the gene library was greater than 98%,and the value of Clean Reads Q30?%?was greater than 95%,indicating that the sequencing quality was good.3.A total of 17 differentially expressed genes were screened out,7 of which were significantly down-regulated,10 were significantly up-regulated,and only 5 of the differential genes were known genes:Sgk1,Pnpla1,Grm2,HCRTr1,RT1-CE16,other genes are unknown new genes.GO functional enrichment analysis revealed that the differentially expressed genes were highly enriched in the behavioral function?P=0.02281?.Pathway functional enrichment found that differential genes are enriched in neuronal ligand-receptor interactions,glutamatergic metabolism and other neurotransmitters,as well as the FoxO signaling pathway,PI3K-Akt signaling pathway,mTOR signaling pathway and other apoptotic pathways.4.According to gene function and interaction effect,five genes of HspB1,Rem2,Oprd1,ATF5 and TRHr may be involved in the pathogenesis of manganese poisoning.The interaction scores of the above genes and their interaction genes are:726,907,231 191,900.RT-PCR verification experiments were consistent with gene sequencing results,but there was no significant difference between Sgk1,Pnpla1,TF5 and TRHr?P>0.05?.5.Correlation analysis between differential gene and interaction gene revealed that Rem2 and TRHr were significantly correlated with HCRTr1,and Rem2 was negatively correlated with HCRTr1?r=-0.782,P<0.05?.TRHr was positively correlated with HCRTr1?r=0.707?,P<0.05),there was also a negative correlation between Rem2 and TRHr?r=-0.651,P<0.05?.There was a negative correlation between RT1-CE16 and Oprid1?r=-0.502,P<0.05?.There was no correlation between the two interaction genes HspB1 and ATF5 of the unknown gene novel_G001016.?Conclusions?1.The 17 differential genes were screened,and the gene function was determined by GO analysis and Pathway function analysis.The differential genes involved PI3K-Akt,mTOR,Apoptosis-related pathways such as FoxO,glutamate metabolism,and neuroactive receptor-ligand interactions.2.Using the String database to observe the interaction genes of differential genes,it is speculated that HspB1,Rem2,Oprd1,ATF5 and TRHr genes may be involved in the pathogenesis of manganese poisoning.3.The whole genome sequencing and RT-PCR verification results are basically the same,but the results of individual gene verification are not statistically different.Considering the individual differences of the samples,it is necessary to repeat the experimental verification.Screening differentially expressed genes of manganese poisoning requires further functional experiments to determine its expression regulation mechanism and its effect on manganese poisoning.