The Expression Profile Of Circ RNA After Intracerebral Hemorrhage In Rats

Objective:The circular RNA(circ RNA)expression profile of brain tissue after intracerebral hemorrhage(ICH)in rats was screened by high-throughput sequencing technology,and then the differentially expressed circ RNA was verified.Bioinformatics analysis was conducted to identify the function of the altered circ RNA after ICH,exploring the key regulatory targets of the molecular biological mechanism in the pathological process of intracerebral hemorrhage from the perspective of transcriptomics,providing a new strategy for the treatment of intracerebral hemorrhage.Method:(1)Establishment of an intracerebral hemorrhage(ICH)model: a model of cerebral hemorrhage was established according to the method of Rosenberg et al.,and the caudate putamen was located with reference to Bao Xinmin’s “stereotactic map of rat brain”(B+1.0mm,R3.1mm,H6.0mm),and a total 2ul of the type VII collagenase(0.2 U/ul)was injected in the injection point.(2)Experimental group: 18 male Sprague-Dawley rats were randomly divided into a 24-hour sham operation group,a 24-hour model group and a 48-hour model group,with 6 rats in each group.(3)Material: According to the time point,The perihematoma was obtained freshly and quickly,and was preserved with liquid nitrogen.(4)Circ RNA sequencing: using high-throughput sequencing technology,extracting total RNA from the brain tissue,removing ribosomal RNA(r RNA)linear RNA after quality control,constructing circ RNA sequencing library,and sequencing on Hiseq Xten sequencer.The data was analyzed and processed,and the circ RNA was annotated with circbase and exorbase to screen for circ RNA with significant differences.(5)Circ RNA-Seq data accuracy verification: Four cir RNA were randomly selected from the differentially expressed circ RNA using real-time quantitative PCR(RT-PCR)to verify whether they were consistent with the high-throughput sequencing results.(6)The bioinformatics analysis of the differentially expressed circ RNA: using the Gene ontology(GO)analysis and Kyoto Gene and Genomic Encyclopedia(KEGG)pathway analysis to identify the possible function of differentially expressed circ RNAs;(7)Differential expression of circ RNA and micro RNA(mi RNA)interaction prediction: using Target Scan,mi Randa and RNAhybrid software to explore the Circ RNA-micro RNA interaction prediction,and the network map of interactions of 40 differentially expressed circ RNAs(10 down-regulated and 30 Up-regulated)with the top 5 micro RNAs that may be combined with them were constructed using Cytoscape software.Result:(1)This study successfully constructed a rat model of intracerebral hemorrhage with collagenase injection,and the neurological function score was effective.The gross specimens of the model group were located in the caudate nucleus and the hematoma was obvious.(2)The sample quality test results showed that the sample quality was good,and the sequencing tissue samples were highly correlated.The comparison between the sham group and the model group brain tissue samples showed that there were 13330 circ RNAs were expressed at the detectable level,mostly from exon regions of the genes(13165)in the sample of the sham annimals.Compared with the sham group,346 circ RNAs were differentially expressed after 24 hours of intracerebral hemorrhage,of which 198 were up-regulated and 148 were down-regulated.After 48 hours of intracerebral hemorrhage,389 circ RNAs were differentially expressed,of which 248 were up-regulated and 141 down-regulated.Among the circ RNAs with differential expression,86 circ RNAs were differentially expressed at 24 hours and 48 hours after intracerebral hemorrhage.There was a significant difference in the expression of circ RNAs in the brain tissue of intracerebral hemorrhage group.Differentially expressed circ RNAs are widely distributed on all chromosomes,and most of the differentially expressed circ RNAs are derived from exonics,of which a small fraction is derived from splicing.(3)GO analysis revealed that the biological and molecular functions controlled by the parent genes of circ RNAs differentially expressed after intracerebral hemorrhage are mainly the regulation of biological processes,transportation,the composition of cellular components,the establishment of cellular localization,integration,catalytic activity,nucleotide binding,the combination of protein and small molecular substances,etc.(4)KEGG pathway analysis revealed that the predominantly enriched pathways of circ RNAs differentially expressed 24 hours after intracerebral hemorrhage were dopaminergic synapses,retrograde endogenous cannabinoid signaling pathway,glutamatergic synapses,ubiquitin-mediated proteolysis Signaling pathway,MAPK signaling pathway,endocytosis,GABAergic synapse,etc.However,after 48 hours of intracerebral hemorrhage,KEGG analysis showed that the most abundant pathway of differentially expressed circ RNAs were retrograde endogenous cannabinoid signaling pathway,suggesting that with the passage of time after brain injury,the retrograde endogenous cannabinoid signaling pathway may play an important regulatory role after brain injury.(5)RT-PCR results showed that the verification of Circ Fhod3 was consistent with high-throughput sequencing result.(6)The circ RNA-micro RNA network map shows that the verified circ RNA(rno_circ: chr18: 17224244-17264715)Circ Fhod3 binds to mi R-211,and studies have shown that mi R-211 is involved in the regulation of apoptosis as a "pro-survival micro RNA",which attenuates the stress-dependent expression of the proapoptotic transcription factor chop/gadd153.Mi R-211 directly targets the proximal chop/gadd153 promoter,increasing histone methylation and inhibiting chop expression,playing a role in inhibiting apoptosis.(7)The mi RNA-m RNA network map shows that mi RNA-211 can bind with a large number of m RNA,it may involve in the regulation of inflammation,oxidative stress and autophagy,affecting the pathophysiological process after intracerebral hemorrhage.Conclusion:(1)The expression of circ RNAs in rats’ brain tissue after intracerebral hemorrhage was significantly different,suggesting that differentially expressed circ RNAs is involved in specific pathological processes after cerebral hemorrhage and affects neurological function.(2)Bioinformatics analysis suggests that the interaction between circ RNAs differentially expressed and mi RNAs,mi RNAs and m RNA may be one of the pathophysiological mechanisms of brain injury after intracerebral hemorrhage.(3)Circ Fhod3 expression was significantly down-regulated after intracerebral hemorrhage,suggesting that it may be a key molecular target in the pathological process after intracerebral hemorrhage.