Bioinformatic Analysis Of MiRNA Expression Profile Based On High-throughput Sequencing Of Intracerebral Hemorrhage In Rats

Objective and background:As a common subtype of stroke,intracerebral hemorrhage has high morbidity and mortality.It has always been a public health concern of WHO.How to alleviate the neurological impairment caused by bleeding has also been the focus of the treatment of cerebral hemorrhage.MiRNA is a noncoding small molecule RNA composed of 18-22 bases.It is ubiquitous in plants and animals.Recent studies have found that miRNA participates in many physiological processes after intracerebral hemorrhage.In order to further clarify the regulation mechanism of miRNA after cerebral hemorrhage,this study uses animal and cell model of cerebral hemorrhage to detect the difference of miRNA expression in animal and cell level,and further carry out bioinformatics analysis,establish the protein interaction network in vivo,discuss the mechanism and find the intervention measures.Methods:By injecting autologous arterial blood into the basal ganglia of the rat model of cerebral hemorrhage,SD rats were randomly divided into 2 experimental groups:normal group,ICH = 3,sodium pentobarbital anesthesia,by stereotaxic apparatus(coordinate before Simon: at 0.3cm,forward 0.1cm)base section the caudate nucleus,50 ul injected into the femoral artery blood,collected after rat brain tissue specimens,the expression of miRNA in brain tissue was detected by high-throughput sequencing,establish differential expression degree of screening criteria,screening significant differences in expression of miRNA.After the expression was verified by RT-PCR,the bioinformatics analysis was carried out,and the PPI network analysis was established,and the target gene site was detected by luciferase.Results:(1)616 small miRNA molecules were found in animal models,including 282 miRNA expression,283 up-regulated the expression of miRNA and 30 only havesimple hemorrhage group and sham operation group did not appear in the miRNA,there is only 20 in sham operation group and miRNA did not appear in the hemorrhage tissues.(2)Using foldchange as an index,80 differential miRNAs were screen from the blank group and miRNA expression profiles of intracerebral hemorrhage alone.Compared to the blank group,there are significantly increased in rno-miR-10a-3p and rno-miR-20a-3p.rno-miR-19b-1-5p,rno-miR-489-5p;significantly decrease compared to the blank group: rno-miR-451-3p,rno-miR-223-5p,rno-miR-147,rno-miR-144-5p,rno-miR-10b-5p,rno-miR-21-5.(3)Randomly select 11 miRNA detected by RT-PCR,Delta CT value calculation,and the results of cerebral hemorrhage group was significantly increased,and the sequencing results were consistent(P<0.05).(4)A total of 121 mouse-derived differential miRNA target genes are predicted,and the enrichment pathways:MAPK,Ras,and Cancer,and the protein interaction network PPI is established after intracerebral hemorrhage.(5)Luciferase gene reporter gene: mir-21+DUSP8/mir-21 plasmid transfected into 293 T cells,Luc/renilla ratio results 1:0.77(P<0.05),indicating that miR-21 and DUSP8 have targeted regulatory relationship.Conclusion:1.There were 616 small molecules RNA differential expression in the rat cerebral hemorrhage group and the blank control group.2.Through differentially expressed miRNA,we predicted and analyzed the target gene loci.Through KEGG and GO analysis,we found that the major regulatory pathways were MAPK,cancer,Ras and other signal pathways.3.Construction of biological information network map,miR-21 was one of the key regulatory elements,regulate the target gene loci of DUSP8,FGF18,NFT3,RASGRP and FASLG in MAPK.4.Luciferase gene report showed that there was a target regulation relationship between miR-21 and UDSP8.