Study On The Regulation Network Of CircRNA-miRNA-mRNA Related To Ischemic Stroke

Objective:IS is a very common cerebrovascular disease with high mortality and disability rate.Most of the studies used high-throughput sequencing technique to explore the association between single type of RNA and IS,the more widely studied RNA involve lncRNA,miRNA and mRNA,but the relationship between circRNA and IS was not clear.In addition,the study of single type of RNA may lead to one-sided results.Therefore,based on high-throughput sequencing technique,this study explored the differential expression of circRNA,miRNA and mRNA in 3 IS patients and 3 healthy controls,then according to the regulatory and regulated relationship among the three kinds of RNA,combined with the ceRNA theory,we further constructed the circRNA-miRNA-mRNA regulatory network,so as to understand whether circRNA was associated with human IS as ceRNA,as well as the molecular mechanism and regulatory mode involved in the pathogenesis of IS.Methods:From July to December 2017,three patients with IS who were first diagnosed in the second Clinical Hospital of Jilin University and three controls who were treated in orthopedics?except for without IS,other related features were balanced with IS patients in the control group?were involved in this study.Then collected information of age,behavioral characteristics?smoking,drinking?and the history of related chronic diseases.And the fasting venous blood 3ml of the study subjects was retained in the early morning of the next day in hospital in accordance with the relevant requirements of this study.Then based on the high-throughput sequencing technique,the differential expression of RNA was screened by DEseq based on negative binomial distribution.All samples were clustered into ischemic stroke group?A1,A2,A3?and control group?D1,D2,D3?by hierarchical clustering analysis.Based on the principle of ceRNA,the circRNA-ceRNA regulatory network was constructed according to the regulation and regulated relationship between circRNA,miRNA and mRNA and the expression of three kinds of RNA sequencing,the regulation network was visualized with cytoscape3.7.0 software.The GO and KEGG functions and signal pathway enrichment of mRNA in the network were analyzed.Then,according to the frequency of gene appearing in each pathway,the key gene which may be closely related to IS was screened out,and the miRNA and circRNA regulating the key gene were found in the ceRNA regulatory network.Results:1.High throughput sequencing showed that there were 309 differentially expressed circRNA?151 up-regulated and 158 down-regulated?,116 differentially expressed miRNA?60 up-regulated and 56 down-regulated?and 144 differentially expressed mRNA?70 up-regulated and 74 down-regulated?.2.According to the relationship between the regulation and regulation of RNA,the circRNA-miRNA-mRNA regulatory network was established.The network consists of 63 circRNA?33 up-regulated and 30 down-regulated?,8 miRNA?5 up-regulated and 3 down-regulated?and 7 mRNA?3 up-regulated and 4 downregulated?.3.The main pathways of mRNA enrichment in regulatory networks are:Type I diabetes mellitus,CAMs,Intestinal immune network for IgA production and IBD and so on.4.ENSG00000179344 is the highest frequency of gene on every enrichment pathway,the circRNAs which can be as ceRNA involved in the regulation of ENSG00000179344 in the network were: hsa_circ₀001910?hsa_circ₀003039?hsa_circ₀007360?hsa_circ₀000175?hsa_circ₀000042?novel_circ₀025531?novel_circ₀024610?novel_circ₀014500?hsa_circ₀011173?hsa_circ₀011174?hsa-circ-0006140;And the miRNAs were hsa-miR-45-5P and hsa-miR-4672.Conclusion:1.circRNAs?hsa_circ₀001910 ? hsa_circ₀003039 ? hsa_circ₀007360 ?hsa_circ₀000175?hsa_circ₀000042?novel_circ₀025531?novel_circ₀024610?novel_circ₀014500?hsa_circ₀011173?hsa_circ₀011174 and hsa-circ-0006140?may be associated with IS by regulating miRNA?hsa-miR-45-5P and hsa-miR-4672?as ceRNA,and have indirect effect on the gene of HLA-DQB1.These RNAs may be related with IS.2.Type I diabetes mellitus,CAMs,IBD and Intestinal immune network for IgA production pathways may be related to IS.