Construction Of CeRNA Network Of LncRNA-miRNA-mRNA In Peripheral Blood Of Patients With Intracerebral Hemorrhage And Its Mechanism

Objective: Intracerebral hemorrhage(ICH)accounts for about 10% to 20% of all strokes.Compared with ischemic stroke,ICH has a higher mortality and disability rate.Patients with ICH have different degrees of dysfunction and neurological complications,resulting in huge economic impact and social burden.At present,the treatment of ICH is limited,and there is no specific therapy for ICH to improve the prognosis.Long non-coding RNA(lncRNA)is a non protein coding transcript longer than 200 nucleotides.It has similar functions with messenger RNA(mRNA)in transcriptional regulation and biogenesis.Most of them cannot be translated into proteins,and some encode small peptides.The number of identified lncRNA is far less than that of genes that encode proteins,namely mRNA,but they have higher specificity in tissue and organ.LncRNA can regulate the expression and function of micro RNA(miRNA)in central nervous system injury.LncRNA can also act as a miRNA sponge,known as competitive endogenous RNA(ceRNA),which can recognize cytoplasmic miRNA and isolate miRNA from mRNA,thus regulating the expression of protein targeted by miRNA.In addition,lncRNA can compete with miRNA in binding to mRNA.At present,our understanding of the mechanism of lncRNA in ICH is still limited.The regulatory network composed of lncRNA,miRNA and mRNA has attracted more attention.In this study,peripheral blood samples from patients with ICH and healthy controls were used for ceRNA microarray.The results were analyzed to try to find the differential mRNAs expression profile and lncRNAs expression profile in ICH compared with healthy controls.Cluster analysis and functional enrichment analysis were carried out.And we constructed a ceRNA regulatory network of lncRNA-miRNA-mRNA,so as to explore the possible regulatory mechanism of non coding RNA in ICH.In addition,we verified some differentially expressed lncRNAs by qRT-PCR to provide a basis for further study of mechanism in ICH,and a possible new target for the treatment of ICH.Research methods: Peripheral blood samples of ICH patients and healthy controls were collected and detected by ceRNA microarray.The original images were processed by feature extraction software(version 12.0.3.1,Agilent Technologies),and the original data were extracted.Genespring software(version 14.8,Agilent Technologies)was used for standardization and follow-up processing.The screening criteria was P < 0.05 and FC ?2.0.The different expression profiles of mRNAs and lncRNAs were analyzed in ICH.Using Graph Pad Prism 8.4.2,metascape,MCODE plug in STRING database and R package clusterprofiler,the differentially expressed mRNAs and genes targeted by lncRNAs were clustered and enriched by their functions and pathways.The sequences of miRNAs in ceRNA network were from the miRBase.Mi Randa and Targetscan were used to determine the interactions between miRNA-mRNA and miRNA-lncRNA.The intersection of the two prediction softwares was taken as the interaction pairs among the three types of RNAs.The ceRNA network of lncRNA-miRNA-mRNA was constructed and visualized by cytoscape 3.8.2.Then,10 differentially expressed lncRNAs were selected and verified by qRT-PCR in peripheral blood samples of ICH patients and healthy controls.The subcellular localization of differentially expressed lncRNAs was predicted by lnc Locator database.In the lncRNA-miRNA-mRNA ceRNA network of ICH,we found certain lncRNA-miRNA-mRNA axes related to immune and inflammation.Results: 1.In comparison of the clinical data between ICH group and controls,the number of white blood cells in the ICH group was significantly increased,and the difference was statistically significant.While,there was no significant difference between the two groups in gender,age,history of hypertension,history of diabetes,history of smoking,history of drinking,and other blood biochemical tests(platelet count,hemoglobin count,coagulation function,blood lipid,homocysteine,blood uric acid,C-reactive protein,procalcitonin,erythrocyte sedimentation rate).2.In comparison of the data of ceRNA microarray in peripheral blood between ICH group and controls,a total of 101 differentially expressed mRNAs were obtained,of which 31 were significantly up-regulated and 70 were significantly down regulated.Gene Ontology(GO),Kyoto Encyclopedia of genes and genome(KEGG),protein-protein interaction(PPI)and gene set enrichment analysis(GSEA)of differentially expressed mRNAs in ICH group were analyzed.The immune system processes,immune and inflammation related pathways were enriched significantly.3.There were 8 immune inflammation related genes in ICH group by crossing with Immport immune database,including UCN,LIF,LEPR,HTR3 E,DEFB121,CMTM8,CD8 B,CD8A.4.In comparison of the data of ceRNA microarray in peripheral blood between ICH group and controls,a total of 211 differentially expressed lncRNAs were obtained,of which 40 were significantly up-regulated and 171 were significantly down regulated.Go and KEGG enrichment analysis of genes targeted by differentially expressed lncRNAs in ICH group showed that immune reaction process and related pathways,signal pathways regulating stem cell pluripotency,lipoic acid metabolism and lysine degradation pathways were significantly enriched.5.After the analysis of ceRNA microarray data in peripheral blood samples of ICH group and controls,the differentially expressed lncRNAs and mRNAs in ICH group were obtained.The miRNAs sequences were obtained from the miRbase,and the interactions between miRNA-mRNA and miRNA-lncRNA pairs were obtained by target gene prediction softwares.Finally,the lncRNA-miRNA-mRNA ceRNA network was constructed.6.Compared with controls,10 differentially expressed lncRNAs were verified by qRT-PCR.LOC101927210,LOC100131626,FAM182 B,LINC00472,RP11-222A5.1,XIST,MOXD2 P were significantly down regulated and HOXB-AS3 was significantly up-regulated in patients with ICH.Although there was no significant difference in the expression of LINC00266-4P and PCBP1-AS1 in patients with ICH,an up-regulated trend was shown.LOC101927210,LOC100131626,RP11-222A5.1 and HOXB-AS3 were mainly located in cytoplasm and might be post transcription regulation.FAM182 B,XIST and MOXD2 P were mainly located in nucleus and might play a role in transcriptional regulation.Linc00472 was mainly located in exosomes,and might be transported to the distal end by exosomes,affecting the function of recipient cells.7.The axes of differentially expressed lncRNAs verified by qRT-PCR in ICH were found in the ceRNA network,including LOC100131626-miR-129-5p-MARC1 axis,FAM182B-miR-128-1-5p/miR-128-2-5p-RPS6KL1 axis,LINC00472-miR-211-5pTMEM237/PRAMEF12 axis,LINC00472-miR-338-3p-SLC2A3 axis,RP11-222A5.1-miR-328-3p-SLC5A9 axis,RP11-222A5.1-miR-328-5p-RPS6KL1 axis,RP11-222A5.1-miR-330-5p-ABAT/PRAMEF12 axis,RP11-222A5.1-miR-370-3p-CLEC4 C axis,RP11-222A5.1-miR-423-5p-CD8A/FAM222 B axis,XIST-miR-143-5p-TMEM 237/SHQ1/SMDT1 axis,XIST-miR-18a-5p/miR-18b-5p-SLC22A8 axis,XIST-miR-214-3pRPS6KL1 axis,MOXD2P-miR-211-3p-LIF/COA7/RFT1 axis,MOXD2P-miR-361-3p-ETV4/PRAMEF12 axis.8.In the ceRNA network of peripheral blood samples from patients with ICH,the regulatory relationships associated with immune inflammatory response and lncRNAs verified by qRT-PCR included RP11-222A5.1-miR-423-5p-CD8 A axis and MOXD2 PmiR-211-3p-LIF axis.Conclusion: 1.We analyzed the mRNAs expression profile in peripheral blood of patients with ICH,and found that there was significant difference between patients with ICH and healthy controls.The enrichment analysis in biological process,pathway,PPI,GSEA of differentially expressed mRNAs in ICH group,was enriched in immune inflammatory response and related pathways,suggesting that immune inflammatory response was involved in the pathophysiology of ICH.Eight differentially expressed immune and inflammation related genes in peripheral blood of patients with ICH,including UCN,LIF,LEPR,HTR3 E,DEFB121,CMTM8,CD8 B,CD8A might participate in the immune and inflammation response of ICH.2.We analyzed the lncRNAs expression profile in peripheral blood of patients with ICH,and found that there was significant difference between patients with ICH and healthy controls.The enrichment analysis of biological processes and pathways of genes targeted by differentially expressed lncRNAs in ICH group showed that immune inflammatory response,lipoic acid metabolism,signal pathways regulating stem cell pluripotency and lysine degradation pathway were significantly enriched.The above processes and pathways played an important role in ICH.3.We constructed a ceRNA regulatory network of differentially expressed lncRNA-miRNA-mRNA in peripheral blood of patients with ICH,and explored the underlying mechanism of non-coding RNAs in ICH.RP11-222A5.1-miR-423-5p-CD8 A axis and MOXD2P-miR-211-3p-LIF axis were possible to related to immune inflammatory response of patients with ICH.RP11-222A5.1 and MOXD2 P were expected to be new targets for ICH treatment.