Construction And Analysis Of The LncRNA-miRNA-mRNA Network Based On CeRNA Identified Key LncRNA Biomarkers In The Neutrophil Spontaneous Apoptosis Process

Objective: Polymorphonuclear leukocytes(neutrophils or PMNs)are the most abundant white blood cell in human peripheral blood,constitute the first line of host defense against invading pathogens(bacteria,fungi,etc.),and play crucial role in the innate immune system.PMNs are terminally differentiated cells with an extremely short lifespan(8–20 hour in circulation and 1–4 days in tissue),having powerful toxic weapons that are highly effective at killing pathogens,but they also can cause damage to normal tissues.Therefore,PMN's death must be precisely regulated,and this task is mainly completed by programmed spontaneous neutrophil apoptosis,which plays an important role in immune system homeostasis and inflammation resolution.In recent years,it has been found that long non-coding RNA(lncRNA)plays regulatory roles in various biological processes.With relatively longer nucleic acid chains lncRNA can regulate the mRNA expression by binding to miRNA of specific sequences,so lncRNA is called miRNA sponge.Accumulating evidence shows that lncRNA,as competitive endogenous RNAs(ceRNAs)may be closely related to neutrophil apoptosis,but its specific regulatory mechanism is largely unknown.In this research,we identified differentially expressed microRNA,lncRNA,mRNA in the PMN spontaneous apoptosis process,constructed lncRNA-microRNA-mRNA endogenous competitive RNA network,and screened key lncRNA which may regulate PMN spontaneous apoptosis,using bioinformatics methods.Methods :(1)neutrophils were isolated from healthy volunteers and cultured in different groups(0 hour as control and 12,24 hour as the experimental groups).(2)Total RNA was extracted from different groups at each time point.(3)miRNA microarray was used to detect and screen out differentially expressed miRNA.(4)Four differentially expressed miRNAs,namely,hsa-miR-487 b,hsa-miR-4538,hsa-miR-4417 and hsa-miR-4485 were selected from the screened miRNA for verification the reliability of the chip results by RT-PCR.(5)Search and download the time series transcription data of mRNA and lncRNA of PMN from GEO,and screen out the differentially expressed lncRNA and mRNA.(6)Target mRNAs with differentially expressed miRNAs were predicted by three highly reliable online miRNA reference databases,miRWalk,Miranda and Targetscan.The miRNAs' putative lncRNAs targets were predicted by an intersection of three bioinformatics algorithms including miRWalk,RNAhybrid and Targetscan.(7)The differentially expressed lncRNAs and differentially expressed mRNAs were intersected respectively with the target lncRNA and mRNA of differentially expressed miRNA to obtain the miRNA-mRNA gene pair library and the miRNA-lncRNA gene pair library(8)Cytoscape software was used to map the miRNA-mRNA gene pair and miRNA-lncRNA gene pair into the interaction network,and finally construct the lncRNA-miRNA-mRNA ceRNA interaction network.(9)The lncRNA-miRNA-mRNA interaction network was further analyzed to screen the key lncRNAs in the process of PMN spontaneous apoptosis.And the key lncRNAs were verified with the chip results by RT-PCR.Results:(1)miRNA chip results: taking group 0h as the control group,the fold change >2 as up-regulated,and fold change < 0.5 as down-regulated,the data showed that there were 81 differentially expressed miRNA,including 49 up-regulated and 32 down-regulated.There were 21 upregulated miRNA and 21 down-regulated miRNA in the 12 h group,41 upregulated miRNA and 22 down-regulated miRNA in the 24 h group,13 upregulated miRNA and 11 down-regulated miRNA in the 12 h and 24 h groups.RT-PCR analysis of has-miR-487-b,hsa-miR-4538,has-miR-4417 and hsa-miR-4485 were consistent with the miRNA microarray results;(2)GSE37416 mRNA-lncRNA microarray data mining results: taking group 0h as the control group,using the LIMMA analysis,p values < 0.01 and fold change > 2,a total of 176 differentially expressed lncRNA and 1966 differentially expressed mRNA were detected in the process of the spontaneous apoptosis.(3)A network of lncRNA-miRNA-mRNA was constructed and then visualized by Cytoscape,composed of 117 lncRNA nodes,671 mRNA nodes,59 miRNA nodes,and 3636 edges.(4)The GO interaction network was constructed by Cytoscape plug-in BinGO.The biological processes(BP)and Pathway were analyzed by DAVID.The high-enrichment GO terms included: regulation of I-kappaB kinase/NF-kappaB cascade,intracellular signaling cascade,positive regulation of programmed cell death and more.Pathway analysis manifested that 8 pathways were significantly enriched particularly,Apoptosis,Toll-like receptor signaling pathway,and Chemokine signaling pathway.(5)Topological analysis was conducted for the lncRNA-miRNA-mRNA network,and three key subnetworks centered by three lncRNA(AC021016.2,LINC00909,TTN-AS1)were screened out.The expression of the three lncRNAs was validated by RT-PCR being largely consistent with previous microarray results.Conclusions:(1)Differential expression profiles of mRNA,lncRNA and miRNA in neutrophil spontaneous apoptosis were obtained.(2)We firstly constructed lncRNA–miRNA–mRNA global triple ceRNA network in PMN apoptosis process,(3)Moreover,three key subnetworks were screened out,and three lncRNAs(AC021016.2,LINC00909,TTN-AS1)may be involved in regulating the neutrophils spontaneous apoptosis by regulating the expression of miRNA,potentially providing new clues for understanding spontaneous neutrophil apoptosis mechanism.