Differential Expression Of Long Non-coding RNA In Neonatal Necrotizing Enterocolitis Based On High-throughput Sequencing

Objectives:Long non-coding RNAs(lnc RNAs)play important role in gene regulation and development of human,and lnc RNAs are involved in a wide aspect of biologic processes.Dysregulated expression of lnc RNAs is involved in many human diseases.There was seldom study on the relationship between lnc RNA expression and neonatal necrotizing enterocolitis(NEC).The change of intestinal expression of lnc RNA potential roles in NEC are unclear.In this study,the difference of lnc RNA and m RNA expression profiles between intestinal tissues of NEC patiens and normal controls was studied with high-throughput sequencing technology.Bioinformatics technology was used to explore lnc RNA associated with NEC which may provide new ways for prevention,diagnosis and treatment of NEC.Method: 1.Necrotizing enterocolitis models in SD rats of 2 hours were established by hypoxia,cold stimulation,artificial feeding and LPS gavage.2.High-throughput sequencing was applied for small intestine tissues from human and necrotizing enterocolitis models.Differential expression profiles of lnc RNA and m RNA in NEC group and control group were obtained by Illumina Hi Seq platform.3.To verify the high-throughput sequencing results,differentially expressed Inc RNAs and m RNAs were selected from the sequencing data,and were detected by q RT-PCR.The results were compared with the the high-throughput sequencing results.4.Gene Ontology(GO)analysis and KEGG signaling pathway analysis were performed on differentially expressed genes.Result:1.Neonatal rats at the age of 2h were artificially fed under hypothermia and hypoxia with or without LPS gavage were established a rat model of NEC with clinical manifestations and histological evidence.2.We filtered expression profiles of lnc RNAs and m RNAs and identified aberrantly expressed lnc RNAs and m RNAs with absolute fold change of altered expression levels more than 2 times and the Adjusted Pvalue less than 0.001.There were 3457 m RNAs and 5257 lnc RNAs differences between the control group and the NEC group from human..3.Three lnc RNAs and three m RNAs were selected for verification of the sequencing data by q RT-PCR.The q RT-PCR assay showed that NDUOXA2,OLFM4,REG1 A,lnc-CXCL-1-1-1,ABHD11-AS1 were all up regulated and lnc TLR10-2:1 was down regulated in the NEC group compared with their expression in the control group.The results were consistent with the high-throughput sequencing.4.GO analysis showed that the different regulated m RNAs were involved in the multicellular biological development,antibacterial humoral response,neutrophil chemotaxis,innate immune response activation signal transduction,inflammatory response,regulation of cytokine secretion,regulation of cell proliferation,Toll-like receptor signaling pathway,body response to bacteria,activation of innate immune response,granulocyte chemotaxis,lipopolysaccharide receptor activity.KEGG analysis identified several biological pathways in Ig A production-related intestinal immune system,Fc?R-mediated phagocytosis,B cell receptor signaling pathway,PI3K-Akt signaling pathway,inflammatory bowel disease(IBD),NF-?B signaling pathway,Fc epsilon RI signaling pathway.The intestinal tissue associated with NEC patiens and NEC rat model showed that the KEGG pathway associated with enteropathy was inflammatory bowel disease.The expression of TTC39C-AS1 in NEC intestinal tissue decreased,and the expressions of IL4,IL6,IL17,IL10,IL13,IL1,CCL5 and IFN-? in intestinal tissues increased,suggesting that down-regulation of TTC39C-AS1 may promote inflammatory response.Conclusion:1.Neonatal rats at the age of 2h were artificially fed under hypothermia and hypoxia with or without LPS gavage were established a rat model of NEC.2.The expressions of some lnc RNAs and m RNAs were significant differences in small intestinal tissues in NEC group compared with control group.3.Inflammatory bowel disease(IBD)were most important pathways which associated with the NEC disease,while TTC39C-AS1 may play an important role in the inflammatory bowel disease pathway.