| Background and ObjectiveNecrotizing enterocolitis(NEC)is a kind of intestinal mucosal damage with ischaemia and hypoxia,caused by various of high risk factors,and is a disease causing diffuse or local necrosis of the small intestine or colon.About 90%of patients are premature babies,especially for those whose birthweight less than 1500g,its mobility is about 7%.It is an acute gastrointestinal disease that seriously affects the prognosis and life of premature infants.But the exact etiology and pathogenesis of the disease have not been fully elucidated,it is considered that it may be a cascade inflammatory reaction caused by high risk factors such as intestinal immature,formula feeding,hypoxia,ischemia,microbial dysbiosis and infection.In the absence of effective treatment,researchers have been working on specific biomarkers and pathogenesis of NEC.As a non-coding RNA small molecule of 18-24 nucleotides,miRNA is widely found in eukaryotes,and involved in regulating a variety of biological behaviors,such as development process,stem cell differentiation,cell proliferation and apoptosis,systemic stress and immunity.miRNA has been shown to play an important role in human acute and chronic diseases,such as cancer,chronic infection,inflammatory response and acute organ damage.And,specific miRNAs have been found in the ileum and colon mucosa,widespread and significant miRNA expression dysregulation was found in the intestinal tissues of NEC children.Therefore,miRNA may play an important role in the pathogenesis of NEC.This paper hopes to explore the possible role of miR-429/200a/b and miR-141/200c clusters and their target genes(VEGFA,SELE,KDR,FLT1 and HGF)in the pathogenesis of necrotizing enterocolitis.MethodsPart Ⅰ,We obtained differentially expressed miRNAs and differentially expressed genes(DEGs)of NEC through miRNA chip GSE68054 and gene chip GSE46619 from GEO database,the miR-429/200a/b and miR-141/200c clusters as well as their target genes(VEGFA,SELE,KDR,FLT1 and HGF)were proved to be abnormally expressed by systematic bioinformatics analysis.Part II,We constructed a NEC model of SD rats by formula feeding,hypoxia(5%O2,5 min),cold stimulation(4℃,7 min)and LPS(2 mg/kg)intraperitoneal injection.With the specimens of animal blood,feces and tissues,blood routine and stool routine were tested.And clinical manifestations,intestinal histopathology and inflammatory factors are detected to evaluate NEC modeling.Part III,firstly,the expression of miR-429/200a/b and miR-141/200c clusters and their target genes(VEGFA,SELE,KDR,FLT1 and HGF)at mRNA and protein levels was detected by real-time PCR and western-blot using successfully modeled blood samples and intestinal tissues of pups.Secondly,IEC-6 cells were selected as transfection objects.The expression of target genes(VEGFA,SELE,KDR,FLT1 and HGF)at the mRNA level was further detected after transfection of miRNAs,which were significantly differentially expressed,and then verified the targeting relationship between miRNA and target genes.Thirdly,the expression of target genes(VEGFA,SELE and HGF)was further detected by immunohistochemistry with intestinal tissue sections from newborns suffering NEC surgery.ResultsPart Ⅰ,Fifteen miRNAs were differentially expressed in NEC in newborn,among which miR-429/200a/b and miR-14 1/200c clusters were poorly expressed.Besides,target genes of miR-429/200a/b and miR-141/200c clusters were enriched in 11 signaling pathways.VEGFA,SELE,KDR,FLT1 and HGF were highly expressed in NEC in newborn,which were negatively regulated by miR-429/200a/b and miR-141/200c clusters and shared close association with disease genes.Part Ⅱ,The NEC model of newborn rats was successfully constructed by formula feeding,hypoxia(5%O2,5 min),cold stimulation(4℃,7 min)and LPS(2 mg/kg)intraperitoneal injection,the survival rate was about 64%,and molding rate was 56%.Part Ⅲ,The expressions of miR-429/200a/b and miR-141/200c clusters in both blood and intestinal tissues of NEC newborn rat model were down-regulated compared with that of normal group,in which miR-141 and miR-200a were obviously down-regulated(p<0.05),and there were also statistical differences in expression between severe and mild groups(p<0.05).While its target genes VEGFA,SELE,KDR,FLT1 and HGF expression increased to varying degrees at both mRNA and protein levels.The cell transfection experiment found that the FLT1,KDR,SELE and HGF expression levels decreased significantly when the miR-141 and miR-200a mimics were transfected into the IEC-6 cells.And immunohistochemistry of NEC tissues from surgical newborns showed that the expression of VEGFA,SELE and HGF in pathological tissues of intestine was increased comparing with that in adjacent normal tissues.ConclusionmiR-429/200a/b and miR-141/200c clusters are down-regulated in both NEC blood and intestinal tissues,and may exist as protective forms in NEC pathogenesis by regulating their target genes VEGFA,SELE,KDR,FLT1 and HGF,which may be related to growth factors’ promoting cell proliferation,survival and angiogenesis,among which expressions of miR-141 and miR-200a are significantly down-regulated and correlated with disease severity,which may play a major role. |
