Effects And Mechanism Of MicroRNA-200b On The White Matter Damage Induced By Intrauterine Infection

With the development of NICU care technique,the survival rate of premature infant raised,the encephalopathy of premature infant is receiving more and more attention.The white matter damage due to immuno-inflammatory responses caused by intrauterine infection during the perinatal period is closely related to the onset of encephalopathy in premature.In the white matter damage,researchers observed reactive astrogliosis,microglia infiltration,oligodendrocyte injury and myelin sheath damage as well as axon injury.The activation of oligodendrocytes,microglia and astrocytes induced by the inflammation of intrauterine infection is believed to be one key point in the mechanism,yet the precise molecular biological mechanism is unclear.MicroRNA(MiRNA)is an endogenous small RNA,it plays an important role in the regulation of life activity.Recently,it has been found that miRNAs exists almost in every central nervous system disease.The expression level of miRNA in microglia and astrocyte has been observed,demonstrating that miRNA participate in the process of brain injury induced by activation of microglia and astrocyte.We constructed the animal model of white matter damage induce by intrauterine infection,detected the changed expression level of GFAP with immunohistochemistry and PCR,and the miRNA expression profile in the brain of the animal model,and screened out the most obviously changed miRNA:miR-200b.We use the bioinformatic analysis to predict the possible target gene,and then to verify the internal mechanism between miR-200b and the target gene in astrocyte,and observe the modulation of miR-200b on the proliferation,astrogliosis and the inflammatory cytokines expression level of astrocytes.Objectives:To explore the characteristics of miRNA expression in immature rat's brain tissue after intrauterine infection/inflammation,screen out miRNA related to white matter damage,predicte the target gene,and illuminated the mechanism of miRNA in regulating the astrogliosis and inflammation in the brain.Method:1.Establish animal model:the intrauterine infection group:E.coli suspension were endocervically inoculated to the pregnant SD rats;the control group:sterile normal saline were endocervically injected to the pregnant SD rats.2.The brain tissues were stained with HE.The expression levels of GFAP and TNF-a in brain tissue of fetal rats and neonatal rats were qualified by quantitative RT-PCR techinique.3.MiRNA chip was applied for the detection the miRNA expression profile in brain tissues of neonatal rat aged Id and 3d after birth in the group of intrauterine infection group and control group,screened out the miRNAs which were related to white matter damage and whose differential expression was significant.Taqman fluorescence quantitative PCR technique was used to verify the result.Besides,the expression level of the 2 screened out miRNAs were qualified in rats brain at E17?E19?E21?P1?P3?P7?P14,and finally locked on miRNA-200b for further research.4.The astrocytes were cultured and purified,and then were added with LPS,the expression level of miR-200b were tested;and then the astrocyte were transfected with miR-200b mimics/inhibitor,then the proliferation of astrocyte and the expression level of GFAP in astrocytes were detected with CCK-8 or PCR technique,and the expression level of inflammatory cytokines were detected with ELISA.5.Bioinformatic analysis were used to predict possible target gene of miR-200b.The expression level of c-jun was tested in astrocytes stimulated with LPS;the expression level of c-jun nd the activation of astrocyte was tested in astrocytes transfectecd with miR-200b mimics/inhibitor.Luciferase reporter gene was used to verify c-jun as the target gene;in the meanwhile,synthesized siRNA,solely transfected astrocytes or co-transfected astrocytes with miR-200b inhibitor were used to verify the function of c-jun on the activation of astrocytes.Results:1.The fetal and neonatal brain tissues histomorphological examination showed that:in group of intrauterine infection the dyed color of white matter was light,with structure being sparse and changed into screen mesh shaped,with especially significant differences in groups aged 7d;in the control group,the white matter tissue had normal structure and clear dyed color;GFAP immunohistochemical examination for brain tissue shows that GFAP positive cell plasma was obvious and processes increased dramatically in white matter area beside cerebral ventricle in group of intrauterine infection,especially in neonatal rats aged 7d after birth,compared to the control group.2.GFAP mRNA and TNF-amRNA detection of brain tissue:the expression level of GFAP mRNA in rats at P3,P7 and P14 showed significantly increased level compared with the control group(P<0.05);the expression level of TNF-a mRNA for fetal rats at E17,E19 and E21 and neonatal rats at P1 and P3 in intrauterine infection group showed significantly increase level compared with control group(P<0.05).3.MicroRNA chip analysis of neonatal rats brain tissure revealed 63 miRNAs with differential expression in total.In P1 group,43 miRNAs were screened out(7 up-regulated significantly,and 36 down-regulated significantly);In P3 group,20 miRNAs were screened out(2 up-regulated significantly,and 18 down-regulated significantly).The expressions of miR-199a,miR-200b in neonatal rats with age of Id and 3d after birth in intrauterine infection group all down-regulated,and among which,the change of miR-200b was the most significant one.4.Taqman fluorescence quantitative PCR technique verified the results of the microarrays showed that compared with the control group,the expression of miR-199a and miR-200b in neonatal rats with age of Id and 3d after birth in intrauterine infection group all down-regulated,with miR-200b changed most obviously,which was consistent with the chip detection result.The detection result of taqman fluorogenic quantitative PCR at 7 different time points shows that:the expression levels of miR-199a,miR-200b in fetal rats with age of 17d and 19d all up-regulated,especially the 17d group with statistic significance in difference from the control group(p<0.05),the expression level of miR-199a?miR-200b in fetal rats with age of 21d and neonatal rats with age 1d,3d,7d,14d all down,regulated,with miR-200b in neonatal rats with age 3d with statistic significance in difference from the control group(p<0.05);wherein the expression difference of miR-200b was the most significant.We locked miR-200b as the object of study.5.Relationship between miR-200b and astrocytes:The expression level of miR-200b in astrocytes stimulated by LPS down-regulated,compared with the control group,with the time and the concentration of LPS,the down-regulation is obvious(p<0.05);compared with group of miR-200b inhibitor,the proliferation of astrocytes in group of miR-200b mimics dereased markedly,with statistical significance in difference(p<0.05);compared with group of miR-200b inhibitor,astrogliosis degree of astrocytes in group of miR-200b mimics decreased dramatically,with statistic significance in difference(p<0.05);compared with group of miR-200b inhibitor,the expression of inflammatory cytokines TNF-?and IL-6 in group of miR-200b mimics down-regulated obviously than the control group,with statistic significance in difference(p<0.05).6.Expression of c-jun and the regulation of miR-200b on c-jun:The expression level of c-jun mRNA and protein in the astrocytes stimulated by LPS up-regulated,compared with control group,and with the time and the concentration of LPS,the upregulation change obviously(p<0.05);compared with the group of miR-200b inhibitor,the expression level of c-jun protein in the group of miR-200b mimics downregulated obviously,with statistic significance in difference(p<0.05).7.c-jun is the target gene of miR-200b:Constructed luciferase reporter gene bearer and conducted detection of luciferase reporter gene,with result showing that:Compared with cotransfection group of miR-200b mimics scramble+c-Jun 3'UTR,the luciferase activity in astrocytes of cotransfection group of miR-200b mimics+c-Jun 3'UTR dropped significantly(p<0.01);compared with cotransfection group of miR-200b mimics+c-Jun 3'UTR,the luciferase activity in astrocytes of cotransfection group of miR-200b mimics+c-Jun Mut 3'UTR increased significantly(p<0.01),and has no significant differences from that for cotransfection group of miR-200b mimics scramble+c-Jun 3'UTR.At the meanwhile,c-jun siRNA was synthesized,and solely transfected astrocytes or co-transfected astrocytes,with result showing that:The expression level of c-jun protein and inflammatory cytokines in si-c-jun group all down-regulated significantly compared with the control group,with statistical significance in difference(p<0.05);The expression level of c-jun protein and inflammatory cytokines in group of si-c-jun+miR-200b inhibitor all up-regulated significantly compared with the group of si-c-jun,with statistical significance in difference(p<0.05).Conclusions:1.Intrauterine infection/inflammation makes white matter tissue sparsificated,and lead to the changed expression level of astrogliosis of astrocytes and inflammatory cytokines.2.Intrauterine infection/inflammation leads to change of miRNA expression profile in immature rat's brain tissue,and the change is time dependent.Wherein the expression level of miR-200b changed significantly.The expression of miR-200b upregulated obviously at first,and then downregulated gradually,and go up until 7 days after birth,then gradually go down.3.The expression of miR-200b in astrocytes in vitro is downregulated.c-jun is the target gene of miR-200b.miR-200b inhibits proliferation,astrogliosis and expression of inflammatory cytokines(IL-6?TNF-?)of astrocytes via c-jun/JNK signal pathway.