The Preliminary Study Of Relationship Between Virus MiRNA And Premature Infant With Periventricular Leukomalacia

BackgroundPeriventricular leukomalacia(PVL)is a common cerebral injury in premature,caused by lack of oxygen which result in progressive ischemia of ventricle peripheral artey,infarct and eventually cerebral tissue softened.PVL is the main cause of premature cerebral palsy and mental retardation.As a result of the development of medicine and perinatal medicine,the survival rate of premature and low birth weight increased significantly,however,the incidence of PVL in premature is on the rise.A survey of seven major cities in China in 2011 reported an incidence rate of 2.3%.Some studies reported there are 63,000 low birth weight premature infants borned in the United States every year,including 10%～15%of which with cerebral palsy and 25～50%of which with cognitive disorder or other neurological sequelae.At the present,there are about 400 to 500 millions of children with cerebral palsy in China,the disability rate is about 42%and an annual increase of children with cerebral palsy is up to 40 thousand,which has been a new problem of public health and society.In 1962,Banker and Lacrroche firstly proposed the term of "periventricular leucomalacia".The pathology of PVL usually shows symmetrical distribution in the surrounding of the cerebral ventricles,especially in the anterior horn of lateral ventricle,lateral,triangle,and after the angle of the lateral.The pathogenesis of PVL is complex and has not been clarified up to now.Except for hypoxia-ischemia,infection and inflammation are aslo important causes of PVL.Intrauterine infection is an important cause of neonatal brain injury,can lead to cerebral palsy,hearing impairment and intelligence damage and has close relation with preterm birth.With the social environment becoming complicated,a growing number of bacterias,viruses,chemicals are exposed to the environment,the exposure during pregnancy also becomes complicated.Intrauterine infection can increase levels of cytokines and due to the characteristic of the immature brain,whose ventricular blood vessels around the development is not mature and whose immature oligodendrocytes precursors are susceptible to inflammation and hypoxia-ischemia,both not only do damage to the autoregulation of cerebral blood flow,aggravate ischemic,but aslo induce activation of microglias in the white matter of brain,which further release inflammatory reaction factors,generate a mass of reactive oxygen species and reactive nitrogen,result in damage and lose of oligodendrocytes precursors in periventricular white matter(PVWM),and eventually promote the occurrence and development of PVL.Since Lee et al discovered the first microRNA(miRNA)inside samsonite small elegansin 1993,miRNA has been the focus of researches in various fields.miRNAs are a kind of non-coding single-stranded RNA molecules with a length of about 22bp(21-25bp),spliced and produced by miRNA precursors of length of 70～80bp with hairpin structure.MiRNA has a wide range of genes regulating function,by its target messenger RNA(mRNA)complementary base pairing specificity,thereby cause degradation of the target mRNA or inhibit its translation to achieve regulation of gene expression on transcription level.As is known,miRNAs are involved in the regulation of many fundamental biological processes of life,about one third of human encoding mRNAs are regulated by miRNAs negative regulation in order to achieve a wide range of gene regulation at all levels.The brains of human and animal contain many miRNAs,and their expression in the nervous system usually have a certain timing and specificity of cells,tissues and diseases.Numbers of studies have reported that miRNAs have close correlations with the development of nervous system as well as the incidence,progress and outcome of parts of nervous system related diseases.Smirnovaet al found that miR-181a,miR-9 and miR-125b expression levels were low in the early embryos,and with the increased expression of developmental processes,but its expression decreased after birth.They further found that miRNA expression in the brain aslo presented the specificity of tissues,such as miR-128 and miR-124 expression had advantage in neurons,while miR-29,miR-26 and miR-23 just expressed at high levels in oligodendrocytes and astrocytes.miRNAs aslo play important role in the inflammation of brain,involved in the innate immunity as well as adaptive immunity.One study of miRNA target genes found that miRNA could regulate the generation of antibodies and cytokines,involved in the Toll-like receptor(TLR)and T cell signal transduction cascade,and decreased activation of T cells and innate immune response.It has been reported that miR-125b can directly target the pro-inflammatory cytokine TNF-a in 3’-UTR,negatively regulates the expression of TNF-a.miR-146a/b negatively regulate NF-κB signaling pathwayby inhibiting the target protein of TRAF6 and IRAKI.Cullen found that miRNA of human can have impact on life cycle of the virus,selectivity of tissues from virus and related diseases caused by viruses.Like EBV(Epstein-Barr virus,EB virus)and HCMV can coding miRNA,then lowered the level of target genes in vivo,including a large number of inflammatory cytokines,signaling molecules,thus can cause an inflammatory immune response.Virus infection can changes miRNA of human lead to promote viral infection,in the course of HIV infection,the human miR-17-5a and miR-20a were downregulated,resulting the increased expression of the target PCAF,and PCAF happens to be cofactors of Tat,which benefit to HIV infect,suggests that virus infect have impact on human miRNA,result in progression of the disease.There are few studies on the role of miRNA in pathogenesis of PVL in domestic and overseas.Kai Guo et al found that miRNA played an important role in the regulation of lipopolysaccharide(LPS)-induced pathogenesis of PVL in rat model.The study adopted gene chip to screen differentially expressed miRNA in the rat brain,filtered out significantly differentially expressed miRNA and made biological analysis,and it was found these miRNA were closely associated with the inflammatory response pathway and the differences of expression were further tested with RT-PCR.The results showed significant changes in expression of miRNAs related to the inflammatory,and this suggested that the miRNAs related to inflammation played an important role in the pathogenesis of PVL.As miRNA in the peripheral blood circulation system is detected,the role of miRNA in serum clinical diagnosis of various diseases and drug treatment has gradually been a concern.Some studies reported that miRNA was stably expressed in serum and plasma and could be used as biological markers of cancer or other diseases to provide a reliable basis for diagnosis of clinical diseases.Li Shuqiang compared the miRNA expressions in plasma samples from 13 hypertensive patients and 5 healthy control subjects.Twenty-seven miRNAs were found to be differentially expressed.The expressions of selected miRNAs hcmv-miR-UL112(a human cytomegalovirus HCMV encoded miRNA)were validated independently in plasma samples from 24 hypertensive patients and 22 control subjects.The absolute expression level of hcmv-miR-UL112 were further determined in 127 patients and 67 control subjects by using qRT-PCR.Additionally,we demonstrated that interferon regulatory factor 1 is a direct target of hcmv-miR-UL112.Increased HCMV seropositivity and quantitative titers were found in the hypertension group compared with the control group.Suggest that hcmv-miR-UL112 contribute a lot to hypertension disease.The miRNA microarray is a rapid developing high-throughput test item,which can not only observe hundreds of miRNA gene expression changes in a single same sample,but aslo detect the quantitative of miRNA.It can accurately reflect differential expression of miRNA and provide a reliable basis for pathophysiology studies of nervous system,cardio vascular system,cancers or other diseases.Our previous studies applied miRNA microarray to analyze differentially expressed miRNA in serum of preterm infants with periventricular leukomalacia,screened 4 virus code miRNA associated with inflammation,infection,immune:sv40-miR-S1-5p,hsv1-miR-H6-5p,hsv2-miR-H10,ebv-miR-BART8-3p.In this study,bioinformatics methods and qRT-PCR technology are using to confirm this four differentially expressed miRNAs.Designed to confirm the significant differences expression of four viral miRNA between preterm infants with PVL and healthy preterm children,to provide an accurate and early biological markers of PVL,and to further explore the specific miRNA function in PVL and find out effective treatment strategies.Objective:1.Using miRNA microarray to analyze differentially expressed miRNA in serum between preterm infants with periventricular leukomalacia and healthy preterm children,to filter out significant different expressed miRNA,providing an accurate basis for the function of specific miRNA in the pathogenesis of PVL.2.Using bioinformatics methods to predict target gene of the specific miRNA,then filter out the PVL closely related miRNA for GO analysis and Pathway analysis.3.Using qRT-PCR to verify the the specific miRNA,to confirm the significant differences expression of four viral miRNA between preterm infants with PVL and healthy preterm children,to provide an accurate and early biological markers of PVL,and to further explore the specific miRNA function in PVL and find out effective treatment strategies.Method:1.miRNA microarray detect miRNA expression between preterm infants with periventricular leukomalacia and healthy preterm children.(1)Collect 5ml peripheral blood in 7 cases of premature infants with PVL and 5cases healthy preterm children.The experimental group of premature infants with PVL had no clear perinatal risk factors exist,and excluded genetic metabolic,chromosomal disorders.The experimental group confirmed the presence of PVL disease typical imaging findings by the head MRI,and line with the clinical manifestations of cerebral palsy spastic paralysis of the diagnostic criteria.(2)Blood samples were put in two EDTA anticoagulant tubes at low temperature low-speed centrifuge centrifuged for 5 minutes 1800 rev/min,then took the homogenate supernatant,placed in no RNA enzymes EP tubes stored in low temperature refrigerator at-70 ℃.(3)After extraction of RNA,miRNA microarray detected miRNA in serum between preterm infants with periventricular leukomalacia and healthy preterm children,screening differentially expressed miRNA(fold change≥ 2.0 or ≤ 0.5 and P<0.05).2.Selected the miRNA which value obviously difference and fluorescent expression valuesis in the highest expression in premature infants with PVL,using software(miRanda and targetscan)to predict target gene,and integrated the two software as a result of prediction candidate miRNA target genes.Using GO analysis to enrich analyse and classify the specific miRNA mentioned before,screening out the target gene which closely related to infection,inflammation and immunity,then predicted target genes related signaling pathways through Pathway analysis.3.Using qRT-PCR to verify the miRNA filtering out by GO analysis and Pathway analysis.(1)Total RNA was extracted from peripheral blood specimens in the experimental group and control group,then tested its purity and integrity.(2)Reverse transcription:compounded reverse transcriptase mixed reaction liquid,then took reverse transcription in PCR amplification.(3)Quantitative PCR:The real-time reverse transcription PCR reaction samples were configured system,placed in the PCR reaction on quantitative PCR instrument.Result:1.There are significantly different miRNA between premature infants withPVL and healthy preterm children,there are 100 miRNA differences in expression(P<0.05,Fold change≥2.0 or ≤0.5),among them,66 were down regulation,including:hsv2-miR-H10,hsvl-miR-H6-5p,ebv-miR-BART8-3p,sv40-miR-S1-5p,hsa-miR-4472,hsa-miR-920,hsa-miR-4462,hsa-miR-4478,hsa-miR-3915,hsa-miR-4656,hsa-miR-4710,hsa-miR-574-5p,hsa-miR-4666a-3p,hsa-miR-4443,hsa-miR-3665,hsa-miR-4450,hsa-miR-638,hsa-miR-4447,hsa-miR-4456,hsa-miR-29b-1-5p,hsa-miR-4755-5p,hsa-miR-4508,hsa-miR-1299,hsa-miR-3620-3p,hsa-miR-4292,hsa-miR-711,hsa-miR-149-3p,hsa-miR-193b-5p,hsa-miR-4690-5p,hsa-miR-3656,hsa-miR-4645-5p,hsa-miR-4750-5p,hsa-miR-4667-5p,hsa-miR-25-5p,hsa-miR-4685-3p,hsa-miR-1268b,hsa-miR-483-5p,hsa-miR-498,hsa-miR-4484,hsa-miR-371 a-5p,hsa-miR-4268,hsa-miR-1469,hs a-miR-1228-5p,hsa-miR-4784,hsa-miR-3976,hsa-miR-1915-3p,hsa-miR-645,hsa-miR-3148,hsa-miR-4514,hsa-miR-1291,hsa-miR-3924,hsa-miR-4768-5p,hsa-miR-373-5p,hsa-miR-4661-3p,hsa-miR-3189-3p,hsa-miR-2392,hsa-miR-622,hsa-miR-4709-3p,hsa-miR-585,hsa-miR-4687-3p,hsa-miR-5004-3p,hsa-miR-365b-5p,hsa-miR-4707-5 p,hsa-miR-147b,hsa-miR-5572,hsa-miR-4459.34 were up regulation,including:hsa-miR-654-5p,hsa-miR-125b-5p,has-miR-1270,hsa-miR-130a-3p,hsa-miR-758-3p,hsa-miR-3928,hsa-miR-369-3p,hsa-miR-382-3p,hsa-miR-1976,hsa-miR-133a,hsa-mi R-454-3p,hsa-miR-3675-5p,hsa-miR-133b,hsa-miR-4328,hsa-miR-379-3p,hsa-miR-3074-3p,hsa-miR-4662b,hsa-miR-4659b-3p,hsa-miR-328,hsa-miR-409-5p,hsa-miR-3688-3p,hsa-miR-1304-3p,hsa-miR-323a-3p,hsa-miR-671-3p,hsa-miR-100-5p,hsa-miR-4659b-5p,hsa-miR-889,hsa-miR-424-3p,hsa-miR-330-3p,hsa-miR-485-5p,hsa-miR-144-3p,hsa-miR-3173-5p,hsa-miR-4454,hsa-miR-548ag.2.Using the target gene prediction software to predict target gene for four virus-associated miRNA which obvious differences and had highest expression values fluorescence(sv40-miR-S1-5p,hsvl-miR-H6-5p,ebv-miR-BART8-3p,hsv2-miR-H10),the GO analysis results:sv40-miR-Sl-5p had 906target genes are associated with infection,inflammation and immune-related,and 549are associated with development and physiological activity of nervous system,and 300are associated with apoptosis-related;hsvl-miR-H6-5p had 579 target genesare associated with infection,inflammation and immune-related,and 257 are associated with development and physiological activity of nervous system,and 48 are associated with apoptosis-related;hsv2-miR-H10 had 99 target genes are associated with infection,inflammation and immune-related,and 103 are associated with development and physiological activity of nervous system,and 32 are associated with apoptosis-related;ebv-miR-BART8-3p had 114 target genes are associated with development and physiological activity of nervous system,and 15 are associated with apoptosis-related.The result of Pathway analysis:Toll-like receptor signaling pathway,MAPK signaling pathway,T cell receptor signaling pathway,HIF-1 signaling pathway and mTOR signaling pathways are predict to associated with PVL.The target gene(MyD88,TRAF6,IL1R,TLR6,IFN-αβR,TGFB,ERK,p38)of four virus play ankey role in the above inflammation related pathways.3.Using qRT-PCR technology to further verify the above four virus associated miRNAs(sv40-miR-S1-5 p,hsvl-miR-H6-5 p,ebv-miR-BART8-3 p,hsv2-miR-H10),among them,sv40-miR-S1-5p,hsvl-miR-H6-5p,hsv2-miR-H10 were significantly expressed(P<0.05).Conclusion:Some virus-associated miRNA significantly differently express in peripheral blood of premature children with PVL,target genes of these differentially expressed miRNA involved in infection,inflammation and immunity,suggesting that virus-associated miRNA may be involved in the development and progression of PVL.