Study On The Gene Regulatory Network In The Pathogenesis Of Anorectal Malformations

Part 1 Mi RNA-mRNA Interaction Network in AnorectalMalformationsObjective:we aimed to assess the miRNA and mRNA expression profiles in the anal terminal sections of patients with anorectal malformations,and evaluate the obtained results by bioinformatics means,and preliminarily explore the mechanisms of the gene network contributing toanorectal malformations.Methods: a total of 7 samples containing anal terminal tissues from the children with anorectal malformations from May to October 2013 were collected in our hospital.Of them,5 were from male,remaining 2 from female;4 were middle-high atresia,remaining 3 low atresia.Additionally,another 3 samples containing anal terminal tissues from autopsy children(dead from non-intestinal diseases)were collected as control.Gross RNA was extracted and qualified.Using miRNA-mRNA chip,we assessed and screened miRNA and mRNA significantly different expressed among 3 groups.The expression tendency of the gene expressed significantly was also evaluated.We then predicted the target genes of different expressed miRNA.Subsequently,we intersected the target genes and the screened genes to obtain intersection-gene 1,which was forward evaluated in terms of genetic function and biological pathway profiles.After integrating these databases including KEGG,HPRD,BIOGRID,INTACT,MINT,via Cytoscape software,we evaluated the interaction profiles among significantly different GO and all genes under the pathway item,then attempted to find out the crucial regulating genes.Of the obtained crucial regulating genes,4 were chosen and verified using Real-Time PCR.Data were analyzed by One-Way ANOVA.Results: by comparison of the data amongthe control,low atresia and middle-high atresia groups,we totally screened 8011 different expressed genes and 88 mRNA.There were 4 kinds of significant tendency in mRNA,another 2 in the miRNA.A total of 2705 significance genes were obtained after miRNA target genes prediction and further negative intersection with mRNA.GO of the significance genes analyses revealed that the GO items included transcription and transcription regulation,signal transduction,apoptosis process,embryogenesis,nervous system development,muscle cell differentiation and so on.The KEGG signal pathway analyses revealed that significance genes were mainly located in tumor-related signal pathways,nervous nutrition factors signal pathways,EB virus infection,cytoskeleton regulation,calcium ion signal transduction,and other pathways such as tumor regulation.Interaction analyses of the genes and their production of significantly different GO as well as those under the pathway items developed Signal-Net analyses.We found that the crucial genes included MAPK1,FGFR2,EP300,SRC,PLCB1,CDC42,MLLT4,TJP1 and so on.Based on the regulation interaction between mRNA and target genes,we constructed micro RNA-Gene-Network.Crucial micro RNA included has-miR-124-3p,has-miR-29c-3p,has-miR-1185-2-3p,has-miR-103a-3p,has-miR-4795-3p and so on.MAPK1,FGFR2,EP300,LRP6 were chosen and verified using Real-Time PCR.Statistical significance was found between any 2 of them(P<0.05).Conclusions: after in-depth explorations of the related genes contributing to anorectal malformations by combining mRNA-miRNA chip with bioinformatics,we finally screened crucial genes correlated with the disease initiation.We demonstrated that the gene network played an important regulatory function in the initiation and development of anorectal malformations.Besides,the screened significance gene may represent a novel direction in investigating anorectal malformations.PART 2 EFFECTS OF VITAMIN A AND ITS RELATEDGENES IN THE ANORECTAL MALFORMATIONSDEVELOPMENTObjective:we aimed to measure vitamin A level in serum,assess related receptors expression in tissues and explore the association between a new HA117 gene related with vitamin A as well as DPF3 gene expression and congenital anorectal malformations development in children,which furtherly helped us to investigate the effects of vitamin A in the anorectal malformations development.Methods:(1)we randomly enrolled 35 children diagnosed as anorectal malformations in the gastrointestinal surgery and newborn surgery departments of Affiliated Children Hospital,Chongqing Medical University from May 2012 to September 2013.Of them,22 were male,remaining 13 were female.The age ranged from 1 day to 8 months plus 23 days.11 cases were low atresia,12 middle atresia,another 12 high atresia.The anal terminal tissues obtained after surgery were collected as experimental group.Additionally,another 7 samples containing anal terminal tissues from autopsy children(dead from non-intestinal diseases)were collected as control.Gross RNA was extracted using the Trizol method.Using the Real Time Fluorescence Quantitative PCR,the expression levels of HA117,DPF3 b,RARα,RARβ,RARγweredetected in experimental and control groups,respectively.Proteins in the tissues were extracted.The expression levels of DPF3 b,RARα,RARβ proteins were also measured in experimental and control groups using the Western-blot method.(2)venous blood of 2 ml was obtained from each children when admitted to our hospital for the first time.The liquid supernatant was restored until analyses.Additionally,another 10 serum samples from healthy children were collected as control.The vitamin A levels were assessed in both groups by High-phase liquid chromatography.(3)all data were analyzed by SPSS 19.0 software.One-Way ANOVA was performed to compared the differences between 2 groups.A value of P<0.05 was considered as statistically significant.Results:(1)Q-PCR results showed that the RARαexpression levels were much lower in the children with anorectal malformations than those in the control group(P<0.01).The higher the anal terminal was located,the lower RARα was expressed.There was a statistical significance(P<0.05)in comparison of RARβamong middle-high located anorectal malformations,low located anorectal malformations and control groups.The expression levels of RARγ in all types of anorectal malformations were not significantly different from control groups(P>0.05),while there was a significant difference in the expression levels of HA117 among middle-high located anorectal malformations,low located anorectal malformations and control groups(P<0.01).The expression levels of HA117 fell in turn in the 3 groups.By contrary,the expression levels of DPF3 b increased in turn in the 3 groups.(2)the expression levels of DPF3 b,RARα,RARβ proteins were assessed by the Western-blot method.Of them,the expression levels of DPF3 b fell in turn in the order of control,low located anorectal malformations and middle-high located anorectal malformations groups.There was a significant difference when compared among the groups(P<0.01).The expression of RARα markedly decreased in the middle-high located anorectal malformations group;its expression in the low located anorectal malformations group was also much lower than that in the control group(P<0.05).There was no significant differences between three groups in the expression levels of RARβ(P>0.05)(3)the serum vitamin A levels from both high located and low located children were significantly lower in the experimental group than that in the control group(P<0.01);the actual data were 0.37 ±0.11,0.38 ±0.14,0.90±0.09μmol/L,respectively.Grouped according to pathological type,there was no statistical significance between middle-high located and low located anorectal malformations groups(P>0.05).Conclusions:vitamin A and its receptors played a crucial role in intestinal development in embryonic phase.Vitamin A deficiency was likely to be one of the mechanisms contributing to anorectal malformations development.A novel vitamin A related HA117 gene may be involved with the disease process,probably by negative regulation with DPF3.PART 3 ONSTRUCTION OF INTESTINALMALFORMATIONS MODEL IN MOUSE VIASLOW-VIRUS VECTOR AND TRANSPLACENTALRNAIRARΑ GENEObjective:we aimed to first interfere the expression of RARα gene by appling slow-virus vector and transplacental RNA interference technique,furtherly explore optimal techniques which could partly down-regulate genes in mouse and also observe the intestinal development in the mouse with interfered RARα gene.Methods:we first constructed RARα interfering slow-virus which was a vitamin A-specific receptor gene,then the recombined interfering slow-virus was transfected to the mesenchyma stem cell C3H10T1/2 derived from mouse embryo.The interfering effects of slow-virus on RARα were validated via the RT-PCR and Western-blot methods.The RARα-RNAi slow-virus vector,empty-virus vector and Ringer’s liquid were injected into the tail vein of the mice with 9-week gestation.Part of these mice were killed 96 hours after the injection,with embryo removed.The changes of the embryo were observed.Fluorescent expression was evaluated by stereo fluorescence spectrometer.Also,the expressions of RARα,Sonic hedgehog(Shh),HA117 were assessed via Western-blotting and RT-PCR.After natural delivery of the remaining mice,the intestinal developmentsof offspring mice were observed and evaluated in proper ways.Results:(1)RARα-RNAi slow-virus vector and LV-vector empty-virus could be effectively transfected into C3H10T1/2.(2)in the experiments,96 hours after the virus injection into tail vein,fluorescence could be detected by the spectrometer,which could be more easily done when injected with a dose of 108 Tu each mouse than other doses(P<0.05).(3)the expression levels of mRNA and proteins in the RARα-RNAi group were significantly down-regulated compared with those in the empty-virus group and Ringer’s control group(P<0.01).(4)no developmental abnormities were found in the empty-virus group and Ringer’s control group,as well as part of offspring mice.In the RARα-RNAi group and some other offspring mice,we found some malformed cases with atresia anus,developmental abnormalities of the extremities,spine,head and face.(5)compared with other groups,the intestinal tissues of the RARα-RNAi groupdeveloped more slowly,with a thinner muscular layer and lower expression levels of RARα,Shh,HA117(P<0.01),as well as weakly developed gangliocyte.Conclusions:transplacental RNA interfering via slow-virus vector was feasible and effective in affecting the expression of part genes of animal bodies and thereafter constructing experimental models with down-regulated genes.RARα gene played an important role in embryonic development.Part interfering of RARα gene could lead to intestinal and other malformations in mouse;the underlying mechanisms may be related with RARα/Shh signal pathway.PART 4 XPLORATION ON THE ROLES OF RELATEDGENES IN ANORECTAL MALFORMATIONS BY THE2NDGENERATION SEQUENCE TECHNIQUEObjective:we aimed to explore the roles of RARα,RARβ,HA117,DPF3,MAPK1,FOXO3,Shh,EP300 and FGFR2 in anorectal malformations by the 2ndgeneration sequence technique.In addition,these genes were screened for mutation.Methods:blood samples were collected in 50 children diagnosed as anorectal malformations from May 2013 to December 2014 in our hospital.The whole genome DNA was extracted from peripheral blood.After designing the probes specific to the exon regions of RARα,RARβ,HA117,DPF3,MAPK1,FOXO3,Shh,EP300 and FGFR2,prime was multiply amplified by the multiplex PCR amplification method.We then performed sequence with Hiseq2500 and evaluated the SNP and mutation information of samples by SOAPsnpas well as GATK software.Based on database comparison and analyses,mutant sites were identified.Results: the design-integrated capture probes which were specific to target genes could effectively concentrate the exon regions of genome DNA.Mutant sites were found to exist in 31 of 50 samples,15 were missense mutation,and remaining 16 were nonsense mutation.Compared with the incidence of these mutation in Asian population,significant differences were found in 10 sites: 7 were missense mutation and were all located on Fox O3 gene.Of the 7 missense mutation,3 were identified for the first time as c1582 T(29)G,c 874 C(29)T and c1088 T(29)A;5 mutations,including rs199833934/rs9635679,rs201947198/rs9635700,c 1241 T(29)A,c1088 T(29)A and c 874 C(29)T,could cause hydrophobic changes of amino acid.Conclusions:multiplex amplification sequence technique,as a rapid and effective method,could be applied to excavate the mutation related with anorectal malformations.Mutant sites located on Fox O3 may be closely associated with development of anorectal malformations.Fox O3 represents a novel direction to study anorectal malformations.