| Background:Congenital anorectal malformation(ARM)is a congenital malformation caused by neonatal anorectal developmental disorder,with an incidence of 1/5000-1/1500 and male preponderance.The arrest in the development of the urorectal septum towards to the cloacal membrane may be the main event leading to ARM.Great progresses have been obtained in the etiology of congenital anorectal malformations.Both genetic aberrations and environmental influences have been studied in recent decades.The related genes and signal pathways are complex.Many genetic and signal pathways aberrations that affect this normal development have been studied in recent past.It is suggested that Shh,Gli,Bmp4,Wnt5a,Wnt3a,Fgf,Fgf10,Hox and Cdx1 play an important role during the process of ARM.With the advent of the post-genomic period,proteomics has played a critical role in exploration of life.Proteomics focuses not only on the regulation of the transcription level but also on the regulation of the translation and post-translational level.Proteomics is crucial for protein post-translational modifications,subcellular localization,migration,and protein-protein interactions.The amino acid residues are much larger than those of nucleotide residues,and contain a great of post-translational modifications such as phosphorylation and glycosylation,research of proteins must rely on high-throughput and highly sensitive technologies.Accumulating evidence has revealed that proteins are not only the products of gene but also mediate cell processes such as proliferation,migration,differentiation,and apoptosis.The etiology of ARM in protein level is rare relatively present,so this research intends to make a preliminary exploration of the pathogenesis of ARM at the protein level.Methods:18 ARM infants were treated in our department,and sera were collected and saved.Age and gender matched 18 infants without digestive malformations or infectious diseases were also tested as cases.Quantitative analysis of proteins in samples was performed and differentially expressed proteins were screened out by iTRAQ technology.VCP,PCDH24 and Fibulin-1 were selected based on the fold change and P-values.Western Blot verification was performed on the population samples.According to the appeal results,VCP and PCDH24 were selected for cell experiments.RT-PCR was used to select the interference sites with the best efficiency.After si-RNA was used to silence PCDH24 and VCP,the number of changes in Hcope cells about proliferation and apoptosis were observed.Results:A total of 701 differentially expressed proteins related to ARM were screened by protein microarray,of which 112 up-regulated proteins and 77 down-regulated proteins were observed with the difference of ≥1.5 or ≤0.67,and 73 significant proteins were further selected with the difference of ≥3.0 or ≤0.33,containing 44 up-regulated and 29 down-regulated.1 protein up-regulated and 2 down-regulated with the difference of ≥4.0 or ≤0.25.The Gene Ontology annotation involved molecular functions(binding,catalytic activity,enzyme regulator activity,molecular transducer activity),cellular components(extracellular region,organelle),and biological pathways(cellular process,biological regulation,metabolic regulation).COG annotation indicated the top five proteins as follows:a,post-translational modification,protein turnover,chaperones;b,general function prediction only;c,signal transduction mechanisms;d,cytoskeleton;e,translation,ribosomal structure and biogenesis;KEGG pathway annotation showed 12 up-regulated proteins were mainly involved in phagosomes,while 21 down-regulated proteins were involved in complement and coagulation cascades.In differential protein enrichment analysis:Gene ontology(GO)enrichment analysis mainly involved as follows:a,Component Ontology:extracellular region part,extracellular region,vesicles,membrane-bounded vesicle and membrane attack complex;b,Function Ontology:serine-type endopeptidase inhibitor activity,pattern binding,polysaccharide binding,double-stranded RNA binding and collagen binding;c,Process Ontology:response to external stimulus,regulation of chemotaxis,response to wounding,inflammatory response,and regulation of leukocyte chemotaxis.Pathway enrichment:The number of up-regulated and down-regulated statistical proteins in the Complement coagulation cascade response pathway are the largest of all,7 and 21 respectively.In Huntingtons disease pathway,only 4 up-regulated proteins are statistical.In Western Blot,The relative grey of VCP in the serum of the ARM group was 0.74 ± 0.07,and that of the control group was 0.48 ± 0.05,suggesting that the expression level of VCP in ARM was significantly higher than that of the control(P-value<0.05).The relative gray of PCDH24 in the serum of ARM group was 0.41 ±0.08,which was lower than control(0.78 ± 0.09),suggesting the expression level of PCDH24 in ARM was lower than that of the control(P-value<0.05).The validation results are consistent with those of the protein chip.Experiments in vitro,after PCDH24 was silenced by si-RNA,the relative numbers of Hcope cell at the 48 hours were:mock:2.634 ± 0.054,control:2.522 ± 0.096,and case:2.157± 0.062.The P-value between case and control was<0.05,statistically.After VCP was silenced by si-RNA the relative cell numbers of the Hcope cell was as follows:mock:2.607±0.094,control:2.464±0.049,and case:2.098±0.037,P-value between case and control was<0.05,statistically.Apoptosis experiment:After silencing PCDH24 and VCP by si-RNA,the number of apoptotic cells were increased..Conclusions:Protein microarray can screen out the proteins in the pathogenesis of anorectal malformation effectively.Many proteins are involved in the pathogenesis of ARM.The VCP was highly expressed in the serum of children with ARM,and PCDH24 was lower,correspondingly.The results above are consistent with the expression trend of the protein chip,suggesting this experiment was reliable.Both VCP and PCDH24 promoted cell proliferation and inhibited apoptosis in Hcope cells,considering VCP and PCDH24 might be related to the development of ARM. |
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