The Study Of Hirschsprung Disease Related Genes And Potential Intervening Molecules Based On Microarray And CMAP Database

PART ONE Identification of HD related genes based on microarrayObjectIn this study, we screened the differential expression of genes between diseased tissues and normal tissues of Hirschsprung disease (HD) by using microarray to obtain differential gene expression profiles. Bioinformatics analysis was carried out on the significantly differential expression of genes to search genes and signaling pathways associated with the pathogenesis of HD. Then we analyzed their possible roles in the development of HD, providing basic datas further for HD related gene function research. By comprehensive understanding of gene expression of HD, we hope to find out HD related genes and explore its role and mechanism in the occurrence and development process of HD. With the result that theoretical basis will be provided to reveal the pathogenesis of HD and search HD related biomarkers.Materials and MethodsWe selected 8 patients for study who were confirmed as HD and accepted one-stage definitive operation in Neonatal Surgery Department of Guangdong Women and Children Hospital in the period of January,2015 to June,2015. Eight pairs of specimens confirmed as diseased tissue and normal tissue by pathology were collected. Agilent human genome expression profile chips of 60110 probes were used to detect for gene expression on 8 pairs of specimens (8 were diseased tissues,8 were their corresponding normal tissues of cut edge). After RNA extraction, purification, reverse transcription and labeling of Cy5 and Cy3, hybridization and scan were performed. According to signaling value>70% and cases of chips with gene expression>80%, expressed genes were selected. We analyzed the expressed genes with the use of SAM software to screen out differentially expressed genes of HD, and then the bioinformatics analysis were performed. These genes were analyzed with Gene ontology (GO) and KEGG pathway to obtain possible genes and signaling pathways associated with the pathogenesis of HD. Through literature retrieval, we found out some of these genes and signaling pathways relevant to the biological process of HD. Finally, RT-qPCR and immunohistochemical staining were performed to verify the test results.Results:1、Microarray According to the standards above, we have selected expressed genes of 11949. Among them,253 differential expression of genes were obtained, of which 213(84.19%) were down-regulated and 40(15.81%) were up-regulated in diseased tissues, with the use of SAM software according to the differentially significant standard of fold changes>2.0 times, or<0.5 times, and q-value<0.05. Generally, total gene expression in diseased tissues is down regulated than in normal tissues. Genes in diseased tissues show degenerative expression based on differential gene expression profiles.2、Bioinformatic analysisSeveral important gene functions and signaling pathways enrich together, such as muscle contraction, muscle system process, G-protein coupled receptor protein signaling pathway, et al, analyzed by Biological Process, Molecular Function and Cellular Component of Gene ontology. On the other hand, some important pathways enrich together, such as vascular smooth muscle contraction and calcium signaling pathway, analyzed by KEGG. These gene functions and pathways are considered related with the pathogensis of HD and have important biological function during the process.Since it is believed that HD development is concerned with migration, differentiation and proliferation of ENCC, we speculate regulation of neuron differentiation and regulation of neuron projection development among these signaling pathways may be related to the pathogenesis of HD the process of ENS development in HD. We combined these two pathways for the neurons differentiation and project related signaling pathway. Five genes (PHOX2B TLX2, CDH2, TNFRSF12A, NEFL) in this pathway showed significant difference(fold-changes were 0.15,0.15,0.34,2.01, 0.25,respectively).They are supposed to be HD related genes,of which PHOX2B, TLX2 and CDH2 had been reported previously but TNFRSF12A and NEFL are found for the first time.3、PCR and immunohistochemical verificationExpression of CDH2, TNFRSF12A and NEFL on the specimens of diseased tissues and normal tissues were tested by RT-qPCR. On average CDH2 was down-regulated 4.6 times in diseased tissues compared with normal tissues (2.2times in the microarray), NEFL 14.9 times(4 times in the microarray);whereas, TNFRSF12A was up-regulated 2.3 times(2.01 times in the microarray). The results of their significantly differential expression imply they may be related to pathogenesis of HD, which is consistent with the results of microarray. The immunohistochemical result of the above 3 genes showed that CDH2 and NEFL expression were negative or weakly positive in nerve fibers of diseased tissues but strongly positive in myenteric plexus of normal tissues; whereas, TNFRSF12A expression was strongly positive in nerve fibers of diseased tissues but weakly positive in myenteric plexus of normal tissues. Our RT-qPCR data and immunohistochemical results were consistent with the findings from microarray in this study.Conclusions:1. There are obviously difference in the level of mRNA between diseased tissue and normal tissue of HD by Genome-wide expression chip which generally showed degenerative expression. The obtained differential expression genes may refer to some important biological process in the development of HD. More study is needed to verify the function of these genes in ontogenesis of HD.2. Apparently unusual gene expression of neuronal differentiation and project related signaling pathways in HD tissues verified by routine molecular biological techniques, indicates that the signaling pathways involved in the occurrence and development process of HD, of which PHOX2B, TLX2, CDH2 had been reported previously but TNFRSF12A, NEFL were found for the first time.3. Microarray is a method of high throughput analysis for screening the differential gene expression profiles which might be of benefit to efficiently select the HD relative genes. Genes and pathways obtained from this study provides a theoretical basis for reveal the pathogenesis of HD and for the future exploration of HD related biomarkers and potential targeted intervention measures.PART TWO Screening of potential intervening molecules of HD based on CMAP databaseObject:We screen the small molecular compounds that are negatively related with the HD gene expression based on the gene differential expression from diseased tissues to normal tissues of HD by using Connectivity Map (CMAP) database and bioinformatics method in order to investigate potential drugs for HD in the future, further to open up a new treatment method for HD.Materials and Methods: Differentially expressed genes of diseased tissues and normal tissues of HD were converted into query signature format documents, and then logined on CMAP database website, obtained compounds associated with HD. We screened the small molecular compounds that were most negatively related with the HD and analyzed their possible mechanism in the development and treatment of HD through literature retrieval.Results1. The top ten small molecular compounds that most negatively related with the HD according to the enrichment of negative scores from high to low were HC-toxin, Phenacetin, Etifenin,5666823, PHA-00665752, Tubocurarine chloride, AH-6809, Monobenzone, Fenspiride, Selegiline, which were intervening molecules that reverse effected HD gene expression.2. Through literature retrieval, the main mechanism of these small molecular compounds are in the order. HC-toxin is a kind of histone deacetylase inhibitor which can induce nerve cell differentiation. Phenacetin is acetanilide antipyretic analgesics. Etifinin can be used as liver developer.5666823, a not yet named small molecule compounds, has the effect of promoting nerve regeneration, PHA-00665752, a c-Met inhibitors, commonly used in cancer research. Tubocurarine Chloride is a kind of non depolarizing muscle relaxant. AH6809 is a kind of specific prostaglandin E2 (PGE2) receptor antagonist. Monobenzone is a kind of local used decolorizing agent. Fenspiride is a-adrenaline receptor blockers. Selegiline, a kind of monoamine oxidase inhibitors, has the nerve protective effect.3. Through index, we found that HC-toxin (histone deacetylase inhibitor) has the effect of inducing neuronal differentiation, Selegiline (monoamine oxidase inhibitors B) have nerve protective effect. HC-toxin and Selegiline were intervening molecules that have reverse effect for abnormal gene expression in neural differentiation and protect signaling pathways of HD. The functional mechanism of pathways above have a close relationship with HD biological processes, so we speculated that these two compounds have the prospect of becoming the potential drugs for HD.Conclusions:1. HC-toxin and Selegiline were intervening molecules that have reverse effect for abnormal gene expression in HD due to their promoting neural cell differentiation and protecting neural cell, revealing these compounds have potential applications outlook in the treatment and prevention for HD.2. CMAP database can be used as effective tool in prediction of potential intervening molecules of prevention and treatment for diseases. These obtained HD potential intervening molecules, based on expression profile and CMAP database, will provide theoretic foundation for further open up new methods of the treatment for HD in the future.