| Objective:To explore the effect of5-fluorouracil (5-Fu) on reproduction of the Caenorhabditis elegans (C.elegans), and the different expression of relative genes. Discuss the mechanism of the effective of the5-fluorouracil in the experiment.Methods:C. elegans was explored as a model organism in this paper, and added the different concentrations of drugs into the NGM plates and incubated the C. elegans about24hours. Then the numbers of alive C.elegans were counted checked whether there were some eggs on the plates each day. After that the total RNA of the C.elegans was isolated, and we did digital gene expression of the samples to find out the difference of gene expressions and the relative pathways. The Real time RT-PCR was done to confirm the results from the digital gene expression.Results:1. The number of C.elegans embryos were decreased and defected in development. The embryos were decreased after the worms were cultured by petri dish with5-Fu, which has three concentrations as5uM, lOuM and20uM. The worms exposed to20uM5-Fu which had less embryos than the normal worms. The worms which cultured by the concentration of the5uM and10uM group had less embryos than the control group (P<0.05), but had more embryos than the worms in concentration of20uM group (P<0.05)2. Digital gene expression library sequence and mapped sequences to the reference (http://www.ncbi.nlm.nih.gov/). Solexa DGE analysis was performed to get a general idea of the C.elegans samples which cultured by5-Fu and control. Two DGE libraries were sequenced after the cultured with5-Fu and had no treatment with another. We obtained approximately4207526total clean tags and56471distinct clean tags in control; Meanwhile, approximately4167565total clean tags and56530distinct clean tags in5-Fu treatment; There were71.16%and71.78%of the distinct tags were unique mapped to the reference.3. Different gene expression in two libraries. The sample of5-Fu had1147genes which up-regulated and had1067genes which down-regulated genes. There were101genes from up-regulated genes which the value of log2Radio were higher than8while the absolute value of log2Radio of141genes from down-regulated genes were higher than8. When it came to the result that the log2Radio≥1, we could consider the gene expression had the significant differences between5-Fu and control.4. Identification of different expressed genes and the signal pathways analysis. We have found that10067genes had annotated in database and1114genes of them had significant difference in expression. The1114unambiguous transcripts were located in248pathways. The pathway involved the most creditable was Protein digestion and absorption (ko04974) with Q-value of0.000023which followed by Amoebiasis (ko05146) and Focal adhesion (ko04510). We’ve selected28pathways according to the p-value<0.05and Q-value<0.05which could be considered have statistical significance. And four pathways were significant enriched in genes associated with the change of C.elegans cultured by5-Fu.5. Expression of genes mapped to the TGF-beta signaling pathway and ECM-receptor interaction pathway.On the basis of the KEGG database and the analysis of the mapped genes, we have found that several gene expressions had changed in some pathways. TGF-beta signaling pathway and ECM-receptor interaction pathway were the representative for the reason of the change after the treatment with5-Fu. There were19genes had different expression compared with control and the selection rule was that p-value<0.05and FDR<0.05. And these genes also had the relationship with the other two pathways which were Focal adhesion and Hypertrophic cardiomyopathy. The genes in these pathways had upregulated or downregulated, and there were interactions in these pathways caused of the different expressions of these genes6. The confirmation of different gene expression by real-time RT-PCR. In order to verify the reliability of this method,19genes that we have selected randomly from the4pathways had been confirmed. The results indicated that the expressions of19genes were consistent between the real-time RT-PCR and DGEConclusion:The number of the embryos had been declined after the usage of the different concentration of5-fluorouracil. Compared with the control, there are2214genes in C.elegans group of20uM of5-fluorouracil. After mapped to the reference database,4pathways had been confirmed involved in this phenomenon and19genes had been participated in them had the significant meaning in statistics, including ECM-receptor interaction pathway, TGF-beta signaling pathway, Focal adhesion and Hypertrophic cardiomyopathy, which could involved in declining the embryos of the C.elegans. There were14genes participated in both ECM-receptor interaction pathway and Focal adhesion, and these genes were all from collagen family genes. It may had relationship with the cuticle of the vulva of the Ceanorhabditis elegans. daf-14belonged to the TGF-beta signaling pathway and regulated SMAD2and SMAD3, it may had relationship with the differentiation of the germocyte. The mechansim of the CELE_C24B9.3to Hypertrophic cardiomyopathy was unclear. |
PDF Full Text Request