Genes Associated With Metastasis Of Mouse Dendritic Cell Sarcoma And Bioinformatics Analysis

It is a common feature that the tumor cells metastasize to other tissues or organs, and the metastasis is the principal reason leading to death in individuals with tumor. Yet its molecular basis is not well understood. Especially, the recent studies have indicated that the key genes associated with metastasis and the mechanisms are diverse among the different tissues or of tumor types. Metastasis is a complex process involving multiple stages, such as local invasion, transit through the bloodstream, extravasation, proliferation and survival within a favorable target organ. We use the model of highly and poorly metastatic cell lines from the same parent (mouse Dendritic Cell Sarcoma, DCS) to examine differences of their gene expression profiles with Affymetrix high-density microarrays. We try to screen the genes related with lung metastasis of mouse DCS by the high-throughput method. The followings are the main points of our studies.1. The study of the genes associated with metastasis of mouse DCSMouse dendritic cell sarcoma (DCS) was a cell line that established and identified by Institute of Basic Medical Sciences at Chinese Academy of Medical Sciences. They isolated the highly and poorly metastatic cells (DD1 and DG6) from the parent cell line DCS by limited dilution method. It is a good model to study the metastasis of mouse DCS to lung for the nearly same genetic background between DD1 and DG6 subclones. We use the Affymetrix high-density microarrays to screen the genes that may be associated with metastasis by comparing the differences of genome-wide expression.The results show that expressed genes are quite different in transcription factors, RNA process, cell proliferation and apoptosis, transport, protein synthesis and modification, and other aspects between the DD1 (highly metastasis to lung) and the DG6 (poorly metastasis to lung) cells. Furthermore, several expression-difference genes have identified by the methods of RT-PCR and Immunocytochemistry, such as the transcription factor Ehf that influences the cell differentiation, potential tumor suppressors and apoptosis related gene Pedf, adhesion molecule Nectin-3 and the chemokine Mcp-1(CCL2). The results suggest these genes might be associated with the DCS metastasis.2. DNA methylation and the gene expression of mouse DCSWe choose some obvious up or down-regulated genes of transcription factors Smcy and Ehf, potential apoptosis molecule Pedf and adhesion molecule Nectin-3 to detect the DNA methylation status. It is to find whether the DNA methylation status in promoter region controls these genes over or lower-expression and try to explore the possible mechanism of gene expressions associated lung metastasis of mouse DCS. The genome DNA of DD1 and DG6 cells are transformed by bisulfite and as a template for PCR amplification using the primers that sequences not contain CG sequences. The DNA methylation status can be obtained by sequencing for conversion of the unmethylated cytosine is transformed into thymine with regions of interest. The result demonstrates that there is a site of CG difference in Pedf gene between DD1 and DG6 cells (changing C into T), and not obvious alteration in Smcy and Ehf genes between the two cells. From the results, we tendencious consider that the DNA methylation is not the main element to influence the three gene expression. There may be some other elements, such as the interaction with other transcription factors, to impact on the up or down-regulation expression.3. Gene expression analyses of mouse DCSWe isolated and screened two tumor cell clones DD1 and DG6 with different capacity of metastasis from the same parent cell line, a mouse dendritic cell (DC) sarcoma, using limited dilution method. The genome-wide expressions of DD1 and DG6 cells are done by Affymetrix's MOE-430A microarray. The expressionprofiles related with mouse DC development are downloaded from GEO at NCBI and ArrayExpress at EBI database. In order to compare expression of DC sarcoma and DC developmental arrays which performed by MG-U74av2, we have screened the best matched probesets between MOE-430A and MG-U74av2 according to the probe identities from Affymetrix technical annotation. After the normalization of 11 housekeeping genes across the 34 arrays (2 DC sarcomas and 32 DC developmental arrays), all these expression profiles are analyzed by the methods of hierarchical clustering, principal component analysis, nearest-neighborhood and self-organizing maps. The results indicate that expression profiles of DC sarcoma are closer to the DC progenitors and hematopoietic stem cells from bone marrow compared with the sorted DCs from spleen. The results support the hypothesis that cancers (tumors or sarcomas) arise from stem cells. Furthermore, the results also suggest cells isolated from mouse are DC sarcoma cell lines for they are similar to the DC progenitors in gene expressions.4. Bioinformatics analysis on the transcription factors containing homeoboxThe results of Affymetrix microarray show that En1(Engrailed 1 homeobox)and Hlxb9(homeobox gene HB9)are up-regulated in DD1 cell, lhx2(LIM homeobox protein 2)and prrx2(paired related homeobox 2)are down-regulated in the cell. The result indicates that the homeobox gene may be related with metastasis of mouse DCS. The proteins with 'homeobox' (HOX) domain play an important role in embryonic development, gene regulation, cell differentiation and tumorigenecity. Hydrophobic and positively charged residues in HOX domain are conservative, and the proteins with HOX bind to the rich A or T nucleotides in the gene promoters. Some other domains are found in the proteins together with HOX, such as PAX, POU, LIM, OAR, ELK, SIX, bZIP, PHD-finger, Engrailed, hexapeptide domains. These"complex homeobox"also take part in tumorigenesis by the way of gene fusions and deregulation. Recently, the new homeobox gene Nanog and OCT4(POU complex homeobox)are discovered that they are essential in self-renewal of embryonic stem cell.5. Finding binding sites by comparing the homology of promoter sequencesInsulin promoter factor-1(IPF1 or PDX1,pancreatic duodenal homeobox-1)is an important transcription factor containing homeodomain which takes part in regulating the expression of insulin gene as well as neurogenic differentiation factor 1(NDF1 or NeuroD) and hepatocyte nuclear factor 4(HNF4). All of them are MODY (maturity onset diabetes of the young) genes. The dysfunction of these factors is the key genetic element for type 2 diabetes. The sequences and domains of the three factors are homologous by the residues alignment of human, mouse and rat. It is supposed that the nucleotide binding sites are similar in the promoters of insulin gene. The ClustalW program of multiple alignment is used to compare the promoters of insulin genes. Meanwhile, the nucleotide binding sites of NDF1, IPF1 and HNF4 are searched in the TRANSFAC gene regulation database. The conservative nucleotide sequences are found in insulin promoters and some sequences are identified as the binding sites of several known transcription factors. There are also other conservative DNA sequences in the promoters perhaps are the binding sites of unknown factors. The alignment could offer a simple and applicable method to find and confirm the nucleotide binding sites of DBPs for the molecular biology experiments.