Molecular Biology And Bioinformatics Analysis Of Colorectal Liver Metastasis

Part one: Detections of related-genes and interacted-proteins for ST14, a tumor metastasis-related geneColorectal cancer (CRC) is one of the most popular cancers today. The incidence rate of the colorectal cancer has increased to top three or five these years in China. The liver is the most common site of colorectal cancer metastasis. About 15%~25% colorectal cancer patients were confirmed liver metastasis when they were diagnosis. An additional 20 percent will develop liver metastasis subsequent to treatment of their primary cancer.From the invasion into the extracellular matrix (ECM) to the proliferation in the liver, many genes and proteins were involved in the whole procedure. ST14 gene was found as a tumor metastasis-related gene because it can degraded ECM. ST14 was expressed at different level in the different tumor. The expression of ST14 in colorectal cancer was not detectable or at lower level, however up-regulated in the other cancers. Different cleavage forms of ST14 were reported by different research groups. Several proteins inhibited the activity of ST14, such as Hepatocyte growth factor Activator Inhibitor-1 (HAI-1) and Aprotinin;and other proteins can active ST14, such as Proteinase-Activated Receptor 2(PAR2), the precursor of Hepatocyte Growth Factor/Scatter Factor(HGF/SF) and the precursor of urokinase-type Plasminogen Activator(uPA). ST14 contains a trypsin-like serine protease catalytic (SP) domain and an N-terminal transmembrane signal anchor with two regulatory modules: two tandem repeats of the complement sub-component Clr/s (CUB) domain and four tandem repeats of the lowdensity lipoprotein receptor (LDLR) class A domain. Domains in the ST14 were important for the activation of ST14 and the details were under construction. It is very helpful to understand the role of ST14 in the rumor metastasis if we can find related-genes and interacted-proteins of ST24. The following works were done.1. Detection of gene expression profiles in RKO-ST14 cell line using microarray method, confirmed the up-regulation of ITGB1, MMP1, MALAT1, AP1S3 and Oxidative Phosphorylation Pathway after ST14 was transfected into the RKO cell line.2. Construction of GST fusion protein for the SP, CUB and LDLR domains in the ST14 and their corresponding mutants, confirmed the new interacted protein TMEFF1 binding to CUB domain of ST14 using a modified GST pull down method with gradient elution. Quantitative real-time PCR revealed that the expression of the TMEFF1 gene was dependent on the transfection of the ST14 gene in the RKO cell line. These two genes were co-upregulated in kidney tumors versus normal tissues, consistent with our results that showed in RKO cells.Part two: Integration analysis of cytoband, pathway and gene expression profileChromosomal instability is also an import feature of CRC. Many chromosome copy number aberrations (gains or losses) were confirmed in colorectal cancer and colorectal liver metastasis. Pathway information revealed the interaction and relationship of genes. All genes in the tumor metastasis located in a cytoband or belonged to a pathway. ST14 gene was one of them and several genes had been confirmed to inhibit or active it However, it is difficult to confirm the relationship between the phenotype and the genotype (changes of genes, proteins, or chromosomes) because cancer is an extremely complex disease. In consideration of the breakthrough of high-throughput technology, such as comparative genomics hybridization (CGH) and microarray method, and the whole is great than the sum of parts, we can shift traditional point of view in cancer research and analyze it more integrated.We detected ST14-related genes in the RKO cell line using microarry method and detected interacted proteins of ST14's domains using GST pull down method. Several genes and a protein were confirmed to relate or interact with ST14. Based on the gene expression profiles data of the primarycolorectal cancer and liver metastasis tissue, we applied singular value decompositions (SVD) method, then integrated with cytobands and pathways information, and found the role of cytoband Ilq24 (ST14 located in this cytoband) and ST14-related genes in the colorectal liver metastasis. The following works were done.1. Based on the gene expression profiles data of the primary colorectal cancer and liver metastasis tissue, we applied singular value decompositions (SVD) method, then integrated with cytobands information, we confirmed the down regulation of 4ql3 in the primary colorectal cancer and dominant gene of this cytoband was UGT2B15, cytoband 4q22 was up regulation in the liver metastasis tissue and dominant gene of this cytoband was Osteopontin, cytoband Ilq24 that involved ST14 gene had not show the relationship with colorectal liver metastasis, and several new colorectal liver metastasis-related cytoband were found.2. Integration analysis of pathways and gene expression profiles, confirmed the up regulation of ST14-related genes in the primary colorectal cancer, the up regulation of ECM-related genes (ECM chip, superarray) in the primary colorectal caner and liver metastasis, tumor metastasis-related genes (Tumor metastasis chip, superarray) and uPA that was activated by ST14 were up-regulated in the primary colorectal cancer and down-regulated in the liver metastasis. These results revealed the different gene groups plays different roles in the colorectal liver metastasis. The cancer cells that involved the invasion in the ECM and the proliferation in the liver could be different. Down regulation of ITGB1, ST14 and Oxidative Phosphorylation Pathway in the primary colorectal cancer was consistent with the result in the RKO cell line.Conclusion1. Integration analysis of chromosome information and gene expression profile data can representthe whole expression level of genes in a cytoband, and find the genes that cannot be identified by the traditional methods. The results of integration analysis for pathway information and gene expression profile data were similar with the former results, however the complicated information of pathway reduced the validity of this method.2. Application of gradient elution in GST pull down method and construction of mutants that without biological activity an negative controls can reduce the false positive results and get theinteracting proteins indeed.3. The genes in the cytoband 4ql3 were down-regulated in the primary colorectal cancer and the dominant gene in this region was UGT2B15. The genes in the cytoband 4q22 were up-regulated in the liver metastasis tissues and the dominant gene in this region was Osteopontin. The former cytoband and genes were related with the colorectal cancer genesis and the latter were related with the colorectal liver metastasis. These results consistent with the existent reports.4. TMEFF1 maybe participate in the activation of ST14 by interacting with the CUB domain. Quantitative real-time PCR revealed the co-upregulation of TMEFF1 and ST14 in colorectal cancer cell line RKO and kidney cancer tissues. These results implicated the relation of co-upregulation of these two genes and tumor genesis of kidney cancer.5. ST14-related genes (HAI-1, ITGB1, uPA and PAR2) were up-regulated in colorectal primary cancer. In these genes, uPA was up-regulated in primary tissues and down-regulated in liver matastasis tissues, however ITGB1 just down-regulated in the colorectal primary cancer tissues. These results revealed the heterogeneity of cancer cells in the tumor genesis and metastasis and implicated the different gene groups plays different roles in the colorectal liver metastasis.