rker genes were overexpressed in U3-C and U3-DT. U3-A has the potential to differentiate into several types of cell, including adipocytes and osteoblasts. Gene expression of markers characteristic of these cell types was down-regulated in U3-DT cells. Expression of adhesion-related genes was strongly suppressed in U3-C and U3-DT, with the exception of COL4A5, a member of the collagen family. Ecadherin is one of the most important molecules in cell-cell adhesion in epithelial tissue. CDH1 expression was markedly reduced in U3-C. Suppression of CDH1 expression is one of the primary molecular events responsible for dysfunction in cell-cell adhesion, such as that which occurs in tumor progression. On the other hand, N-cadherin expression remained high. Other adhesion molecules were also 13 / 23 Alteration in Gene Expression on Transformation expressed at low levels comparable to those in U3-A, as observed in tumor cells. Expression levels of integrins, receptors that mediate attachment of cells to extracellular components such as collagen and fibronectin, were normal; however, collagen expression reduced considerably with tumorigenic progression. These results suggest that reduced expression of these adhesion molecules plays an important part in promoting invasion into surrounding stroma. Angiogenesis, invasion, and metastasis Oxygen and nutrients supplied by vasculature are crucial for cell function and survival. Beyond a certain size, tumors induce blood vessel growth by secreting various growth factors, thereby allowing the tumor to obtain oxygen and essential nutrients in vivo. In UE6E7T-3, genes encoding major contributors to angiogenesis were expressed at remarkably low levels, although the expression of genes encoding their receptors was high. Another major contributor to angiogenesis PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19785914 is matrix metalloproteinase. These proteases were also expressed at low levels, as were the MMP inhibitors . Similar low levels of expression were observed in invasion- and metastasis-related genes. Cell-cell and cell-matrix adhesion molecules play an important role in cell migration. E-cadherin, which is considered an important suppressor of invasion and metastasis, rapidly decreased in gene expression at Stage III. This reduction is likely to represent a key step in the acquisition of invasive properties. Furthermore, integrin family genes were also expressed at low levels. These results suggest that even U3-DT may still be in the earliest stages of tumorigenesis. Alternatively, in terms of angiogenesis-, invasion- and metastasis-related gene expression, tumorigenesis in fibroblastic cells may differ from that of epithelial cells. Validation of RNA-Seq To evaluate of RNA-Seq analysis, we chose 29 genes at Stage IV in each pathway identified by RNA-Seq for qRT-PCR analysis. The expression pattern of the genes determined by qRT-PCR DCC 2618 chemical information analysis is similar to the results of RNA-Seq analysis, although slight differences in expression level were detected between two methods. In addition, to confirm the accuracy of RNA-Seq data we compared the expression levels of 41 samples according to RNA-Seq and qRT-PCR analyses, including 29 genes at Stage IV analyzed by Method A and 4 genes at Stage PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 II-IV analyzed by Method B, and obtained 
a high concordance between these two sets of data. A coefficient of 0.76 corresponds to a significance level of slight less than 0.1%. This indicated that our RNA-Seq could reliably measure gene expression differences. GPC5 expression a