Identification of the differentially expressed genes between primary breast cancer and paired lymph node metastasis through combining mRNA differential display and gene microarray

Abstract

To identify and clone the genes related to breast cancer metastasis through comparing the mRNA expression profiling between primary breast cancer and paired lymph node metastasis. First, primary breast cancer cells and paired lymph node metastasis tissues were used to identify their differentially expressed genes by using mRNA differential display, and fragments of differentially expressed genes were obtained by gel cutting and cloning to pGEM-T vector, then sequencing and blasting with the GenBank for their homologous genes. Then, the obtained genes were validated by using reverse dot blot hybridization and gene microarray. Finally, the mRNA expression levels in the 7 breast cancer cell lines with different metastasis behaviors were detected by real-time RT-PCR. The mRNA expression profiling of the metastatic breast cancers in lymph node was similar to that of their primary cancers. 16 differentially expressed gene fragments were obtained from mRNA differential display; four of them were found to be unknown genes, and had been accepted by the GenBank, with the accession numbers BG518428, BG518429, BM005520, and BM005521. Of the other 12 genes, 8 were known genes, and 4 were genes with known sequences but unknown functions. The kinesin-like DNA binding protein (KNSL4) and dihydropyrimidine dehydrogenase (DYPD) were down-regulated in the metastatic tissues in comparison with the paired primary tissues, which was identified by both radioactivity-labeled reverse dot blot hybridization and fluorescence-labeled gene microarray hybridization. KNSL4 mRNA expression had a down-regulation trend with increasing metastatic ability of breast cancer cell lines. The gene expression profiling of the lymph node metastasis is almost similar to that of their primary breast cancer, indicating that the metastatic cancer cells in lymph node are the subclone with higher metastasis ability of the primary cancer cells, and that some differentially expressed genes between them may be involved in the change of metastasis phenotype. KNSL4 and DYPD are potential metastasis-related genes, and KNSL4 mRNA expression is correlated with metastasis behaviors of the breast cancer cell lines, suggesting that KNSL4 may play an important role in the metastasis process of breast cancer.