Screening Of Differential Expression Genes Between Primary Breast Cancer And Its Lymph Node Metastasis By Microarray And Verification Of Clincal Cases

Background:Breast cancer is one of the frequent malignant cancers in female. Despite important advances in diagnosis and therapy, breast cancer patients' overall survival have not been improved markedy. Recurrence and metastasis are important death reasons of breast cancer patients. The key of improving survival rate and outcome is to evaluate accurately metastasis potential and prognosis early to guide clinical therapy. Aquiration of metastasis ability is regulated by changes of metastasis related genes. The development of cancer is a multifactor, multistep complex process, and involves in coopperation of many genes. Gene microarrays, the emerging high-throughput molecular technologies, make it possible for us to explore gene expression profile on a genome wide scale. Gene microarrays provide a powerful tool for studying the molecular mechanism of gene changes during the oncogenesis and processing and have speficial values in research of metastasis and prognosis. Objective:To screen genes related to breast cancer metastasis by comparing difference of expression profile between primary breast cancer and lymph node metastasis. To verify difference gene in the level of mRNA using clinical cases, and analyze the relation of differential gene mRNA expression level with metastasis and clinical factors. Methods:Comparing difference of gene expression profile between primary breast cancer and its lymph node metastasis of 10 breast cancer patients, using SPA to prepare fluorescence-labeled targets and then having them hybridized with Oligomicroarray concluding 21,000 human functional genes, to screen out 1.5 fold or more differential expression genes in at least 5 pairs of samples. To analyze differential gene function and gene network using GoMiner and String software. Average cluster was used by cluster software. Identified the genes which was screened out by real-time PT-PCR. Results:1. Preparing targets using SPA method reduced initial total RNA to 0.25ug.2. There are 57 genes screened out, of which 19 were up-regulated expression and 38 were down-regulated expression in metastasis tissues. 8 genes were related to cell migration and adhesion, 14 genes to signal trasduction, and 14 genes to cell growth or metabolism. 17 genes of 57 differential expression genes including in FN1 COL1A1 COL1A2 COL6A1 COL11A2 GZMK, EVI2B LCP1 POU2AF1 LGALS1 IGF1 IL7R, CD48 IL6ST RGS1 MEF2C PTGDS build up a network.3. Cy3 and Cy5 fluorescence intensity in 10 microarrays were analyzed using average cluster after global normalization. As a result , in 8 cases, the paired primary tumor and metastasis samples were closely matched; in the remaining 2 cases, the difference between primary tumor and metastasis samples was more evident.4. Differential genes OSF-2, SELL, IGFBP-5 was vertificated in large clinical cases. As a result, expression of OSF-2 mRNA in lymph node metastases is lower than primary breast cancer(t=2.938, P=0.006); in primary cancers with c-erb-2 positve is lower than primary cancers with ER negative 2.4-fold (t=2.258, P=0.027) . There is no correlation between SELL mRNA expression level and lymph node clinic stage, tissue grade ,pothologic type , ER and PR. Expreesion of IGFBP-5 mRNA in primary cancers with lymph node metastasis is higher than...