Regulation Of S100A4 Gene Expression Under Hypoxic Microenvironment In Gastric Cancer Cell Line

ObjectiveGastric cancers incidence and mortality is among the highest in China, and its development is a multi-step and multigenetic process. Although a large number of investigation, their molecular mechanisms are not clear. S100A4 is an important gene related to the invasiveness and metastasis of tumor. It plays a key role in the process of metastasis via promoting the migration of tumor cells, expression of protein hydrolase, adhesion of cells to matrix, and escaping of tumor cells from primary tumor. The S100A4 expression is significantly increased in tumors, such as esophageal cancer, lung cancer, colorectal cancer, and pancreatic cancer etc. Our researches have confirmed the close correlation between the Overexpression of S100A4 gene and gastric cancer's infiltration, lymphode metastasis and in vitro invasiveness of gastric cancer cells. LEE and Cho YG also reported the similar results.Recently the relationship between tumor microenvironment and its invasiveness and metastasis is well recognized in oncology, especially the hypoxic microenvironment. Hypoxia leads to the expression of a number of gene products that are involved in tumor progression, invasion, and metastasis formation. The most important one is thought to be HIF-1 (hypoxia inducible factor 1), a key factor in this process. HIF-1 is activated under hypoxia and then regulates the expression of metastasis-associated genes, accordingly affects the biological characteristics of tumor cells. We predicted a putative hypoxia responsive element (HRE) in the first intron of S100A4 gene by using bioinformatics software. So we speculate that microenvironment hypoxia may up-regulate the expression of S100A4. Therefore, we treated gastric cancer cell line BGC823 cells with hypoxia and found that hypoxia could up-regulate the S100A4 gene expression. In this study we tried to explore the mechanism by which hypoxia regulate S100A4 gene expression. Our research will be not only useful for clarifying the molecular mechanism of gastric carcinogenesis and development, but also for instructing the diagnosis and therapy of gastric cancer.Materials and methods1. Materials: Gastric cancer cell line BGC823 (derived from low differential adenoma); reagents for cell culture; hypoxia inducer CoCl₂; reagents for electrophoresis mobility shift assay (EMSA); chromatin immunoprecipitation (ChIP) and PCR etc.2. Methods: BGC823 cells were conventional cultured and then treated with CoCl₂ The expression of HIF-1 protein was analyzed by Western Blot. Bioinformatics analysis suggested a HIF-1 binding site in S100A4 gene. EMSA was carried out to identify the binding of HIF-1 to hypoxia responsive element (HRE) within S100A4 gene in vitro conditions. Furthermore ChIP assay were used to test the binding of HIF-1 to this site. Analysis of the HRE sites and around was done by sequencing in order to find mutation or SNP.Results1. The results of Western Blot showed that the expression of HIF-1 protein began to increase from 6th to 48th hour after treatment with CoCl₂, in BGC823 cells.2. EMSA showed that there was a HRE in the position +329～+334 of the S100A4 gene in vitro. ChIP assay obtained the same results in vivo.3. Sequencing and Blast analysis showed that there was no variation in the HRE and its surrounding sequence in S100A4 (+44～+550) from 25 gastric cancer samples.Conclusion1. We have learnt the characters of HIF-1 protein expression after treatment with CoCl₂.2. The position +329～+334 of the S100A4 gene was a hypoxia responsive element (HRE).3. No sequence variation was found in the region +44～+550 of S100A4 gene in 25 gastric cancer samples.