Preliminary Study On The Biological Function And Mechanism Of AGR2

AGR2is a secreted protein and widely found in the lung, breast, prostate, pancreatic gland and colon. It is also overexpressed in the tumor tissue of these glands and regulates tumor cell survival, growth and metastasis. The expression of AGR2protein is found to be relative to the progress of tumor development in breast cancer, prostate cancer and pancreatic cancer. Thus, AGR2is considered to be a promising biomarker for early diagnosis and prognosis on cancer.The previous studies explored the effects of AGR2on cellular process by overexpressed or deleted its protein in cancer cell lines. However, the biological regulation and underlying mechanism of AGR2as a secreted protein are poorly understood.In this study, we firstly purified AGR2protein from prokaryotic lysate by using Glutathione Agarose protein purification technology, and then checked the activity of AGR2by stimulating breast cancer cell line MCF7. A clear signal was detected by the xCELLigence System in cells with AGR2treatmented after20min, and the signal could be suppressed by some cytoplasmic protein kinase inhibitors specificitily. When cells were treated with AGR2, the activation of P38and AKT were also observed with specificity antibody by Western blotting. The results suggested that purified AGR2protein could quickly activated the signal in target cells.To further understand the signaling mechanisms of AGR2, we stimulated MCF7by AGR2protein, and then performed the gene expression profile chip assay to evaluate the expression level of its target genes. Meanwhile, bioinformatics methods were used to predict the signaling pathways through which AGR2regulated the expression of target genes. After12hours stimulation, the expression patterns of lots of signal molecules were significantly changed. Then,20of them (VEGFA, VLDLR, FZD10, CCL2, AXIN2, etc.) were validated with realtime RT-PCR and found these genes expression were increased or decreased in mRNA level, which was consistent with the results of chip assay. AGR2protein may paly a role through the mTOR and other signaling pathways by GO term enrichment analysis, pathway analysis and cluster analysis.This study demonstrated that secreted AGR2protein can quickly evoke the signal in target cell. The deeply discovery of the regulation of AGR2will promote the understanding of the role of AGR2as a molecular marker on diagnosis and prognosis of cancer.