The Expression Profiling And Function Of Gastric Cancer Related Gene GDDR(GKN2/TFIZ1)

GDDR was a novel gene obtained by RACE(rapid-amplification of cDNA ends) from suppression subtraction library established for screening down-regulated genes in gastric carcinoma by Ph.D Du Jianjun in 2003,whick is down-regulated in gastric cancer. GDDR localizes at the site of chromosome 2p13.3. The whole length of its cDNA is 751bp which includes a 555bp open reading frame and thus encodes 184 amino acids with the Molecular Weight of about 20 KD. Bioinformatics study predicts that GDDR contains a BRICHOS domain and a signal peptides sequence which locates in the 1-22aa of N-terminal. Previously study has showed that transient transfection of GDDR could suppress the proliferation of gastric cell lines.GDDR was specifically amplified only in stomach from the 11 tissues cDNA library.GDDR was one of the most obviously reactivated genes after eradiation of Helicobacter pylori and its loss is associated with shorter overall survival in the intestinal subtype. Previously data has also established that GDDR gene was down-regulated by pro-inflammatory cytokines(TNF-α,IL-1β,IL-6,IFN-γ) in gastric cancer and colonic cancer cell lines while up-regulated by TGF-β1 in the colonic cell line. GDDR expression was significantly reduced in gastric adenocarcinoma cell line by the active form of NFκB transcription factor. Down-regulation by IL-6 was mediated by C/EBPβtranscription factor in case of HT-29 cells. GDDR and TFF1 can form a heterodimer via a disulfide bond, resulting in the reference of GDDR as TFIZ1 (for trefoil factor interactions(z) 1).In view of the above data, this subject aims to provide more information of GDDR's biological function and its role in tumorigenesis and development through study on its expression profiling and the correlation of its expression with cell proliferation. To provide a powerful tool for further study of GDDR, we expressed GDDR in prokaryotic system and then purified the protein for antigen to immune mice for monoclonal anti-body.The main results are as follows:1. Elucidating the expression pattern of a novel gene should help to explain its function. First, we analyzed the expression of GDDR by Northern blotting in multiple tissues. Two human normal multiple tissues membranes containing 8 tissues each were used for analysis of the expression of GDDR and the internal control wasβ-actin. GDDR is highly expressed in the GI tract, including the stomach, small intestine, pancreas, colon and rectum, while it is absent in the heart, skeletal muscle, liver, spleen, and brain. In other words, GDDR is specifically expressed in the GI tract. Determining subcellular localization is a prerequisite for identifying the function of a gene. Expressing vectors, in which GDDR were fused in-frame with flag and EGFP at the C-terminus respectively , were constructed to transfect into the SGC-7901 cell lines transiently. The GDDR fusion proteins were visualized as dot like granules distributed in the cytoplasm and pericellular membrane under the confocal fluorescence microscopy field. It is highly possible that the GDDR product is a secreted protein. We then tested the correlation of the expression of GDDR and TFF1 in 31 clinical gastric cancer samples. The data suggested the similar expression pattern of GDDR and TFF1. From the above data, we asked whether GDDR expressing could affect the expression of TFF1. Strikingly, we found that the expression of TFF1 was up-regulated by GDDR over-expression in SGC-7901 cell line.2. Based on the above rough comprehension about the expression profiling of GDDR and TFF1, we next decide to find GDDR's role in the gastric cell line and xenotransplantation model. SGC-7901 cells were stably transfected with either pcDNA3.1-GDDR or the corresponding vector as functional model. These cells were cultured parallel and then subjected to FACS and MTT assay. We found exogenous expression of GDDR resulted in G1/S arrest and a significantly reduced of proliferation rate. In addition, compared with the control, SGC-7901 cells stably transfected with GDDR displayed fewer and smaller colony forming and xenotransplantation study demonstrated reduced tumorigenesis by GDDR over-expression. Over-expression of GDDR decreased the sensitivity of gastric cancer cells to chemotherapy such as doxorubicin. Notably, compared with the control, siRNA-TFF1 nearly blocked the effects of over-expression of GDDR, suggesting that the function of GDDR is dependent on the TFF1.3. Prokaryotic expression of GDDR and purification for immuning mice to product monoclonal anti-body. We successfully gained 10 strains of hybridoma which can produce specific anti-GDDR mAbs. The primary identifications by Western blotting and immunohistochemistry showed high titer and specificity of the mAbs targeting GDDR.In summary, this subject profiled the expression of GDDR in multiple tissues and cell lines for the first time. The similar expression patterns of GDDR and TFF1 in gastric cancers suggest that the two factors should cross talk with each other. The functional study demonstrated GDDR's biological roles on cell growth and sensitivity of gastric cancer cells to chemotherapy such as doxorubicin are dependent on TFF1 expression. High titer and specific mAbs targeting GDDR were successfully produced, which as a power tool would contribute to the further study on GDDR.In light of these finding, GDDR could be a useful indicator of gastric cancer, making it importance in the diagnosis and prognosis of disease. It also suggests that this novel gene might be used as a tumor suppressor. No apoptotic cell death was observed in our current experiment. Thus, it is reasonable to conclude that GDDR is important in the maintaining the homeostasis of the gastric epithelium in the GI tract and that its downregulation contributes to the high proliferation rate of gastric cancer. In other words, GDDR should protect the gastric epithelium from malignant transformation by growth inhibition.Our study presented here suggests that, in the process of gastric cancer, cytokines, such as TNFα, released by the inflammatory microenvironment down-regulate GDDR, which in turn contributes to the onset or progression of gastric cancer. Our results point to the possibility of reactivation of GDDR in the treatment of gastric cancer.