Exploring The Function Of Tiam1 In Development Of Colorectal Cancer In Vivo

BACKGROUND & OBJECTIVEColorectal cancer (CRC) is the second worldwide leading cause of cancer death in the world. During the past decade, the incidence rate of colorectal cancer in China was rising year by year.. Metastasis, one of the basic characteristic of malignant tumors, affects the therapeutic efficacy and leads to the death of patients with tumors. Surgery is very important when the disease is confined to the bowelwall.70% to 80% of patients can be cured by resection of the lesion. After surgery,the five-year survival rate for Patients with localized disease is 90%, decreasing to 65% in the case of metastasized disease to the lymph nodes. Therefore early detection of CRC is critical. Tumorigenesis is a multistep process. Cancer gene mutation and inactivation of tumor suppressor genes is the main reason.Tiam1 is one of guanine nucleotide exchange factors (GEFs), which activate GTPases by promoting the exchange of the GDP-bound forms from inactive to active. Although Tiam1 displays GEF activity towards all three Rho-like GTPases Rac1, Cdc42 and RhoA in vitro, it specifically activates Rac in vivo. Tiam1 was identified in 1994 by Proviral tagging in combination with in vitro Selection for invasiveness from murine lymphoma cells. It had been reported that Tiam1 was extensively expressed in a variety of cancers, such as T-cell lymphoma, B cell lymphoma, pancreatic cancer, breast cancer, bladder cancer and lung cancer and so on, especially in adenocarcinoma and squamous cell cancer with local infiltration and distant metastasis.In our previous studies, we prepared a cDNA microarray consisting of 447 tumor metastasis-associated candidate genes to screen metastasis-associated genes and obtained 51 candidate genes closely related to metastasis of colorectal cancer, four genes of which were selected as candidate metastatic genes of colorectal cancer by literature mining. Tiam1, one of the four candidate metastatic genes, was validated in the RNA level. Preliminary studies in our laboratory showed that, Tiam1 was higher expressed in adenoma compared with normal colorectal epithelium.Tiam1 expression was significantly stronger in lymphatic metastasis in comparison with colorectal carcinoma. Furthermore, the Tiam1 expression in colorectal carcinoma with lymph node metastasis was higher than that in colorectal carcinoma without lymph node metastasis.Tiam1 gene was highly expressed in LoVo and SW620 cells and moderately expressed in SW480, HCT116, HRT-18, LST and Hce8693. Low level expression was seen in LS174T and no expression was found in HT29.Transfection of Tiam1 significantly increased the proliferation of HT29 cells along with markedly enhanced in-vitro invasion and metastasis. These studies suggest, Tiam1 is closely related to the evolution of colorectal cancer. but studies in vitro often can not fully simulate genes in the body. In order to fully reveal the role of Tiam1 in development of colorectal cancer, Tiam1 transgenic mice, combined with the model of colorectal cancer induced by DMH, were applied in the study.The incidence rate of colorectal cancer, tumor growth index and metastasis was observed in Tiam1 transgenic mice and wide-type.METHODS1. Screening of Tiam1 transgenic mice homozygousFive founders were mated with wild-type mice for reproduction. When offspring were 3 weeks old, the tail were scissor for extraction of DNA. PCR were used to explore the Tiam1 gene. Then, Tiam1-positive mice continued to multiply after identification. By this method, homozygous were obtained. Depending on the combination of fluorescent stereo microscope and immunohistochemistry analysis, the mice, of which Tiam1 expression in colorectal is high, were screen. 2. Construction of mice colorectal tumor model, analysis of the role of Tiam1 overexpression in mice colorectal cancer developmentTiam1 transgenic mice and wild-type mice were 50 respective. Mice were injected intraperitoneally with DMH (20 mg/kg body wt.) once a week for 24 weeks. and sacrificed 8,12,16,20,24and 32 weeks after the 1st DMH injection. The organs were obtained and observed macroscopically and microscopically.3. Statistical analysisData were expressed as the mean±SE, and were analyzed using spss13.0. Student's t-test and Pearsonχ2-test were used for analysis of data.Differences were considered as statistically significant at P< 0.05.RESULTS1. Screening of Tiam1 transgenic mice homozygousFive founder were mated with wild-type mice for reproduction. Then, Tiam1-positive mice Continued to multiply. By this method,13 homozygous were obtained. Depending on the combination of fluorescent stereo microscope and immunohistochemistry analysis,48A (?) and 48H (?)were screen, of which Tiam1 expression in colorectal is high.2. Construction of mice colorectal tumor model, analysis of the role of Tiam1 overexpression in mice colorectal cancer development8 weeks after the 1st DMH injection, focal hyperplastic change in a colonic crypt occurred in transgenic mice and wild-type mice change.12 weeks, the Colorectal epithelium of mice were pronounced proliferation, with an area of focal atypia. Immunohistochemical staining showed that the difference of ki-67 positive cells in transgenic mice and wild-type mice was not significant.16 weeks, colorectal tumor occurred in transgenic mice and wild-type mice.32 weeks, the volume of transgenic mice colorectal tumor was large compared to the wild-type mice. Metastasis occurred in transgenic mice, not in wild-type mice. Immunohistochemical staining showed that ki67 positive cells of colorectal tumor tissue in transgenic mice were more than which in wild type and the difference was significant. CONCLUSION1.5 Tiaml transgenic founder mice for repopulation,13 Tiaml transgenic mice homozygous were obtained.2. Depending on the combination of fluorescent stereo microscope and Immunohis-tochemistry analysis,48A(?) and 48H(?) were screen.3. The model of colorectal tumors were successfully established which lays a solid foundation for research of Tiaml gene in vivo.4. Generally, it suggest that Tiaml overe- xpression in Tiaml transgenic mice may promote proliferation, invasion and metastasis of colorectal cancer.