The Study On The Role Of TFF1and TFF3in Colorectal Cancer Metastasis And The Relationship With EMT

Colorectal cancer is one of common malignant tumor in the digestive system, which is the second cause of death in malignant tumors in Europe and the United States.The incidence and mortality (10.25/100,000) of colorectal cancer rank the third and fifth in malignant tumors respectively. In recent decades, with the improvement of people’s living condition and dietary structure, the incidence and mortality rates of colorectal cancer have been increasing gradually and there is a trend of getting younger. Heterochrony metastasis after radical treatment, especially the liver metastasis is the main cause of poor prognosis. Liver metastasis occurs in Approximately35%of colorectal cancer when confirmed, in which simultaneous Liver metastasis rate is15-25%, while heterochrony liver metastasis rate is40-50%. The liver metastasis rate can be as high as60%～70%when Autopsy. Screening high-risk patients and effective tumor markers is an important link in improving prognosis.Trefoil factor (TFF) family is a group of small peptides that has one or more trefoil structural domain, which is synthesized and secreted mainly by the digestive tract mucous secretory epithelial cells and plays a major role in the process of mucosa damage repair. There are three members:TFF1(breast cancer related peptide, pS2), TFF2(spasmolysis polypeptide, SP) and TFF3(intestinal trilobites factor, ITF). TFF are expressed throughout digestive system and gastrointestinal tract is the main synthesis place for TFF. TFF1and TFF2are mainly expressed in stomach, while TFF3is expressed in large intestine and small intestine.In recent years, the correlation between TFF family and tumorigenesis, invasion and clinical prognosis has widely been drawing attention.There were more studies on correlation between TFF1and TFF3expression and oncogenesis, progression and prognosis of breast, stomach cancer.But reports on relationship between TFF1and TFF3expression and colorectal cancer occurrence, development and clinical prognosis are rare.Twistl is a highly conserved transcription factor hat belongs to the family of basic helix-loop-helix proteins, and it plays an important role in the embryogenesis。Twist gene broadly take part in the process of tumor occurrence and cell proliferation and it also can alter tumor cell apoptosis, inhibit cellular differentiation, induce EMT process, take part in the process of tumor drug resistance and tumor angiogenesis.Part one:expression of TFF1, TFF3and Twistl in different metastatic colorectal cancer tissuesObjectives:To detect TFF1, TFF3and Twistl protein expression in different metastatic colorectal cancer tissues and assess the differences in expression and relationships with clinical pathological characteristics and prognosis of colorectal cancer. Methods:Using immunohistochemical method to detect the expression of TFF1and TFF3and Twistl protein in primary cancer tissue,adjacent normal tissue,metastatic lymph node tissue and liver metastatic tissue of colorectal cancer and Gather clinical and follow-up data.Results:TFF1and TFF3were expressed mainly in the cytoplasm and cell membrane, while Twistl was expressed in both cytoplasm and nucleus. TFF1,TFF3and Twistl expression rates in colorectal cancer were66.7%(50/75),78.7%(59/75) and54.7%(41/75) respectively, while the expressions in adjacent normal tissues were27.3%(13/47),100%(47/47) and17%(8/47) respectively. Compared with the primary cancer tissue, there were no statistical differences between TFF3and Twistl expressions in metastatic lymph node or liver metastases (P=0.879, P=0.105for TFF3and P=0.08, P=0.78for Twistl).But there were significant differences between primary cancer and metastatic lymph node (P <0.05). High expressions TFF3and Twistl were associated with lymph node metastasis and pathological stage and poor prognosis (P<0.05) respectively.The correlation between TFF1and TFF3expressions was statistically significant and were positively related (P=0.014r=0.291)Conclusions:TFF1and TFF3and Twistl genes have different expressions in different colorectal cancer metastatic tissues,although some did not reach the statistical differences. High expressions of TFF3and Twist1are associated with lymph node metastasis, pathological stage and poor prognosis, which can be used as a prognosis indicator.Part two:expression of TFF1, TFF3and Twist1mRNA and proteins in colorectal cancer cell lines with different invasion potentialObjective:To investigate TFF1and TFF3and Twistl mRNA and protein expressions in colon cancer cell lines with different invasion potential and assess the correlation between the three gene expressions and tumor metastasis.Methods:Real-Time RT-PCR and Western Blot assays were applied to detect TFF1and TFF3and Twist1mRNA and protein in colon cancer cell lines (HT-29, SW620, LOVO) with different invasion potential. HIEC (immortalized human intestinal mucosal epithelial cell lines) were used for control. Results:1. QRT-PCR results showed that:expressions of TFF1mRNA in HIEC, HT29SW620and LOVO cell lines were0.595±0.257,3.257±1.424、2.275±0.372. Besides LOVO cell line, the rest of TFF1expressions in colon cancer cell lines were higher than normal intestinal epithelial cells (P<0.05), with the increase of invasion potential, expression of TFF1gradually reduced (P<0.05).expressions of TFF3mRNA were0、0.329±0.181、13.390±2.168and0respectively.TFF3mRNAis not expressed in HIEC, but expressed increasingly in colon cancer cell lines (except for LOVO).The expression in higher metastatic cell line SW620was much greater than that in a lower metastatic cell line HT29(P<0.05).The expressions of Twistl mRNA were0.233±0.093、0.485±0.0535、1.547±0.379and3.410±0.493respectively. Twistl mRNA is expressed lower in the normal intestinal epithelial cell line, but expressed increasingly in colon cancer cell lines.The expression in higher metastatic cell line SW620was much greater than that in a lower metastatic cell line HT29(P<0.05).With the increase of invasion potential, the expression of Twistl mRNA gradually elevated (P<0.05).The expression of Twist mRNA in high metastatic LOVO cell line was significantly higher than that in other two cell lines, the difference between groups was statistically significant (P<0.05). The expression in low metastatic SW620and HT29cell lines are lower.There is no significant difference between the two groups (P<0.05).2. Western blot results showed that expressions of TFF1protein in HIEC, HT29SW620and LOVO cell lines were1371±114.7.26074±1413、8076±1171和343.61±91.47. Besides LOVO cell line, the rest of the expression of TFF1in colon cancer cell lines were higher than that in normal intestinal epithelial cells (P<0.05), with the increase of invasion potential, expression of TFF1gradually reduced (P<0.05).expressions of TFF3mRNA were48.22±10.60、365.6±83.51、929.20±92.95and45.69±7.11. TFF3protein is not expressed in the normal intestinal epithelial cell line, but expressed increasingly in colon cancer cell lines (except for LOVO). With the increase of invasion potential, expression of TFF1gradually elevated.expressions of Twist1protein were48.22±10.60、365.6±83.51、929.20±92.95and45.69±7.11. Twistl protein is expressed lower in the normal intestinal epithelial cell line, but expressed increasingly in colon cancer cell lines (except for LOVO). With the increase of invasion potential, expression of Twistl gradually elevated.Conclusion:It can be concluded that TFF3and Twistl promote the infiltration and invasion,whereas TFF1 inhibits colon cancer metastasis.further function test needed to prove this assumption.Part three:Study on the relationship between TFF1and TFF3gene expression and the EMTObjective:To observe the expressing trend of TFF1, TFF3and target genes linked to the EMT (Twist, Snail, E-cadherin, vimentin,β3-catenin) in different invasion potential colon cancer cell lines and preliminary assess whether TFF1and TFF3is related to the EMT.Methods:Real Time RT-PCR and Western Blot was used to detect the mRNA and protein expression of Twist,snail,E-cadherin,vimentin,(3-catenin that linked to EMT in different invasion potential colorectal cancer cell lines of HIEC、HT29、SW620、LOVO and determine the relationship among them by comparing the expressing trends with that of TFF1and TFF3.Results:The expression of E-cadherin and p-catenin (epithelial markers) mRNA and protein in HIEC were lower or no expression. Expressions in HT29, SW620, LOVO showed a trend of gradual decline (P<0.05). The expressions of Snail and vimentin (mesenchymal cell markers) mRNA and protein were lower in HIEC. Expressions in HT29, SW620and LOVO showed a trend of gradual increase (P<0.05).Conclusion:According to the above trend, we can speculate that TFF1and TFF3plays a certain role in the process of EMT and this effects could be reversed. Part four:Analysis on clinical features and prognosis of colorectal cancer in different nationalities in Xin Jiang regionObjective:To discuss the distribution and clinicopathological characters of different ethnic patients with colorectal cancer(CRC) in Xinjiang region and evaluate the prognosis. Methods:A total of1,421histopathologically confirmed sporadic colorectal cancer patients who were either Han/Chinese or Uyghur were identified and enrolled from a database of both diagnoses and operative procedures from Xin Jiang Tumor Hospital, which is affiliated to Xin Jiang Medical University between2000and2007. The two ethnic groups were compared with regard to clinical features, tumor characteristics, disease stage, overall survival rate, disease-free survival rate and cancer-specific survival rate. Results:Among the1421patients with colorectal cancer enrolled in this study,1210patients were Han/Chinese (mean age,62.3±4.5years; range,19-92years), while211patients were Uyghur(mean age,52.4±15.6years; range,17-87years). There were significant differences in proportions of gender, age, blood type, occupation and histopathological type between the Han/Chinese and Uyghur patients(P<0.05). The median overall, disease-free and cancer-specific survival time were45,62and65months for the Han/Chinese patients and42,49and61month for the Uyghur patients (P=0.000,P=0.005, P=0.007). The cumulative5-year survival of the Uyghur patients was significantly worse than that of the Han patients (P=0.000). A multivariate analysis showed that age, ethnicity, histopathological type, differentiation, T(Infiltration Depth), N(Lymph node metastasis), staging, postoperative metastasis and metastatic site (P＜0.05) were found to be the prognostic factors. Conclusion:Uyghur CRC patients are associated with a much younger age at disease onset, more aggressive histopathologic characteristics and a significantly worse prognosis than those in the Han/Chinese patients. Therefore, more attention should be paid to enhancing health care education and regular screening for the Uyghur population as well as promoting comprehensive treatment techniques in an effort to improve survival for both ethnic groups.