| BackgroundColorectal cancer (CRC) is a common malignant tumor of digestive tract. In the western developed countries, the incidence of colorectal cancer ranks third, the mortality rate ranks fourth, and 5-years survival rate is 50～60%. In our country, the incidence of colorectal cancer in malignant tumor ranks third, the mortality rate ranks fifth. Along with the improvement of continuous progress and social living conditions, the incidence and mortality of colorectal cancer is increasing in the upward trend. Therefore, for colorectal cancer patients, to find the effective early diagnosis and treatment is imminent.At present, chemotherapy is one of the comprehensive treatments of colorectal cancer in clinic, and the FOLFOX4 based on oxaliplatin (OXA), fluorouracil (5-FU) and calcium folinate (CF) has been widely used in the treatment of colorectal cancer, which has the obvious curative effect. However, the failure of the clinical chemotherapy is common, and advanced colorectal cancer to chemotherapy is currently on a poor response, resulting in a poor prognosis. It has been recognized that the same TNM staging of patients also have different prognosis and response to therapy.There are many probable reasons and problems leading to the failure of chemotherapy, such as having a poor sensitivity and even exclusion, the resistance to chemotherapy drugs by tumor, the multidrug resistance to chemotherapeutic drugs (MDR). Among the above possibles, the multidrug resistance is the main reason, which is currently the attention hotspot.Twist gene is one of the highly conserved basic helix-loop-helix (bHLH) transcription factor, which has the characteristics of apoptosis inhibiting gene and can also code apoptosis proteins of inhibitor, so that to cause tumor through a variety of pathways. In Gong Qilin’s study, the expression of Twist was detected in 112 cases of colorectal cancer and 40 cases of normal mucosa tissues, and the results showed that it was positive expression in 78 cases (69.6%) in colorectal carcinoma and 15 cases (17.5%) in normal tissues, and the difference was statistically significant. Moreover, the Twist expression was associated with tumor differentiation, tumor invasion and lymph node metastasis in colorectal cancer. It indicates that the high expression of Twist may cause poor prognosis and metastasis of colorectal cancer in general.Some related research found that Twist is over-expression in kinds of tumor cells and plays a very important role in anti-apoptosis, multidrug resistance, epithelial mesenchymal transition (EMT), the occurrence of angiogenesis and tumor invasion and metastasis. The Meng DW’s study found that cell apoptosis was induced by down-regulation of Twistl expression, enhancing the sensitivity to taxol drug and suggesting that Twistl is a target gene in nasopharyngeal cancer. Cheng pointed out that in breast cancer cells, upregulation of Twist resulted in motility, invasion and resistance to paclitaxel increased significantly in cancer cell. Because Twist induced upregulation of Akt2, which developed resistance to paclitaxel in breast cancer cells, while enhancing invasion and metastasis ability of cancer cell. Therefore, Twist plays an important role in the regulation of tumor metastasis and multidrug resistance, but the specific mechanism is still not very clear.The relationship between Twist gene and drug resistance of tumor is a research hotspot at present. Previous studies have found that Twist can have an effect on chemotherapeutic drugs in tumor cells through different pathway regulation. But, there is no report whether the Twist gene does play a role in resistance to oxaliplatin and fluorouracil in colorectal cancer cells. In this study, we studied on the relationship between Twist gene and biological characteristics and occurrence and development of colorectal cancer, and to explore the role of Twist in resistance to oxaliplatin and fluorouracil in tumor cells, and then to find the therapeutic target of colorectal cancer, to enhance the killing effect on colorectal cancer cells and to provide help for clinical diagnosis and treatment.Object1. To study on the relationship between Twist gene and biological characteristics and occurrence and development of colorectal cancer, and the relationship between Twist and prognosis of patients with colorectal cancer.2. To explore the role of Twist in resistance to oxaliplatin and fluorouracil in tumor cells.MethodPart 1 Expression of Twist gene in colorectal cancer and its effect on prognosisImmunohistochemical SP method was used to detect the paraffin sections of 95 cases with complete clinical and pathological data and follow-up records of patients with colorectal cancer, and to observe the expression of Twist in different tissues, to analyze the relationship between its expression and clinical pathological features, to have the survival analysis and Cox proportional hazard regression model analysis.Part 2 Study on the effect of the Twist gene in colorectal cancer with oxaliplatin and fluorouracil resistant.1. Immunofluorescence, RT-PCR, Western blot were respectively used to detect Twist mRNA and protein levels in SW480, HCT116 and Lovo cells. The Twist overexpression plasmid or Twist-siRNA plasmid was transfected into three cell lines, respectively, leading to Twist gene with high expression or low expression in cells, which was detected the transfection efficiency by RT-PCR, Western blot.2. MTT was used to detect the proliferation of SW480, HCT116 and Lovo cells at different concentrations and times of oxaliplatin and 5-fluorouracil. And different concentrations of oxaliplatin and fluorouracil were added in each experimental group away from light at 24,48,72,96h. The proliferation of oxaliplatin and fluorouracil were respectively screened by MTT in three cell lines at every termination time point, and then the half inhibition concentration (IC50) of oxaliplatin and fluorouracil at 48h were screened in SW480, HCT116 and Lovo cells. With the concentration of IC50 of 5-FU and OXA (volume ratio=1:1) at 48h in three cell lines with different treatments, the role of Twist gene was detected on cell proliferation.3. Flow cytometry was used to detect the effect of Twist gene on cell apoptosis rate with the concentration of IC50 of 5-FU and OXA (volume ratio=1:1) at 48h in three cell lines with different treatments. Three kinds of cells were transfected by Twist overexpression plasmid or Twist-siRNA plasmid, and then cells of number of 5×106 were inoculated in 150mm culture dish. According to grouping and administration measures, drugs were added to every experimental group, and the apoptosis rate of every group were detected by flow cytometry at the termination time point.ResultPart 1 Expression of Twist gene in colorectal cancer and its effect on prognosisThe expression of Twist protein is located in cell cytoplasm and nucleus, mainly in cytoplasm. The positive expression of Twist protein in colorectal cancer was significantly higher than that in the corresponding normal colorectal mucosa (Χ2=6.623, P<0.01). The positive Twistl expression was found to be significantly correlated with histological grade, T-stage, N-stage, M-stage, TNM-stage and recurrence. On the other side, there were no significant differences between groups in age, sex, colorectal cancer site and so on. Kaplan-Meier method was used for overall survival analysis to demonstrate that CRC patients with negative Twist expression are significantly associated with a longer survival time (Log-rank test,Χ2=35.189, P<0.001). In a univariate analysis, expression of Twist and various clinicopathological parameters were evaluated for their impact on overall survival, which was significantly associated with histological grade, N-stage, M-stage, TNM stage and the expression of Twist. A multivariate Cox regression analysis demonstrated that the expression of Twist and N-stage were respectively the significant prognostic factor for overall survival of patients’.Part 2 Study on the effect of the Twist gene in colorectal cancer with oxaliplatin and fluorouracil resistant.1. The results of immunofluorescence, RT-PCR and Western blot showed expression of Twist in HCT116 and Lovo cells was significant higher than SW480 cell, respectively (P<0.05). The expression of Twist mRNA in highly metastatic HCT116 and Lovo cells were significantly higher than SW480 (P<0.05), and the expression of Twist mRNA in Lovo cells was significantly higher than HCT116 cells (P<0.05). The expression level of Twist mRNA in interference group in SW480, HCT116 and Lovo cells was significantly lower than the control group (P<0.05). The expression level of Twist mRNA in overexpression group in SW480, HCT116 and Lovo cells was significantly higher than the control group (P<0.05). There was no statistical difference on the expression of mRNA in the negative control group, the control group of interference and the control group of overexpression (P>0.05). Moreover, the Western blot results suggest that the conditions were the same on the expression of Twist protein and expression of Twist mRNA.2. The results of MTT showed that the inhibition of oxaliplatin and fluorouracil were gradually increased with rising concentration and prolonging time on SW480, HCT116 and Lovo cells, which were in a dose or time dependent way. And The half inhibitory concentration (IC5048h) of oxaliplatin on SW480, HCT116 and Lovo cells were 0.66g/ml,11.86g/ml and 5.75g/ml. The half inhibitory concentration (IC5048h) of fluorouracil on SW480, HCT116 and Lovo cell were 94.83g/ml,1.84g/ml and 27.66g/ml, respectively. After SW480, HCT116 and Lovo cells treated with the concentration of IC5048h of fluorouracil and oxaliplatin at 48h, which were treated with different treatments already, the proliferative inhibition ratio of the interference group was significantly higher than that of negative control group (P<0.05), and the proliferative inhibition ratio of the overexpression group was significantly lower than that of the negative control group (P<0.05).3. The results of flow cytometry displayed that after SW480, HCT116 and Lovo cells treated with the concentration of IC5048h of fluorouracil and oxaliplatin at 48h, which were treated with different treatments already, There were significant differences in the early, late and total apoptosis rates in SW480, HCT116 and Lovo cells between two different treatment groups (P<0.05). On one side, the early, late and total apoptosis rates of the interference group in cells were significantly higher than that of the negative control group (P<0.05). On the other side, the early, late and total apoptosis rates of the overexpression group in cells were significantly lower than that of the negative control group (P<0.05). In the same treatment, there were significant differences in the total apoptosis rate in two cells (P< 0.05).Conclusion1. The expression of Twist could enhance the progression of occurrence, development, metastasis and invasion in colorectal cancer, and it was also the independent risk factor for poor prognosis of patients with colorectal cancer.2. Twist gene plays a positive role in resistance to oxaliplatin and fluorouracil in colorectal cancer. |
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