Study On The Mechanism Of TGF-? Signaling Pathway Related Genes FBXO32,Smad4,PAI-1 And VEGF And FOXC2 In The Pathogenesis Of Colorectal Cancer

Colorectal cancer(CRC),one of the most common malignancies,is the third leading cause of cancer-related deaths in the United States.According to the World Health Organization(WHO),colorectal cancer is the third most common malignancy in male and female.The incidence of CRC is increasing rapidly in Asian countries and is likely similar to western countries.In China,both the morbidity and mortality of CRC are increasing.Overall,about 50%of patients who diagnosed with CRC succumbed to the disease,due to complications associated with distant metastasis.The underlying molecular mechanisms in CRC metastasis are still unclear.In February 2017,the National Cancer Center released the latest cancer data in China,"2017 China City Cancer Update Report",which summarizes the data of 347 cancer registries in China and shows that gastrointestinal cancer is the major burden of Chinese residents.With the process of urbanization in our country and the improvement of peoples' living standards and changes in lifestyle,some cancers have also been reduced,mainly in gastric cancer,liver cancer and esophageal cancer.However,among the top 10 cancers in the city,the incidence of colorectal cancer is still at a high level.The incidence of colorectal cancer is 20.87/100000 in small cities and 44.94/100000 in large cities.The morbidity increased significantly from small cities to large Urban.However,the current molecular mechanism of colorectal carcinogenesis and metastasis is not yet clear.Therefore,the active exploration and research of molecular markers related to the pathogenesis and prognosis of colorectal cancer can provide an effective radiotherapy and chemotherapy strategy for patients with colorectal cancer who have a high risk of recurrence and metastasis in the early stage.At the same time,exploring the pathogenesis of colorectal cancer is also necessary to reduce its morbidity and mortality.The occurrence and progression of tumors are a series of relatively complex multi-factor,multi-step,multi-channel and multi-gene processes.Once the tumor has metastasized and recurred,the prognosis is often poor.With the continuous research on the related genes and signaling pathways involved in colorectal cancer,many of the related factors have been found and play a significate role in the pathogenesis of colorectal cancer,such as micRNA,IncRNA,E-Cadherin,VEGF,MMP.However,there are still few researches in colorectal cancer.Therefore,further study and discussion is needed.TGF-P signaling pathway plays a crucial role in the development of colorectal cancer.However,few study of TGF-? signaling pathway-related genes were performed in the pathogenesis of colorectal cancer,such as FBX032,Smad4,PAI-1 and VEGF.FOXC2,an oncogene,has been shown to be involved in the tumorigenesis,including gastric cancer,esophageal cancer,ovarian cancer and breast cancer.However,there are still few researches in colorectal cancer which need further research,discussion and verification.In this study,we explored the role of FOXC2 and TGF-P signaling pathway-related genes FBX032,Smad4,PAI-1 and VEGF in colorectal cancer through three-part experiment.First,the expression of FBX032 and Smad4 were detected by immunohistochemistry in colorectal cancer tissues and normal tissues.FBX032 and Smad4 were detected by real-time fluorescence quantitative PCR and Western blotting in colorectal cancer cell lines and human colonic epithelial cells.The clinical and pathological data of colorectal cancer patients were analyzed to evaluate the relationship between the expression and clinicopathological data.The survival prognosis of patients with colorectal cancer was evaluated by survival analysis.Secondly,the expression of PAI-1 and VEGF in colorectal cancer cell lines and human colonic mucosal epithelial cells were detected by real-time fluorescence quantitative PCR and Western blotting to explore the possible molecular mechanism of PAI-1 and VEGF in colorectal cancer.Finally,the expression of FOXC2 in colorectal cancer tissues and normal tissues was analyzed by immunohistochemistry.The expression of FOXC2 in colorectal cancer tissues and normal tissues was detected by real-time fluorescence quantitative PCR and Western blotting.Similarly,the clinicopathological data of patients with colorectal cancer analysis was performed to assess the relationship between its expression and clinicopathological data.Part I The Role and Mechanism of FBX032 and Smad4 in the Pathogenesis of Colorectal CancerObjective(1)The relationship between the expression of FBX032 and Smad4 in colorectal cancer tissue and normal colonic mucosa tissues was evaluated by observing the clinicopathological data of colorectal cancer.(2)To evaluate the effect of FBX032 and Smad4 on the prognosis of patients by analyzing the relationship between the expression of FBX032 and Smad4 in colorectal cancer tissues and the prognosis of patients.(3)To explore the role and mechanism of FBX032 and Smad4 in the occurrence and progression of colorectal cancer by detecting the expression of them in colorectal cancer cell lines and normal human colon mucosal epithelial cells.(4)To further explore the role of TGF-?/Smad4 signaling target genes through the expression of FBX032 and Smad4 in colorectal cancer tissues and cells.Methods(1)122 cases of colorectal cancer tissues and 43 cases of normal colorectal tissues were collected.The above samples were confirmed by two or more senior pathologists.The expression of FBX032 and Smad4 were detected by immunohistochemical-SP method,and then the expression differences of them were analysised.(2)According to the clinicopathological data of patients with colorectal cancer,FBX032 and FOXC2 were analyzed in age,gender,clinical stages,differentiation degree and distant metastasis.(3)Kaplan-Meier was used to observe the difference of 5-year survival between FBX032 low expression group and high expression group survival.(4)To culture colorectal cancer cell lines HT-29,HCT-116 and Caco-2 and human normal colorectal cell line NCM-460,and then extract the mRNA and protein from them.(5)The expression of fbxo32 and smad4 in colorectal cancer cell lines HT-29,HCT-116 and Caco-2 and human normal colorectal mucosal cell line NCM-460 mRNA were detected by Real-time PCR(RT-PCR).(6)To detect the expression of FBX032 and Smad4 in colon cancer cell lines HT-29.HCT-116 and Caco-2 and human normal colorectal mucosal cell line NCM-460 by Western blot.Results(1)Immunohistochemistry-SP staining showed that in 122 cases of colorectal cancer tissue,28 cases of FBX032 positive expression,the positive rate was only 22.95%,43 cases of normal colorectal tissue,31 cases were positive,the positive rate was as high as 72.09%.(2)Correlation analysis of clinicopathological features showed that FBX032 was significantly associated with tumor stage,differentiation degree and lymph node infiltration in colorectal cancer(p<0.05).However,there was no significant correlation between FBX032 expression and age and sex(p>0.05).Smad4 was significantly associated with the degree of differentiation and lymph node infiltration in colorectal cancer(p<0.05).However,there was no significant correlation between FBX032 and age,sex and tumor stage(p>0.05).(3)The results of RT-PCR confirmed that the expression of fbxo32 and smad4 in colorectal cancer tissue samples and colorectal cancer cell lines were significantly lower than those in normal colorectal and human intestinal epithelial cells NCM460(p<0.05).(4)Western blot results showed that the expression of FBX032 and Smad4 in colorectal cell lines HT-29,HCT-116 and Caco-2 were significantly lower than those in normal colorectal cell line NCM-460(p<0.05).(5)Survival analysis showed that the overall survival of FBX032-overexpressing patients was significantly lower than that of FBX032-low expressing patients.ConclusionsFBX032 may be the target gene of TGF-?/Smad4 signaling pathway,which is low expressed in colorectal cancer and correlates with tumor stage,lymph infiltration and differentiation in colorectal cancer patients.The high expression of FBX032 indicates the longer survival of patients with colorectal cancer,FBX032 may be used as a target gene to predict the malignant degree of colorectal cancer,which is helpful for the screening of high-risk patients and the formulation of personalized treatment programs.Part ? Expression of PAI-1 and VEGF in Colorectal Carcinoma Cell LinesObjectiveBy detecting the expression of PAI-1 and VEGF in colorectal cancer cell lines and normal human colon mucosal epithelial cells,we explore the role of PAI-1 and VEGF in the pathogenesis of colorectal cancer.Methods(1)To culture colorectal cancer cell lines HT-29,HCT-116 and Caco-2 and human normal colorectal cell line NCM-460,and then extract the mRNA and protein from them.(2)To detect the expression of PAI-1 and VEGF in colorectal cancer cell lines HT-29,HCT-116 and Caco-2 and human normal colorectal mucosal cell line NCM-460 mRNA by Real-time PCR(RT-PCR).(3)The expression of PAI-1 and VEGF in colon cancer cell lines HT-29,HCT-116 and Caco-2 and human normal colorectal mucosal cell line NCM-460 were examined by Western blot.Results(1)The results of RT-PCR confirmed that the expression of pai-1 in colorectal cancer cell lines were significantly lower than those in human intestinal epithelial cells NCM460(p<0.05).However,the expression of vegf in three colorectal cancer cell lines were higher than those in human intestinal epithelial cells NCM460,and statistically significant between HT-29,Caco-2 and NCM460(p<0.05),no statistical significance between HCT-116 and NCM460(p>0.05).(2)Western blot results showed that the expression of PAI-1 in colorectal cell lines HT-29.HCT-116 and Caco-2 were significantly lower than those in normal colorectal cell line NCM-460(p<0.05).However,the expression level of VEGF in three colorectal cancer cell lines is higher than human intestinal epithelial cell line NCM460(p<0.05).ConclusionsPAI-1 is low expressed in colorectal cancer,and VEGF is highly expressed in colorectal cancer,which has guiding significance for the detection and prognosis of colorectal cancer.Part ?:Expression of FOXC2 in Colorectal Carcinoma and the Relationship with Clinical PathologyObjectiveBy detecting the expression of FOXC2 in colorectal cancer tissues and para-carcinoma tissue,we explore the role of FOXC2 in the occurrence and development of colorectal cancer and its relationship with clinicopathological data.Methods(1)83 cases of colorectal cancer tissues and 83 cases of para-carcinoma tissue were collected.The above samples were confirmed by two or more senior pathologists,and the expression of FOXC2 was detected by immunohistochemical-SP method.(2)Extract the mRNA and protein of fresh colorectal cancer tissues and para-carcinoma tissues.(3)Real-time PCR(RT-PCR)was used to detect foxc2 expression in 83 cases of colorectal cancer and 83 cases of para-carcinoma tissues.(4)Western blot was used to detect the expression of FOXC2 in 83 cases of colorectal cancer tissues and 83 cases of para-carcinoma tissues.Results(1)FOXC2 was highly expressed in 45 of 83 colorectal cancer tissues with a positive rate of 54.22%.10 of 83 normal colorectal tissues were highly expressed with a positive rate of 12.05%.(2)There was a significant correlation between FOXC2 and clinical stages and Ki67 expression in colorectal cancer(p<0.05).However,there was no significant correlation between FOXC2 expression and age,gender and lymph infiltration(p>0.05).(3)The results of RT-PCR confirmed that the expression of foxc2 in colorectal cancer tissue was significantly higher than that in para-carcinoma tissues(p<0.05).(4)Western blot results showed that FOXC2 expression in colorectal cancer tissues was higher than that in para-carcinoma tissues(p<0.05).ConclusionsFOXC2 is highly expressed in colorectal cancer and is related to tumor stages and differentiated degree.FOXC2 may be related to the carcinogenesis and malignant progression of colorectal cancer.FOXC2 may be used as a molecular target gene to predict the malignancy of colorectal cancer and contribute to the recurrence of high risk Patient screening,personalized treatment plan.