Effect Of TCN1 On Proliferation And Metastasis Of Colon Cancer And Its Mechanism

Part Ⅰ:Correlation between TCN1 expression and clinicopathological features and prognosis in colorectal cancerObjective:To investigate the correlation between TCN1 expression and clinicopathological features and prognosis of colorectal cancer.Methods:1、The clinical information database of colorectal cancer was established to retrospectively analyze the clinicopathological data of patients with colorectal cancer.Clinical and pathological data were collected,including age,sex,tumor indicators,primary tumor location and tumor size.The tumor tissues were re-graded and graded,and TCN1 immunohistochemical detection was performed.The expression in tumor tissues,nontumor tissues and lymph nodes was analyzed semi-quantitatively,and the 5-year overall survival of the patients was followed up.2、SPSS 23.0 software was used for data analysis.Kruskal-wallis test and MannWhitneyu test were used to evaluate the relationship between TCN1 immunoreactivity and clinicopathological features.Kaplan-meier method was used to describe and generate survival curves,and long-rank test was used to calculate the differences among survival curves.Results:1、The high expression of TCN1 was significantly correlated with the increase of preoperative serum CEA level,tumor differentiation degree,pTNM stage and invasion depth(R=0.460,P=0.002;R=0.353,P=0.024;R=0.331,P=0.011;R=0.446,P=0.003).Other statistically significant clinicopathological variables included regional lymph node metastasis and primary tumor size(Z=-3.845,P<0.001;Z=-2.341,P=0.019).2、In the univariate analysis of influencing 5-year survival,CEA,CA125,CA19-9,degree of tumor differentiation,pTNM stage,primary tumor size,regional lymph node involvement,nerve invasion,and TCN1 immune expression were significantly correlated with the decrease of 5-year survival.In multivariate analysis,age(P=0.031),CA125(P=0.019);Ca19-9(P=0.016),regional lymph node involvement(P=0.007),nerve invasion(P=0.003),TNM stage(P=0.018)and TCN1 expression(P=0.043)were independent risk factors for adverse survival.3、Kaplan-meier survival analysis showed that the overall survival of patients with low TCN1 expression was significantly higher than those with moderate TCN1 expression and high TCN1 expression groups(P<0.05).The 5-year overall survival rates of TCN1 patients were 88.9%,50.0%and 40.0%,respectively.Conclusion:TCN1 is highly expressed in colorectal cancer tissues,which is correlated with CEA level,tumor differentiation degree,tumor size,invasion depth,pTNM stage and lymph node metastasis.High expression of TCN1 suggests a worse clinical prognosis in patients with colorectal cancer.Part Ⅱ:Effect of TCN1 on proliferation and invasion of colon cancer cells and its mechanismObjective:To investigate the effect of TCN1 on the proliferation and invasion of colon cancer cells and its mechanism.Methods:1、In vitro cell experiments:HCT116 and SW480 cell lines with stable TCN1 expression interference were established using lentivirus expression system,and the changes of cell biological behavior were observed.Cell proliferation and growth curves of HCT116 and SW480 cells were measured by CCK-8 and EdU,and apoptosis was measured by flow cytometry.Transwell assayed the invasiveness of each group of cells.The downstream target genes of TCN1 were identified by RNA sequencing,immunoprecipitation and CHIP methods,and verified by Q-PCR and Western blot.The changes of downstream target genes and related proteins were detected by immunofluorescence,and the structure of cytoplasm and basal layer microfilaments were observed by electron microscopy.2.In vivo animal experiment:The model of transplanted tumor was established in nude mice,and the occurrence and growth of transplanted tumor were monitored by in vivo imager,and the tumorigenesis effect of TCN1 low expression and overexpression cell line HCT116 was detected.Q-pcr and Western blot were performed on the tumor tissues to determine the expression level of TCN1 and downstream target genes in the transplanted tumor,and the downstream target genes were further verified in the included clinical samples.3、The data were expressed by mean±standard deviation(SD)and analyzed by Graphpad 6.0 statistical software(Graphpad software).The differences among groups were analyzed and compared by one-way analysis of variance(ANOVA).P<0.05 was the test level.Results:1、TCN1-silenced HCT116 and SW480 cell lines(TCN1-KD1 and TCN1KD2)and TCN1-overexpressed colon cell lines(TCN1-OE)were successfully constructed.Q-pcr and Western blot confirmed that TCN1 knockdown or overexpression was 20%and 25%lower than TCNI-KDC cells,while TCN1 expression was about 10 times higher in TCN1-OE cells than in control(TCN1-OEC)cells.Annexin V/PI staining showed that the apoptosis rate of TCN1-KD1 and TCN1-KD2 cells was significantly higher than that of TCN1-KDC group(P<0.01).The number of EdU positive cells in TCN1-KD1 and TCN1KD2 cells was significantly decreased compared with TCNI-KDC cells(P<0.01 or P<0.05),and the number of EdU positive cells in TCN1-OE cells was significantly increased compared with TCN1-OEC cells(P<0.01 or P<0.05)by EdU and CCK-8.Compared with TCN1-KDC cells,the cell viability of TCN1-KD1 and TCN1-KD2 cells decreased significantly at each time point(P<0.01),and the activity of TCN1-OE cells was significantly higher than that of TCN1-OEC cells(P<0.01).Transwell assay showed that compared with TCN1-KDC cells,the invasion ability of HCT116 and SW480 cells of TCN1-KD2 and TCN1-KD2 cells was significantly decreased(P<0.01),while the invasion ability of TCN1-OE cells was significantly increased(P<0.05).2、Gene profile data showed that 11 common genes were at least twice downregulated and twice up-regulated with low and high TCNI expression.Combined with transcriptional analysis,ITGB4 was the most prominent one in the list of 11 overlapping genes.Western blot analysis revealed co-immunoprecipitation of ITGB4 and TCN1.ChIP analysis using anti-TCN1 antibody on ITGB4 promoter showed that TCN1 gene knockout resulted in a significant decrease in enrichment of ITGB4 promoter by TCN1 in HCT116 cells(P<0.01).In HCT116 cells,TCN1 knockdown resulted in significantly decreased expression of ITGB4 and silk protein A(FLNA),while overexpression of TCN1 resulted in significantly increased expression of ITGB4 and FLNA at protein level.By immunofluorescence staining analysis,ITGB4 fluorescence signal was weak in TCN1KD1 and TCN1-KD2 cells,and PLEC and Phalloidin showed diffuse filamentous distribution.The fluorescence signals of ITGB4,PLEC,ITGA3 and Phalloidin in TCN1OE cells were stronger than those in TCN1-OEC cells.The down-regulation of TCN1 gene also reduced the level of FLNA in HCT116 cells.In TCN1 knockout cells,FLNA signal almost disappeared,while the fluorescence signal of FLNA in TCN1-OE cells was significantly enhanced.Transmission electron microscopy analysis showed that the threedimensional network structure was destroyed in TCN1 knockdown cells,resulting in cytoskeleton destruction.3、Compared with TCN1-KDC group,the tumor size of TCN1 down-regulated cells was significantly decreased(P<0.01),while the tumor size of TCN1 overexpressed cells was significantly increased(P<0.01).Meanwhile,the tumor volume in TCN1-KD1 and TCN1-KD2 groups was significantly lower than that in TCN1-KDC group(P<0.01).The tumor volume in TCN1-OE group was significantly higher than that in TCN1-OEC group(P<0.01).The expression levels of TCN1 and ITGB4 in xenograft tumors were detected by Q-PCR and Western blot.The expression levels of TCN1 and ITGB4 in TCN1-KD1 and TCN1-KD2 groups were significantly lower than those in TCN1-KDC group(P<0.01),and TCN1-OE cells were significantly higher than those in TCN1-OEC cells(P<0.01).Conclusion:1、Low expression of TCN 1 promoted apoptosis of colon cancer cells and inhibited cell proliferation and invasion.High expression inhibits apoptosis of colon cancer cells and promotes cell proliferation and invasion.2、TCN1 can affect the promoter region of ITGB4,affect its transcriptional activity,and cause changes in cell function.TCN1 affects the functions of FLNA and other skeleton-related proteins through ITGB4 signaling pathway,leading to cytoskeleton reconstruction,which is the mechanism of colon cancer cell proliferation and invasion.Part Ⅲ:Effect and mechanism of TCN1 on peritoneal metastasis of colon cancerObjective:To investigate the role and mechanism of TCN1 in peritoneal metastasis model of colon cancer.Methods:1、The peritoneal metastasis model of nude mice was established by direct intraperitoneal injection of tumor cells after peritoneal scratching using HCT116 cell lines with low expression of TCN1 and overexpression of TCN1.2、Peritoneal metastasis models were constructed by colon cancer cells with different TCN1 expression levels.The number of peritoneal metastasis was counted after laparotomy,and the severity of peritoneal metastasis was evaluated by peritoneal metastasis index(PCI)score.The peritoneal metastasis tissues of colon cancer were examined by pathology and immunohistochemistry,and the immunohistochemistry results were compared using Image J software.Data were presented as mean±standard deviation(SD).Graphpad 6.0 statistical software(Graphpad software)was used for statistical analysis.The differences among groups were analyzed and compared by one-way analysis of variance(ANOVA).P<0.05 was statistically significant.3、Q-pcr and Western blot were used to detect the expression changes of ITGB4 and emT-related proteins,e-cadherin,N-cadherin and Vimentin,in metastatic tissues of colon cancer cells with different TCN1 expression levels.Results:1、The peritoneal metastasis model was successfully constructed in nude mice,and no death was observed in all nude mice for 4 weeks.2、After the nude mice were sacrificed under human conditions,abdominal exploration showed that there were fewer metastatic nodules in the TCN1-KD1 group(2.8±2.11)and TCN1-KD2 group(3.3±1.32),which were significantly lower than those in the TCN1-KDC group(6.2±2.35)(P<0.01).Compared with the TCN1-OEC group,abdominal nodules in the TCN1-OE group were significantly more than those in the TCN1-OEC group,and there was a small amount of bloody ascites,the difference was statistically significant(12.5±3.62 VS 8.1±1.17;P<0.05).Peritoneal metastasis index(PCI):there were fewer nodules in TCN1-KD1(13.2±2.41)and TCN1-KD2(12.2±3.22)groups,and the peritoneal metastasis index in PCI was significantly lower than that in TCN1KDC(20.5±3.45)group(P=0.007;P=0.006).PCI in TCN1-OE group was significantly different from that in TCN1-OEC group(28.6±3.84 VS 19.2±1.58;P=0.041).The peritoneal metastasis tissue of colon cancer was confirmed to be cancerous tissue by pathology,and the expression of TCN1 and ITGB4 in the peritoneal metastasis tissue of colon cancer was confirmed to be consistent with the corresponding interfering cell lines by immunohistochemical score.3、Q-pcr and Western blot showed that low TCN1 expression promoted the expression of E-cadherin,and decreased the expression of ITGB4,N-cadherin,and Vimentin.While TCN1 expression significantly enhanced the expression of ITGB4,Ncadherin and Vimentin,and decreased the expression of E-cadherin,with P<0.05,the difference was statistically significant.Conclusion:1、The expression level of TCN1 directly affects the severity of colon cancer peritoneal metastasis,and down-regulation of TCN1 can inhibit peritoneal metastasis of colon cancer cells.2、Overexpression of TCN1 up-regulates ITGB4 and promotes peritoneal metastasis of colon cancer through epithelial mesenchymal transformation(EMT).Low TCN1 expression induces functional degradation of ITGB4,leading to mesenchymal epithelial transformation(MET),which inhibits peritoneal metastasis of colon cancer.TCN1 cascades through ITGB4 signaling and acts on EMT,which is the mechanism of peritoneal metastasis of colon cancer.