Metastasis Of Stage Ⅰ-Ⅲ Colorectal Cancer As Well As Its Molecular Pathogenesis

BackgroundColorectal cancer(CRC)is the third tumor with high incidence rate among cancer patients throughout the world.The most commonly used treatments in real practice for colorectal cancer include combined chemotherapy,radiotherapy,immunotherapy and radical tumor resection.The overall 5-year survival rate of patients with colorectal cancer is between 50%and 60%,and the main causes of death include recurrence and metastasis,which has posed a daunting challenge to the treatment of colorectal cancer.Some studies have tested circulating tumor cells(CTC)in the blood of cancer patients,so as to monitor the metastatic progress of the disease,but the detection rate of these cells restricts their evaluation of patients with colorectal cancer.Thus,with a view to enhance the survival results of these individuals,it is of crucial significance to develop new strategies for diagnosis and postoperative monitoring of patients with colorectal cancer.MUC16 is a mucin associated with cell surface,which may be up-regulated in a number of malignant tumors.However,its role in the pathogenesis of colorectal cancer still remains unknown.PurposesIn light of large numbers of research results in previous period,the present research intends to probe into the correlation between the number of circulating tumor cells and clinical and pathological parameters during operation of stage Ⅰ-Ⅲ colorectal cancer,and further study the influence of MUC16 on invasion and metastasis of colorectal cancer as well as its molecular pathogenesis.MethodsIn this experiment,we collected tumors and paracancerous tissues,peripheral venous blood,and intraoperative intestinal venous blood from 162 patients with colorectal cancer who underwent surgery in the Second Affiliated Hospital of Anhui Medical University from December 2015 to December 2018,as well as peripheral venous blood in 48 healthy volunteers and 72 patients with benign colorectal tumor.We separated and identified the circulating tumor cells of patients by means of negative enrichment-immunofluorescence in situ hybridization(NE-imFISH)technology,and determined the cut-off value of circulating tumor cells via receiver operating curve(ROC)and Youden index;next,we analyzed the survival of patients with colorectal cancer by Kaplan-Meier and Log-rank methods and multivariate Cox regression analysis,in order to comprehensively assess the value of circulating tumor cells in diagnosing stage Ⅰ-Ⅲ colorectal cancer,and then explored the correlation between circulating tumor cells and clinical and pathological parameters as well as prognosis;besides,we detected the expression of MUC16 by immunohistochemistry to explore the relationship between MUC16 and circulating tumor cells,and detected the expression of MUC16 in colorectal cancer cell lines and colorectal normal epithelial cells by real-time fluorescence quantitative PCR;furthermore,after transfection of MUC16 by plasmid knock-down,we studied the changes of invasion and metastasis capability of colorectal cancer cells through CCK-8,Western blot,EdU colormetric detection,flow cytometry,Transwell metastasis experiment,Transwell invasion experiment,and building of models of liver metastasis and lung metastasis in nude mice;in the last step,we detected the regulation of MUC16-related signaling pathway by Western-blot and Co-IP,and exploited the molecular pathogenesis of invasion and metastasis of colorectal cancer cells by MUC16.ResultsThe number of CTCs of intestinal venous blood detected in stage Ⅰ-Ⅲ colorectal cancer is significantly higher than that of peripheral blood CTCs in stage Ⅰ-Ⅲ colorectal cancer and that of peripheral blood CTCs of patients with benign colorectal cancer and healthy volunteers;the number of CTCs of intestinal venous blood detected in stage Ⅰ-Ⅲ colorectal cancer is positively correlated with clinical TNM staging(P<0.001)and CA-125(P<0.001);the number of CTCs of intestinal venous blood detected in stage Ⅰ-Ⅲ colorectal cancer is positively correlated with KRAS(P<0.001)and microsatellite instability(MSI)(P<0.001);the DFS and OS of patients with positive result of CTCs of intestinal venous blood in stage Ⅰ-Ⅲcolorectal cancer are significantly lower than those with negative result of CTC;the DFS and OS of KRAS mutant patients are significantly lower than those of KRAS wild type patients;the DFS and OS of patients with MSS are significantly lower than those of MSI-H and MSI-L;the expression of MUC16 in colorectal cancer tissues is positively correlated with the number of CTCs of intestinal venous blood(P<0.001)and associated with the poor prognosis of patients.Knocking down MUC16 significantly inhibits the growth of colorectal cancer cells in vitro;the capability of invasion and metastasis of colorectal cancer cells with low expression of MUC16 decreases in a significant manner.Knocking down MUC16 inhibits the growth and metastasis of colorectal cancer cells in vivo.MUC16 activates the signal pathway of JAK2/STAT3 by combining with JAK2.Over-expression of JAK2 facilitates malignant biological behaviors of colorectal cancer cells in vitro.ConclusionsThrough regression analysis of univariate and multivariate Cox proportional hazards,the counting of circulating tumor cells has been testified to independently predict TNM staging,microsatellite instability(MSI)and KRAS mutation status.These results suggest that circulating tumor cells are valuable biomarkers,which can be used to monitor and assess the progression of diseases of patients with colorectal cancer.In the meanwhile,MUC16 can facilitate phosphorylation of JAK2 and then activate phosphorylation of STAT3 in combination with JAK2,thereby raising the capability of invasion and metastasis of colorectal cancer cells,which renders it easier for tumor cells to enter the peripheral blood.In the present research,the influence of MUC16 on invasion and metastasis of colorectal cancer cells as well as its pathogenesis are elaborated,laying a solid basis for research on the occurrence and development of colorectal cancer.