Effects Of Cell Division Cycle Associated 2 And Its Mechanism Research In Pancreatic Ductal Adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a common type of malignant tumor in digestive system. It is ranked fourth in the cause of American cancer death and top ten in Chinese cancer death. PDAC is difficult to diagnosis in early stage. It progress rapidly with low resection rate. It is not sensitive to chemotherapy and radiation therapy. Its 5-year survival rate is about 8%. Search for target molecules through genomics to improve early diagnosis and chemotherapy is of great importance. We used laser capture microdissection to enrich cancer cells from surgical resected PDAC samples. Whole exome sequencing was performed to identify CDCA2 as a common differently expresseed protein in PDAC.Cell division cycle associated 2(CDCA2) is the target subunit for phosphatase 1. It accounts for helping PP1 combine with chromatin in anaphase to help finishing cell cycle. The expression of CDCA2 protein is related with malignancy in breast cancer, oral squamous carcinoma, ovarian cancer. It is probably because of promoting cell cycle and inhibiting apoptosis. However, there’s no report about CDCA2 expression in PDAC.Recently, lots of research focused on tumor microenvironment. As the major cell type in tumor microenvironment, macrophages affect the function of cancer cell through different phenotype. It is confirmed on breast cancer. In PDAC, macrophages were also observed affect cancer cell invasion. How macrophages affect cancer cell apoptosis is still not clear.In this research, we performed whole exome sequencing in PDAC. We tried to find the relation between CDCA2 expression and clinical pathological characteristics. We tried to explore the relation between CDCA2 function and chemotherapy sensitivity. At the same time, we tried to identify the CDCA2 changes in cancer cell when co-cultured with M2 macrophage.Objective1 Explore CDCA2 protein expression in PDAC tissues and relationship with clinical pathological characteristics and prognosis of patients with PDAC;2. Study CDCA2 RNA expression in PDAC tissues and relationship with clinical pathological characteristics and prognosis of patients with PDAC;3. Identify CDCA2 expression in pancreatic cancer cell line and find out the effects on cell proliferation, apoptosis and its related mechanism;4. Explore CDCA2 changes in cancer cell when co-cultured with tumor associated macrophage.Methods1. With immunohistochemistry(IHC) technology, we detected CDCA2 protein expression in surgical resected samples. We analysed the relation between CDCA2 expression and clinical pathological characteristics and prognosis of patients with PDAC.2. Through TCGA public database, we analysed the relation between CDCA2 RNA expression with clinical pathological characteristics and prognosis of patients with PDAC.3. Using western blot, we tested basic CDCA2 expression in pancreatic cancer cell lines. Through siRNA transfection technique, we knocked down CDCA2 expression in cell line. We used CCK8 method to test cancer cell proliferation. We used flow cytometry to detect cell cycle apoptosis. We used Transwell method to detect cancer cell invasion and migration ability. We used western blot to detect the mechanism of CDCA2 on cancer cell.4. We induced differentiation of U937 cells to MO and M2 macrophage in vitro. We used the co-culture system to influence cancer cell lines. We used CCK8 method to detect cancer cell proliferation. We used flow cytometry to detect cancer cells apoptosis. We used western blot to detect cancer cell CDCA2 changes in co-culture system.Results1. CDCA2 protein expression is higher in PDAC tissues than in control tissues, p< 0.001. CDCA2 protein expression and N staging is related with patients’overall survival (p< 0.05). Multivariable analysis found CDCA2 expression is not independent risk factors affecting the prognosis of patients with PDAC.2. CDCA2 RNA expression in PDAC tumor is related with T staging (p= 0.010) and tumor staging (p = 0.038). However, statistically, CDCA2 RNA expression is not related with patients’ overall survival, p= 0.067.3. We successfully knocked down CDCA2 expression in BxPC-3 and T3M-4 cell lines. It reduced the proliferation of cell lines, apoptosis of cell lines. But it interestingly increased apoptosis when worked with gemcitabine. Western blot showed CDCA2 expression controls cell cycle by influencing the expression of cell cycle protein such as CyclinD1, CDK4 and CDK6. it also controls cell proliferation ability. CDCA2 expression influenced cell apoptosis by influencing the cleaved caspase - 3 and cleaved RARP expression.4. When co-cultured with M2 macrophages, pancreatic cancer cell lines proliferation was decreased and cell apoptosis was promoted. CDCA2 expression in the process is downgraded.ConclusionsAccording to whole exome sequencing, we successfully identified CDCA2 is a common gene mutation in PDAC in our country. CDCA2 protein expression was significantly higher in pancreatic ductal adenocarcinoma tissues. High CDCA2 expression indicates poor prognosis. In pancreatic cancer cell lines, CDCA2 controls cell cycle, promote the proliferation and also influence apoptosis. Knocking down CDCA2 expression in cancer cells, can increase the apoptosis induced by gemcitabine. When co-cultured with tumor associated macrophages, the ability of proliferation and apoptosis of cancer cell is affected. In the co-culture system, CDCA2 expression was downgraded.