The Effects Of Bit1 On The Biological Behaviors Of Pancreatic Cancer And Related Mechanism

BackgroundPancreatic ductal adenocarcinoma (PDAC) is recognized as one of the most aggressive types of malignant tumors with dormant onset, rapid progress, low resection rate, resistance to radiation and chemotherapy.Because of absence of specific clinical manifestations, early diagnosis of pancreatic cancer is difficult, and lymphatic/blood metastases are common. The average 5-year survival rate of PDAC patients is less than 5%, ranking as the 13rd in global cancer morbidity and 4th in mortality. Since, most pancreatic cancer patients suffer from late stage illness of lymphaticand hematogenous metastasis, missing the only chance of potential radical surgical resection. At present, the invasive and metastatic mechanism of pancreatic cancer is still unknown. It’s urgent to find out the nature and molecular pathogenesis of pancreatic cancer. Moreover, identification of new and specific factors that can provide a possible prognosis of PDAC is of great significance for clinicians to choose the appropriate therapeutic regimen, avoid certain side effect like toxicity caused by unnecessary treatment.Bit1, also namedBcl-2 inhibitor of transcription 1, is found as the important anoikis modulator in 2004, which is located at the inner membrane of mitochondria. At present, the exact biological function and physiological character of Bitl is still not clear. When cells lost contact with extracellular matrix, Bit1 will be released from the inner membrane to outside membrane of mitochondria, then it will compete with TLE1 to bind with AES, forming the transcription factor to affect certain kinds of apoptosis, that is caspase-independent form of apoptosis. Anoikis, which is a normal phenomenon of apoptosis, play essential roles in the maintenance of homeostasis of normal body metabolism. However, when cells escape from anoikis, the cells will become immortalization, which work as the first step of metastasis. During the process, EMT plays important roles in molecular and physiological change of anoikis, which is now believed to be associated with malignant tumors’ invasion, metastasis and drug resistance. Bit1 has been proved to be associated with malignant biological characteristics of lung tumor and breast cancer, but the linkage between Bit1 and pancreatic cancer is still not yet discussed. So it’s necessary to detect the effect of Bit1 on pancreatic cancer’malignant behaviors. And exploring the potential mechanism how Bit1 affect pancreatic cancer cells’behavior is important for us to understand the nature of pancreatic cancer, which is beneficial for us to develop better treatment regimens and possible prognostic markers.Objective1. To observe the expression of Bit1 in six pancreatic cancer and detect the correlation between Bitl withpancreatic cancer cells’ proliferation, migration and invasion.2. To investigate the possible mechanism of Bit1 on pancreatic cancer cells’ biological behaviors.3. To analyze the potential values of Bit1 in pancreatic cancer patients’ tissue microarray with the clinical pathological characteristicsand predicting the prognosis of pancreatic cancer patients.Methods1. The expression of Bit1 in six PDAC cell lines comprising AsPC-1, BxPC-3, Mia paca-2, Panc-1, and SU.86.86 was evaluated by western blotting and real-time PCR.2. Lentiviral vectors in which Bit1 gene over-expressed or short hairpin RNA of Bit1 silenced were transferred into pancreatic cancer cell lines Mia paca2 and Panc-1, respectively. After antibiotic resistant screening, Bit1 stably silenced or overexpressed pancreatic cancer cell lines were established. Both the transfection effects wereverified by western blotting and RT-PCR. Cell proliferation was detected using the CCK-8 kit; cell cycle analysis was performed by fluorescence-activated cell sorting; cell migration and invasion activity assays were performed using would healing and transwellanalysis.The potential mechanism of Bit1 affectingbiological behaviors were detected by western blotting.3. Western blot and CoIP were used to explore the potential mechanism on how Bitl could regulate the behaviors of pancreatic cancers’malignant biological phenotype.4. Theexpression of Bit1 was evaluated using tissue microarray-based immunohistochemistry on tumor tissues and adjacent nontumor tissues from 371 patients with pathology-confirmed pancreatic ductal adenocarcinoma (PDAC). The associations between expression levels of Bitl withclinicopathological factors, and overall survival were evaluated.Results1. Based on validated pancreatic cancer cell lines, pancreatic cancer cell Panc-1 was corfirmed expressing the relatively highest Bitl, Mia paca2 as the lowest. Moreover, Panc-1 with Bitl stably silenced and Mia paca2 overexpressed cell lines were successfully established.2. The prolifetation of Bit1 knockdown cell lines made no difference in Bitl-silenced Panc-1 cells with the control group, however, the migration and invasion abilities were significantly increased in Bitl knockdown group, moreover, the apoptosis rate was decreased in the condition of suspention culture. On the contrary, when Bitl is overexpressed in Mia paca2 cells, the proliferation rate also made no difference, but Bitl overexpression group’migration and invasion abilities were significantly decreased, also the apoptosis rate was enhanced. Western blotting showed that the expression of EMT-associated mesenchymal proteinswere obviously increased as compared with the control group in Bitl knockdown group. While, in Bit1-overexpressionMia paca2 cells, the mesenchymal protein markers were decreased.3. The expression of Bitl was significantly higher in normal pancreatic duct cells of adjacent non-tumor tissuesthan in PDAC cells (P< 0.05). The low expression of Bitl in tumor tissues was significantly correlated with CEA level and Peripheral nerve infiltration. Moreover, drinking, the degree of tumor differentiation, N stage, M stage and TNM stage could significantly affect patients’overall survival (P< 0.05).Drinking, N1 stage and Ⅲ-Ⅳ stage of TNM also work as the independent factors affecting prognosis.ConclusionsBitl may not only work as tumor suppressor gene, but also could significantly affect the sensitivityof chemotherapy and the motility and invasion ability of pancreatic cancer cells through the EMT process, which is potentially associated with Erk activation. Moreover, Bitl protein is significantly associated with malignant tumors’ behaviors.So Bitl played an important role in PDAC progression, and that it could be a practical prognostic marker and potential target in PDAC therapy.