| Pancreatic cancer is one of the most common gastrointestinal malignancy, and have an increasing incidence in this years. Because of the high degree of malignancy, poor prognosis, low survival rate, and lack of clinical features, the diagnosis and treatment becomes extremely difficult. Traditional biological markers, such as CA19-9with sensitivity and specificity of70%-85%and65%-80%, were lack of sensitivity and specificity in the diagnosis and prognosis of pancreatic cancer. The surgery has been considered a cure for pancreatic cancer. However,50%of patients found with distant metastasis, they lost the change of curative surgical resection,5-year survival of postoperative less than5%. Chemotherapy is an important adjuvant therapy for pancreatic cancer. Gemcitabine, as a first-line drug in pancreatic cancer chemotherapy, is widely used in the clinical treatment of advanced cancer. Due to the presence of tumor innate resistance and acquired drug resistance, pancreatic cancer patients are not sensitive to the majority of chemotherapeutic drugs, and it impedes the application of chemotherapy drugs.DJ-1gene was first found by Nagakubo in Japan. It was reported as a new oncogene in mice NIH3cells, and had synergistic transformation of H-Ras. The study confirmed that DJ-1involved in the regulation of gene transcription, cell transformation, anti-oxidation, molecular chaperones and apoptosis inhibition. The mutations and abnormal expression of DJ-1, regulated led to the nervous system and the development of neoplastic lesions. In addition, not only expressed in pancreatic tumor tissue and pancreatic juice, DJ-1protein can also be secreted into the peripheral blood circulation. These studies indicated that DJ-1might be a potential biological marker in pancreatic cancer. The latest proteomics experimental analysis of DJ-1performed that DJ-1may be a chemo-resistance-associated gene. However, the exact mechanism of the DJ-1in pancreatic cancer chemotherapy resistance is unclear.β-catenin is an important component of the Wnt signaling pathway. It plays a key role in a variety of malignant tumors such as leukemia, prostate cancer, lung cancer and pancreatic cancer, and regulates a variety of tumor biological signals and functions. Phosphorylation Akt and GSK-3β enhanced the stability of β-catenin, accumulation of β-catenin followed by translocation into the nucleus. β-catenin combined with nuclear TCF and LEF transcription factors to regulate specific downstream target genes.This study focused on the DJ-1might be a potential biomarker of pancreatic cancer, and analyzed the relationship between DJ-1protein levels with tumor progression and patient survival. We further discussed the function of DJ-1with pancreatic cancer cell apoptosis and cell cycle. DJ-1potentiate enhance sensitize in pancreatic cancer cell apoptosis induced by gemcitabine. We will get more multi-molecular biological information about the mechanism of cancer chemotherapy resistance, which coμld provide new targets and strategies for the treatment of pancreatic cancer.Methods:Part1:Serum DJ-1may be a potential biomarker in pancreatic cancer and correlate with patient prognosisSerum level of DJ-1with pre-operative pancreatic cancer patients were detected by ELISA, the sensitivity and specificity was analysis by ROC curve; furthermore, serum DJ-1levels and patient survival were analyzed by Kaplan-Meier analysis; besides serum level of DJ-1, tumor metastasis, TNM stage, tumor differentiation and tumor respectability had also analyzed with patient survival, using Cox regression univariate analysis and multivariate analysis; Part2:the role of DJ-1gene in gemcitabine-induced pancreatic cancer cell apoptosis and cell cycle arrestFirst, the correlation between DJ-1gene and pancreatic cancer cell proliferation in vitro was detected using the MTT assay and flow cytometry assay, and the role of DJ-1in the pancreatic cancer cell growth inhibition which induced by gemcitabine had also been detected; the function of DJ-1gene with cell cycle regulation in pancreatic cancer cells was examined by flow cytometry analysis;RNA interference technology were used by siRNA and protein over-expression technology via construct p-EGFP-DJ-1plasmid which was over-expression of DJ-1protein in pancreatic cancer cell lines, respectively; the important function of DJ-1gene with gemcitabine-induced pancreatic cancer cell growth inhibition was tested by MTT assay and flow cytometry assay;The expression proteins of Caspase apoptosis pathway and apoptotic proteins such as Bcl-2, etc. was detected by Western blot method, confirm that low expression of DJ-1gene could induce cell apoptosis in pancreatic cancer cells.Part3:p-AKT/(3-catenin signaling pathway was involved in the sensitizing effect of DJ-1in gemcitabine-induced apoptosis in pancreatic cancer cellsThe siRNA-DJ-1and the p-EGFP-DJ-1was transfected into the pancreatic cancer cell line MIA PaCa-2, respectively. The negative control group, the siRNA group, the vector group, and p-EGFP-DJ-1transfected group were given gemcitabine for48h, and then the expression of pAkt and Akt protein were detected by western blot; the nucleus protein was extracted and the expression of nuclear (3-catenin was tested by western blot analysis; the expression of downstream transcription factors, as surviving, c-myc and cyclin D1, was also tested; interference or over-expression of DJ-1gene in pancreatic cancer cell line MIA PaCa-2, the espression of nuclear β-catenin translocation was observed through laser confocal microscopy.Results:Part1:Serum DJ-1may be a potential biomarker in pancreatic cancer and correlate with patient prognosisSerum level of DJ-1in normal healthy and preoperative pancreatic cancer patients were tested by ELISA assay, and the level of DJ-1in pancreatic cancer patients was significantly higher than normal healthy; ROC curves analysis performed serum DJ-1with high specificity and sensitivity; compared with serum CA19-9, DJ-1was superior to CA19-9in the cases-control analysis; Kaplan-meier analysis showed that serum DJ-1level had negative correlation with pancreatic cancer patients survival, the patients with low level of serum DJ-1had longest survival, and also had patient average survival; Univariate analysis indicated that serum DJ-1level, CA19-9level, tumor size, tumor lymph metastasis, distant metastasis, tumor stage, tumor differentiation and tumor resectability was related with patients survival; Multivariate analysis confirmed that serum DJ-1level, CA19-9level and tumor stage may be an independent predictor of prognosis with pancreatic cancer patients. Part2:the role of DJ-1gene in gemcitabine-induced pancreatic cancer cell apoptosis and cell cycle arrestPancreatic cancer cells transfected with DJ-1siRNA, inhibited cell proliferation; While MIA PaCa-2cells over-express DJ-1gene promoted obvious cell proliferation; Pancreatic cancer cells transfected with DJ-1siRNA exposured to gemcitabine, they displayed an enhanced apoptosis effect with time-and concentration-dependent on cancer cells; Instead, when p-EGFP-DJ-1MIA PaCa-2cells exposured to different concentration of gemcitabine, it inhibited gemcitabine toxic effects of the cells;Flow cytometry analyzed cell apoptiosis and the change of cell cycle, pancreatic cancer cells transfected with DJ-1siRNA blocked S phase accumulation, and MIA PaCa-2cells over-expressed DJ-1had obviously increased the capture of S phase;DJ-1gene activated caspase pathway after interference and induced mitochondria apoptosis; the expression of anti-apoptotic protein Bcl-2was reduced, when over-expressed DJ-1, cleaved caspase-3protein expression was reduced, and anti-apoptotic protein Bcl-2was increased; interference pancreatic cancer cells exposured to gemcitabine had a significantly higher expression of cleaved caspase-3protein and anti-apoptotic significantly lower expression of Bcl-2; p-EGFP-DJ-1MIA PaCa-2cells performed an opposite results when exposured to gemcitabine.. Part3:AKT/β-catenin signaling pathway was involved in the sensitizing effect of DJ-1in gemcitabine-induced apoptosis in pancreatic cancer cellsRelative to the si-control group and gemcitabine group, siRNA-DJ-1down-regulated the expression of p-AKT after exposured to gemcitabine48h, and no obvious difference with AKT; it also showed significantly reduced expression with β-catenin, and downstream transcription factor survivin, c-myc and cyclin D1protein; Confocal microscope analysis performed that pancreatic cancer cells after interference weakened β-catenin nuclear translocation induced by gemcitabine cause;MIA PaCa-2cells transfected with p-EGFP-J-1were exposured to gemcitabine for48h. Relative to the control group and gemcitabine treatment group, p-AKT and nuclear P-catenin increased significantly, and the transcription factors as survivin, c-myc and cyclin D1etc. were also high expression; Confocal microscope express clearly enhanced nuclear β-catenin translocation induced by gemcitabine.Conclusions:1. Serum DJ-1level can be used as potential serum biomarker in pancreatic cancer, and it has good specificity and sensitivity; Serum DJ-1level, CA19-9level and tumor metastasis, differentiation, tumor resectability had negative correlation with pancreatic cancer patient survival; Serum DJ-1level may be an independent predictor of prognosis with pancreatic cancer patients;2. Pancreatic cancer cells knock-downed DJ-1sensitized pancreatic cancer cell apoptosis exposured to gemcitabine, but over-express DJ-1weakened the cell apoptosis induced by gemcitabine in pancreatic cancer;3. DJ-1involved in regulating pancreatic cancer cell apoptosis induced by gemcitabine may be through the AKT/β-catenin signaling pathway.
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