| Pancreatic cancer is one of the digestive system neoplasms, It is considered to be a lethal disease due to its high malignancy and traditional therapies resistance. Only a few of patients are able to undergo potentially radical surgery as pancreatic cancer is difficult to be diagnosed at the onset. It accounts for 1-2% of malignant tumors worldwide. In America, pancreatic cancer is the fourth most common cause of cancer related deaths with 44,030 new cases and 37660 deaths in 2011 and the five-year survival rate is only 6%. The morbidity of pancreatic cancer is related to environmental industrialization contributes higher incidence rate. Recent years, with the severe pollution and the changes of life styles accompanied with the rapid economic development, the morbidity of pancreatic cancer is increasing in China. Based on epidemiological studies, the incidence rate of pancreatic cancer is 17.28 per 100,000 for male and 14.04 per 100,000 for female in 2009 in Shanghai.As lack of specific characteristics, the disease is often diagnosed at an advanced stage. No significant improvement of prognosis for patients with pancreatic cancer has been made though active treatments were performed. The tumorigenesis of pancreatic cancer is due to multi-factors with oncogene activation and anti-oncogene inactivation. With the rapid development of medical science, the molecular targeted therapy based on intervention of genetic alterations brings hope for the patients suffered from pancreatic cancer. The combination of molecular targeted therapies and traditional therapies may improve the prognosis of pancreatic cancer and the quality of life of the patients.Aldolase A (ALDOA), is an enzyme that catalyses a reversible aldol reaction: The substrate, fructose 1,6-bisphosphate (F-1,6-BP) is broken down into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). This reaction is a part of glycolysis. Overexpression of ALDOA in malignant tumors has been confirmed to be correlated with differentiation, TNM stage and prognosis. Recent researches show that ALDOA can affect multiple processes of tumor occurrence, proliferation, anti-apoptosis, invasion, metastasis, angiogenesis and drug resistance through the regulation of multiple signaling pathways. ALDOA can play roles in diagnosis, treatment and outcome prediction as a broad tumor maker.The expression and function of ALDOA in pancreatic cancer has not been studied. In our research, we detected ALDOA expression in tissue microarray of pancreatic cancer by means of immunohistochemistry. The results showed that the expression of ALDOA is much higher in cancer tissues than in para-tumor tissues. The staining index of ALDOA in tumor tissues is related to differentiation、TNM staging and is an independent prognostic factor for the overall survival. Then we successfully constructed lentiviral vector carrying ALDOA RNAi and established cell lines of pancreatic cancer with ALDOA stably silenced. We studied the function of ALDOA in proliferation, apoptosis, cell cycle, migration and invasion in vitro. Silence of ALDOA can promote apoptosis and suppress proliferation, invasion and metastasis in pancreatic cancer. Further study is needed to understand the potential functions of ALDOA in tumor treatment.Part Ⅰ. Expression pattern of ALDOA in clinical specimens of pancreatic cancer and its prognostic significancePurpose:This part aimed to evaluate the expression pattern of ALDOA protein in paired tumor and para-tumor tissues and the correlation with clinicopathologic features and clinical outcome of pancreatic cancer.Methods:We detected ALDOA expression in paired tumor and para-tumor tissues by means of immunohistochemistry via pancreatic cancer tissue microarray. The correlation of ALDOA expression and clinicopathologic features was analyzed using rank sum test. Kaplan-Meier analysis and Log-rank test were performed to calculate patient survival. Cox regression was used to screen out single and multiple risk factors.Result:Immunohistochemistry of tissue microarrays indicated increased positivity rate of ALDOA staining in tumor compared with para-tumor tissues. In tumor tissues, ALDOA was expressed in cytoplasm of cancer cells especially in region around the nucleus, while in para-tumor tissues, ALDOA was diffusely expressed in cytoplasm of normal duct epithelial cells. Increased expression of ALDOA (Staining Index> 6) was significantly correlated with short overall survival time (P<0.001) and poor differentiation (P=0.026). Kaplan-Meier analysis and Log-rank test revealed that the survival rate was correlated with ALDOA expression (P<0.001), tumor differentiation (P<0.001) and TNM staging (P=0.004). High expression of ALDOA was demonstrated as an independent predictor of death (HR=2.379,95% CI= 1.357-4.170, P=0.002) for patients receiving radical surgery by multivariate analyses.Conclusion:Results of this part demonstrate that ALDOA expression is higher in tumor tissues than in paired paratumor tissues. Overexpression of ALDOA in tumor tissues often appears in cytoplasm of cancer cells and significantly correlated with short overall survival time, poor differentiation and late TNM staging. ALDIA is an independent predictor of death for patients with pancreatic cancer receiving radical surgery.Part Ⅱ. Effects of ALDOA gene inhibition on the malignancy of pancreatic cancerPurpose:This part aimed to design and construct ALDOA gene specific shRNA eukaryotic expression vector and establish pancreatic cancer cells stably transfected with recombinant vector; evaluate the effects of ALDOA gene inhibition on the activity of proliferation, cell cycle, apoptosis, invasion and migration of PANC-1 and MIAPaCa-2 in vitro, meanwhile, to investigate the changes of common proliferation, cell cycle, migration and invasion makers caused by ALDOA down-regulation.Method:The expression of ALDOA protein in pancreatic cancer cell lines was examed and two cell lines with the highest levels were selected for the next steps. Firstly, we designed and synthesized ALDOA specific shRNA oligonucleotide strands and constructed ALDOA gene specific shRNA recombinant vector based on eukaryotic plasmid pLKO.1 TRC cloning vector. Secondly, we packaged and produced lentivirus for transfectection of the two cell lines. We used CCK-8 kit, flow cytometry, wound healing assay to analyze the impact of ALDOA inhibition on the activity of proliferation, cell cycle, apoptosis, migration and invasion of PANC-1 and MIAPaCa-2 in vitro. We used Western blot to analyze the changes of common proliferation, cell cycle, migration and invasion makers caused by ALDOA down-regulation.Result:Western blot showed PANC-1 and MIAPaCa-2 contained the highest ALDOA protein in 6 pancreatic cell lines. ALDOA specific shRNA oligonucleotides were annealed and ligated to be double-strand shRNA successfully. After indentified by digestion and sequencing, double-strand shRNA were inserted into pLKO.1 TRC cloning vector at right sites. Lentivirus was packaged, produced and transfected into PANC-1 and MIAPaCa-2. CCK-8 assay showed PANC-1 and MIAPaCa-2 declined proliferation activity when ALDOA was inhibited. Cell cycle analysis demonstrated the cells were blocked in G1 phase with more apoptosis by ALDOA down-regulation. Wound healing assay indicated that inhibition of ALDOA suppressed migration of PANC-1 and MIAPaCa-2. The results of Western blot demonstrated that Snail, Cyclin D1 were down-regulated and E-cadherin was up-regulated.Conclusion:Results of this part demonstrated that silence of ALDOA exerts potent suppressive effects on the activity of proliferation, migration and invasion of pancreatic cancer cells with G1 phase arrest and more apoptotic cells. ALDOA inhibition can down-regulate Snail and Cyclin D1, and up-regulate E-cadherin. Immunohistochemistry showed that the expression of ALDOA and E-cadherin showed a negative correlation.Novelty1. For the first time, we analyzed the expression pattern and prognostic value of ALDOA in paired and para-tumor tissues of pancreatic cancer by high-throughout tissue microarray technique.2. For the first time, we systematically evaluated the impact of ALDOA expression on activity of pancreatic cancer proliferation, cell cycle, apoptosis, migration, invasion in vitro and in vivo.3. Combined metabolism and epithelial mesenchymal transition, two popular research fields in pancreatic cancer, together. Potential application1. ALDOA could be a potential candidate of new biomarkers predicting prognosis of pancreatic cancer with radical surgery.2. ALDOA RNAi has potential application for researches and treatments of pancreatic cancer. |
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