Expression Of Oncogene MTDH In Pancreatic Cancer And Its Function

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 for1-2%of malignant tumors worldwide. In America, pancreatic cancer is the fourth most common cause of cancer related deaths with44,030new cases and37660deaths in2011and the five-year survival rate is only6%. The morbidity of pancreatic cancer is related to environmentand industrializationcontributes higher incidence rate. Recent years, withthe severe pollution and the changes of life styles accompanied withthe rapid economic development, the morbidity of pancreatic cancer isincreasing in China. Based on epidemiological studies, the incidence rate of pancreatic cancer is7.26per100,000for male and4.95per100,000for female in2008in 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 therapiesand traditional therapies may improve the prognosis of pancreatic cancer and the quality of life of the patients.Metadherin (MTDH), also called AEG-1, LYRIC or3D3, locates on chromosome8q22that is considered to be related to the development of a lot of malignant tumors. MTDH is a newly discoverd gene with11introns and12exons and is present in many human tissues and organs. Overexpression of MTDH in malignant tumors has been confirmed to be correlated with differentiation, TNM stage and prognosis. Recent researches show MTDH can affect multiple processes of tumor via PI3K/Akt, NF-κB, Wnt/β-catenin and MAPK signal pathways. MTDH can play roles in diagnosis, treatment and outcome prediction as a broad tumor maker.The expression and function of MTDH in pancreatic cancer has not been studied. In our research, we detected MTDH expression in tissue microarray of pancreatic cancer by means of immunohistochemistry. The results showed that the expression of MTDH is much higher in cancer tissues than in paracancer tissues. The staining index of MTDH in tumor tissues is related to smoking, differentiation, lymphatic metastasis and TNM staging and is an independent prognostic factorfor the overall survival. Then we successfully constructed lentiviral vector carrying MTDH RNAi and established cell lines of pancreatic cancer with MTDH stably silenced. We studied the function of MTDH in proliferation, apoptosis, cell cycle, migration and invasionin vitro and in vivo. Silence of MTDH can promote apoptosis and suppress proliferation, invasion and metastasis in pancreatic cancer. Mechanism study showed MTDH can affect processes of pancreatic cancer via Akt/mTor signal pathway. Further study is needed to understand the potential functions of MTDH in tumor treatment.Part Ⅰ. Expression pattern of MTDH in clinical specimens of pancreatic cancer and its prognositic significancePurpose:This part aimed to evaluate the expression pattern of MTDH protein in paired tumor and paratumor tissues and thecorrelation with clinicopathologic features and clinical outcome of pancreatic cancer.Methods:We detected MTDH expression in paired tumor and paratumor tissues by means of immunohistochemistry via pancreatic cancer tissue microarray. The correlation of MTDH 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 MTDH staining in tumor compared with paratumor tissues (58/89,65.2%vs.23/89,25.8%). In tumor tissues, MTDH was expressed in cytoplasm of cancer cells especially in region around the nucleus, while in paratumor tissues, MTDH was diffusely expressed in cytoplasm of normal duct epithelial cells. Increased expression of MTDH (Staining Index>6) was significantly correlated with short overall survival time (P=0.000), smoking (P=0.029), poor differentiation (P=0.014), lymphatic metastasis (P=0.012) and late TNM staging (P=0.036). Kaplan-Meier analysis and Log-rank test revealed that the survival rate was correlated with MTDH expression (P=0.000), tumor differentiation (P=0.002), TNM staging (P=0.036) and CA19-9(P=0.048). High expression of MTDH was demonstrated as an independent predictor of death (HR=2.638,95%CI=1.537-4.528, P=0.000) for patients receiving radical surgeryby multivariate analyses.Conclusion:Results of this part demonstrate that MTDH expression is higher in tumor tissues than in paired paratumor tissues. Overexpression of MTDH in tumor tissues often appears in cytoplasm of cancer cells especially in region around the nucleus and significantly correlated with short overall survival time, smoking, poor differentiation, lymphatic metastasis and late TNM staging. MTDH is an independent predictor of death for patients with pancreatic cancer receiving radical surgery.Part II. Construction of MTDH gene specific shRNA eukaryotic expression vector and establishment of pancreatic cancer cells stably transfected with recombinant vectorPurpose:This part aimed to design and construct MTDH gene specific shRNA eukaryotic expression vector and establish pancreatic cancer cells stably transfected with recombinant vector for further study of the functions of MTDH in pancreatic cancer.Methods:The expression of MTDH protein in pancreatic cancer cell lines was examed and two cell lines with the highest levelswere selected for the next steps. Firstly, we designed and synthesized MTDH specific shRNA oligonucleotide strands and constructed MTDH gene specific shRNA recombinant vector based on eukaryotic plasmid pLKO.1TRC cloning vector. Secondly, we packaged and produced lentivirus for transfectection of the two cell lines. Finally, we selected cells stably transfected with recombinant shRNA vectors by puromycin and indentied the best inhibited effect of MTDH by PCR and Western blot.Result:Western blot showed PANC-1and SW1990contained the highest MTDH protein in6pancreatic cell lines. MTDH 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.1TRC cloning vector at right sites. Lentivirus was packaged, produced and transfected into PANC-1and SW1990. Then, PANC-1and SW1990with stably transfected recombinant vectors were harvested by puromucin. PANC-shMTDH2and SW1990-shMTDH2were the cell lines with best inhibited effect of MTDH identified by PCR and Western blot.Conclusion:We successfully constructed MTDH gene specific shRNA eukaryotic expression vector and established pancreatic cancer cells stably transfected with recombinant vector for further study of the functions of MTDH in pancreatic cancer.Part Ⅲ. Effects of MTDH gene inhibition in pancreatic cancer cell lines and exploration of intrinsic mechanismsPurpose:This part aimed to evaluate the effects of MTDH gene inhibition on the activity of proliferation, cell cycle, apoptosis, invasion and migration of PANC-1and SW1990in vitro and in vivo, meanwhile, to investigate the changes of Akt/mTor signaling pathway plus common proliferation, cell cycle, migration and invasion makers causedby MTDH down-regulation.Method:We used CCK-8kit, flow cytometry, wound healing assay, transwell invasion assay and established subcutaneous tumor implantation model of nude mice to analyze the impact of MTDH inhibition on the activity of proliferation, cell cycle, apoptosis, migration and invasion of PANC-1and SW1990in vitro and in vivo. We used Western blot to analyze the changes of Akt/mTor signaling pathway plus common proliferation, cell cycle, migration and invasion makers causedby MTDH down-regulation.Result:CCK-8assay showed PANC-1and SW1990declined proliferation activity when MTDH was inhibited. Cell cycle analysis demonstrated the cells of G2phase and S phase were reduced and the cells were blocked in Gl phase with moreapoptosis by MTDH down-regulation. Wound healing assay and transwell invasion assay indicated that inhibition of MTDH suppressed migration and invasion of PANC-1and SW1990. In vivo study confirmed the tendency that inhibition of MTDH will turn down the proliferation activity. The results of Western blot demonstrated that Ki67, Cyclin D1, MMP-9were down-regulated and E-cadherin was up-regulated, while the expression of the key molecules of Akt/mTor signaling pathway were significantly changed.Conclusion:Results of this part demonstrated that silence of MTDHexerts potent suppressive effectson the activity of proliferation, migration and invasion of pancreatic cancer cells with G1phase arrest and more apoptotic cells. MTDH inhibition can down-regulate Ki67, Cyclin D1, MMP-9and up-regulate E-cadherin. MTDH can affect processes of pancreatic cancer via Akt/mTor signal pathway.Novelty1. For the first time, we analyzed the expression pattern and prognostic value of MTDH in paired and paratumor tissues of pancreatic cancer by high-throughout tissue microarray technique.2. For the first time, we systematically evaluated the impact of MTDH expression on activity of pancreatic cancer proliferation, cell cycle, apoptosis, migration, invasion in vitro and in vivo.Potential application1. MTDH could be a potential candidateof new biomarkers predicting prognosis of pancreatic cancer with radical surgery.2. MTDH RNAi has potential application for researches and treatments of pancreatic cancer.