Study On The Role Of ARHI Gene And Autophagy In Pancreatic Cancer And The Related Mechanisms

Pancreatic cancer is a highly malignant cancer of digestive system. The mortality rate of pancreatic cancer is almost100%. It is difficult to find with a higher level of invasiveness and metastasis, and it is not sensitive to conventional radiotherapy and chemotherapy. Therefore, prognosis of patients with pancreatic cancer is very poor. The5-year overall survival rate is less than10%, and the median survival only3to6months. Radical resection and adjuvant therapy can only delay the survival to23months.Autophagy is a catabolic process whereby cells digest their intracellular organelles. It is an evolutionarily conserved lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelles. While autophagy has become one of the most attractive topics in cancer research, the current autophagy literature is often viewed as confusing, because of its association with apparently contradictory roles, such as survival and cell death.ARHI, a maternally imprinted gene was first cloned by Yu in1999. Preliminary studies of our laboratory showed that the gene is a tumor suppressor gene, and it is downregulated in pancreatic cancer tissues. We showed that ARHI gene can inhibit the proliferation of pancreatic cancer cell lines, induce apoptosis and cell cycle arrest. It has reported that ARHI could induce autophagy in ovarian cancer, breast cancer cell lines. We also confirmed that transient transfection of the ARHI gene can induce PANC-1cells autophagy in our laboratory.Until now, there is no report about using stably transfected cell line to study ARHI-induced autophagy. This study is based on the pancreatic cancer cell lines stably transfected with ARHI gene, to study the impact of ARHI on pancreatic cancer cell autophagy and the possible molecular mechanisms. We also study the expression of ARHI and the autophagy-related proteins in pancreatic cancer and their relationship at the histological level. Through the above research, we hope to find more theoretical basis for the further study of pancreatic cancer pathogenesis, and the feasibility of autophagy induction treatment in pancreatic cancer. Part Ⅰ:Study on the mechanisms of ARHI gene on autophagy induction in PANC-1Cell LineAim:To investigate if stably transfected ARHI genes can induce pancreatic cancer cell line PANC-1autophagy and the possible molecular mechanisms.Methods:We use the PANC-1cell line stably transfected with ARHI gene constructed in our laboratory. Transfection efficiency can be detected using standard fluorescence microscopy. RT-PCR and Western blot analysis were performed to examine the expression of ARHI in stable transferred PANC-1cells. The growth curve and MTT assay were used to determine the effect of ARHI on the proliferation of stable transfected cell line. Staining with Hoechst33258, we observe the morphological changes of apoptosis. The Apoptosis rate was measured by flow cytometry after staining with propidium iodide (PI). The cells were stained with acridine orange to observe acidic vesicular organelles by fluorescence microscopy. Transmission electron microscopy (TEM) was used to detected scattered double membrane vacuolar structure. We investigate the impact of ARHI gene on LC3, P62, and Beclinl expression by qwstern blot analysis. RT-PCR was performed to detect the changes of LC3, ULK1, Beclinl, PI3KC3, Atg7, Bcl-2gene expression after stable transfected ARHI. We also determine the difference expression of ARHI and LC3protein in the cell cytoplasm and membrane content of stable transfection cell line.Results:1. The ARHI mRNA and protein can be detected in ARHI gene stable transfected PANC-1cells, while the ARHI mRNA and protein can’t be detected in control and vector cells.2. The MTT assay showed ARHI could inhibit proliferation of PANC-1cell in ARHI stable lines with a significant difference (P<0.05) comparing with that in vector group. Apoptotic nuclear morphological changes was found in both ARHI gene transfected group and empty vector group after staining with Hoechst33258, but not in PANC-1cell lines. The Apoptosis rate in ARHI gene transfected group was (4.44±1.96)%, significantly higher than vector group (1.92±0.40)%(P<0.05).3. Cells with the acidic vesicular organelles in ARHI gene transfected group and empty vector transfected group were27.2%and6.3%, respectively(P<0.05). Typical double-membrane of autophagosome were identified with TEM in ARHI gene transfected group and vector group, the number of autophagosome of ARHI group (4.8±3.0) was significantly higher than vector group (0.8±1.0). Western blot analysis showed that the LC3-Ⅱ protein expression was significantly upregulated, and p62protein expression was significantly reduced in ARHI group. RT-PCR results showed that compared with the empty vector group, the expression of ULK-1, Beclinl, PI3KC3, LC3, ATG7gene did not change significantly in ARHI group at the mRNA level, while the BCL-2gene was significantly downregulatd. Western blot results of Beclinl protein expression was no significant difference in ARHI group.5. Western blot analysis showed that the LC3-Ⅱ and ARHI protein was significantly higher in the membrane component than the cytoplasmic component in ARHI stable transfected PANC-1cells.Summary:1. Polyclonal stable transfection of the ARHI gene can inhibit pancreatic cancer PANC-1cell proliferation, induction of apoptosis and autophagy.2. BCL-2mRNA expression was downregulatd, while LC3, Beclinl, ULK-1, PI3KC3, ATG7genes mRNA showed no changes in ARHI stable transfected PANC-1cells and vector cells.3. LC3-Ⅱ and ARHI protein was significantly higher in the membrane component than the cytoplasmic component in ARHI stable transfected PANC-1cells.PART Ⅱ:ARHI and autophagy-related protein expression in pancreatic carcinoma and its clinicopathological significanceAim:Explore the relationship bwtween pancreatic cancer and autophagy, and illustrate that the role of ARHI in the pancreatic carcinoma autophagyMethods:1. Immunohistochemical technique was performed to detect the expression of ARHI, LC3and p62in specimens of39cases of pancreatic cancer,10cases of paracancerous tissues and11cases of normal pancreas.2. We analysis of the relationship between ARHI, LC3and p62expression and clinicopathological factors of pancreatic cancer.3. We also analysis of the relationship among ARHI, LC3and p62expression.Results:1. The positive expression rate of ARHI in normal pancreas, paracancerous tissues and pancreatic carcinoma was100%,80%and35.9%; there is a significant downward trend in these three groups (χ2=39.448, P=0.000). The high expression rate of LC3in normal pancreas, pancreatic carcinoma and paracancerous tissues was27.3%,74.4%and90%; there is a significant upward trend in these three groups (χ2=21.280, P=0.020). The high expression rate of p62in normal pancreas, pancreatic carcinoma and paracancerous tissues was45.5%,70%and66.7%; high expression rate of p62in pancreatic carcinoma and paracancerous tissue is significant higher than normal pancreas (χ2=14.275, P=0.027).2. LC3expression showed no significant correlation with age, gender, smoking history, drinking history, history of diabetes, serum CA199level, clinical stage, tumor size, tumor differentiation and tumor location. p62expression showed a significant correlation with the location of the tumor (P=0.049); but not with age, sex, smoking history, drinking history, history of diabetes, serum CA199level, clinical stage, tumor size and tumor differentiation. ARHI expression showed a significant correlation with tumor differentiation (P=0.005); but not with age, gender, smoking tobacco history, drinking history, history of diabetes, serum CA199level, clinical stage, tumor size and tumor location.3. There was no significant correlation between the expression of ARHI and LC3, ARHI and p62, LC3and p62(P>0.05).Summary:1. ARHI expression in pancreatic cancer is missing or significantly downregulated; LC3and p62showed a significant high expression in pancreatic cancer and paracancerous tissues.2. p62expression had a significant correlation with the location of the tumor; pancreatic head tumor showed a high p62expression. ARHI expression had a significant correlation with tumor differentiation; the lower degree of tumor differentiation, the higher ARHI expression missed.Conclusions:1. ARHI gene can inhibit pancreatic cancer PANC-1cell proliferation, induction of apoptosis and autophagy.2. ARHI gene induced PANC-1cell line autophagy and apoptosis may be related with BCL-2gene downregulated.3. In polyclonal ARHI stable transfection PANC-1cell lines, LC3-Ⅱ and ARHI protein was significantly higher in membrane protein composition than in cytoplasmic composition, suggesting that ARHI may be directly involved in autophagy.4. ARHI expression in pancreatic cancer is missing or significantly downregulated5. LC3expression was higher in pancreatic cancer and paracancerous tissues than in normal pancreatic tissue, suggesting that the autophagy level of pancreatic cancer increased.6. p62expression was upregulated in pancreatic cancer and paracancerous tissues than in normal pancreatic tissue, suggesting that it may be involved in the development of pancreatic cancer.7. The expression of ARHI, LC3and p62have no significant correlation in the pancreatic.