Establishment Of ARHI Monoclonal Stable Transfected PANC-1Cell Line And The Effects Of ARHI In The Metastasis Signaling Pathway Of Pancreatic Cancer

Pancreatic cancer is a highly malignant tumor. The incidence of pancreatic cancer rises in recent years. Right now in US, pancreatic cancer becomes the fourth cause of cancer death. For lacking of effective treatment, pancreatic cancer patients have short survivals and poor quality of life. The5-year overall survival rate is less than5%and the median survival is only6months.A number of signaling pathways involved in development and progression of pancreatic cancer have been well studied. Early metastasis is one of the malignant characteristics of advanced pancreatic cancer. Multiple signaling molecules are known to participate in the metastasis of pancreatic cancer, including the chemokine pathway, vascular endothelial growth factor (VEGF) pathway.ARHI, a maternally imprinted gene that belongs to the Ras superfamily was first identified in1999. However, unlike Ras, ARHI is a tumor suppressor gene in ovarian and breast carcinomas. Re-expression of ARHI inhibits cell growth, motility and invasion in ovarian and breast cancer cells.Previous work of our group also suggests ARHI is a tumor suppressor gene in pancreatic cancer cells. Re-expression of ARHI in pancreatic cancer cells can inhibit proliferation, motility and invasion and induce apoptosis and cell cycle arrest.Until now, all the studies of ARHI are based on the polyclonal stable cell lines. There was no report about the establishment of ARHI monoclonal stable cell lines. Here, we tried to establish the monoclonal stable transfected PANC-1cell line which could express ARHI and find whether ARHI participates in the metastasis signaling pathway in pancreatic cancer.Part Ⅰ:Establishment of ARHI gene monoclonal stable transfected PANC-1cell lineMethods:Recombinant plasmid pIRES2-ARHI-EGFP has been constructed successfully by our group. A new recombinant lentivirus plasmid pLNCX2-ARHI-EGFP was constructed. Both recombinant plasmids were transfected into human pancreatic cancer cell line PANC-1by lipofectamine2000. RT-PCR and Western blot analysis were performed to examine the expression of ARHI in transiently transfected PANC-1cells. G418was used to select the cells which could express neomycin resistant gene. After4weeks of selection and monoclonal purification, monoclonal stable PANC-1cell line was established. ARHI mRNA and protein expression in the stable cell line was analyzed by RT-PCR and Western blot methods, respectively. The growth curve and MTT assay were used to determine the effect of ARHI on the proliferation of monoclonal stable transfected cell line. The crape-motility assay was applied to study the influence of ARHI on motility of PANC-1cells.Results:The ARHI mRNA was detectable in ARHI gene transfected PANC-1cells after48hours transfection. The ARHI protein was detectable in ARHI gene transfected PANC-1cells in24hours,48hours and72hours after transient transfection. ARHI mRNA and protein expression were not detectable in the control cells. After4weeks of G418selection and monoclonal purification, the monoclonal stable transfected PANC-1cell line which could express ARHI was identified. RT-PCR and western blot methods confirmed that expression of ARHI could be detected in monoclonal stable cell line, but not in polyclonal vector group and PANC-1control group. The MTT assay showed ARHI could inhibit proliferation of PANC-1cell in ARHI monoclonal stable lines with a significant difference (P<0.05) comparing with that in vector group. The crape-motility assay showed that ARHI monoclonal stable transfected cells migrated slower than the vector group with a significant difference (P<0.01), and the control PANC-1group with a same significant difference (P<0.01) after12hours,24hours and48hours.Summary:1) The monoclonal stable transfected PANC-1cell line which could express ARHI was successfully established by using two different plasmids which are pIRES2-EGFP and lentivirus plasmide pLNCX2.2) Re-expression of ARHI in monoclonal stable transfected PANC-1cell line could inhibit proliferation and migration of pancreatic cancer cells.PART II:The effects of ARHI in the metastasis signaling pathway of pancreatic cancer in monoclonal stable transfected PANC-1cells in vitroMethods:The total RNA was extracted from monoclonal ARHI transfected PANC-1cell and was converted to the first strand cDNA. The relative mRNA quantification of chemokine signaling pathway (CXCL1, CXCL8) and VEGF pathway (VEGF-A, VEGF-B, VEGF-C and HIFa) was determined by real-time PCR methods. Immunocytochemical stain by VEGF monoclonal antibody was used to study the VEGF expression in cytoplasm of PANC-1cell. Results:The mRNA expression level of CXCL1and CXCL8in monoclonal stable transfected PANC-1of ARHI group was significantly lower than that in polyclonal vector transfectants (P<0.05). With regard to VEGF signaling pathway, the mRNA level of VEGF-A, VEGF-B and HIFa in ARHI group was significantly higher than that in polyclonal vector group (P<0.05), while the mRNA level of VEGF-C did not differ between those two groups (P>0.05). Immunocytochemistry stain of VEGF in cytoplasm showed that there was no significant difference between the ARHI group and vector group (P>0.05).Summary:1) ARHI could inhibit the mRNA level of CXCL1and CXCL8in monoclonal stable transfected PANC-1cell line.2) ARHI may promote mRNA expression of VEGF-A, VEGF-B and HIFa in monoclonal stable transfected PANC-1cell line.Conclusions:1) The monoclonal stable transfected PANC-1cell line which could express ARHI was successfully established by using two different plasmids which are pIRES2-EGFP and lentivirus plasmid pLNCX2.2) Re-expression of ARHI in monoclonal stable transfected PANC-1cell line could inhibit proliferation and migration of pancreatic cancer cells.3) ARHI could inhibit the mRNA expression of CXCL1and CXCL8in monoclonal stable transfected PANC-1cell line.4) ARHI may promote mRNA expression of VEGF-A, VEGF-B and HIFa in monoclonal stable transfected PANC-1cell line.