The Role Of RGC-32 In The EMT Of Pancreatic Cancer And The Underlying Mechanisms

Objective Pancreatic cancer is one of digestive system carcinomas with high malignancy, early metastasis and extremely poor prognosis. The investigation of invasion and metastasis mechanisms of pancreatic cancer is of great importance for controlling progression and improving prognosis of this deadly disease. It has been shown that transforming growth factor-β(TGF-β)-induced epithelial-mesenchymal transition (EMT) is one of the important factors for promoting invasion and metastasis of pancreatic cancer, although the underlying mechanisms are still unclear. Response gene to complement-32 (RGC-32) is a newly discovered gene activated by complement. RGC-32 is extensively expressed in many kinds of human tissues and organs and is involved in cell proliferation and differentiation. RGC-32 is aberrantly expressed in different kinds of tumors, with diverse abundance and effects. However, the role of RGC-32 in pancreatic cancer is still unknown. In the researches of normal cells, RGC-32 was found to mediate TGF-β-induced EMT. However, nothing is known about its role in the EMT of pancreatic cancer. In this study, in vitro, we try to clarify the effect of RGC-32 in pancreatic cancer and explore its possible role in regulating the EMT of pancreatic cancer cells and the underlying mechanisms, so as to further improve the controlling mechanisms of EMT and provide new insight into molecular targets for gene therapy of pancreatic cancer.Methods Immunohistochemical staining of RGC-32 and E-cadherin was performed on pancreatic cancer, chronic pancreatitis and normal pancreatic tissues. The positive expression rate and staining intensity of RGC-32 and the abnormal expression rate of E-cadherin were compared respectively among different kinds of tissues. The relationship between positive expression rate of RGC-32 and pathological features of pancreatic cancer and relationship between abnormal expression rate of E-cadherin and pathological features of pancreatic cancer were analyzed respectively. Furthermore, the correlation between positive expression rate of RGC-32 and abnormal expression rate of E-cadherin was determined. To explore whether RGC-32 is controlled by TGF-βand its possible role in TGF-β-induced EMT, human pancreatic cancer cell line BxPC-3 (Smad4 homozygous deleted) was treated with TGF-β1, and RGC-32 siRNA silencing and gene transfection were performed as well. The expression of RGC-32 and EMT-related markers at mRNA and protein levels was determined by real-time quantitative RT-PCR and western blot respectively. In order to screen the signal pathways by which RGC-32 mediated TGF-β-induced EMT, chemical inhibitors of Smad-independent pathways were used. Transwell cell migration assay was employed to determine whether RGC-32 mediated TGF-β-induced cell migration. Finally, cell counting kit-8 (CCK-8) and flow cytometry were used to determine the influence of RGC-32 on cell proliferation and apoptosis of BxPC-3 cells respectively.Results Immunhistochemical staining and analysis showed that the positive expression rate of RGC-32 in pancreatic cancer tissues was higher than that in chronic pancreatitis and normal pancreatic tissues (P<0.05) and was correlated with lymph node metastasis and TNM staging (F<0.05); the abnormal expression rate of E-cadherin in pancreatic cancer tissues was higher than that in chronic pancreatitis and normal pancreatic tissues (P<0.05) and was correlated with tumor differentiation, lymph node metastasis and TNM staging(P<0.05). Further analysis demonstrated that there was a significantly positive correlation between positive expression of RGC-32 and abnormal expression of E-cadherin (P<0.01, P=0.458).In vitro, we found sustained TGF-βstimuli induced EMT and up-regulated RGC-32 expression in BxPC-3 cells. By means of gene over-expression and RNA interference, we further demonstrated that RGC-32 not only mediated TGF-β-induced EMT, but also induced EMT independently in BxPC-3 cells, which was independent of Smad signal pathways. Signal pathway inhibiting study indicated that RGC-32 could mediate TGF-β-induced EMT at least via Erk-MAPK and p38-MAPK pathways. Finally, transwell migration assays demonstrated that RGC-32 mediated TGF-β-induced cell migration as well. Cell proliferation and apoptosis detection demonstrated that RGC-32 inhibited the proliferation of BxPC-3 cells, but had no obvious effect on cell apoptosis.Conclusion RGC-32 might be a new metastasis promoting factor for pancreatic cancer and it can enhance metastatic phenotype of pancreatic cancer cells by mediating TGF-β-induced EMT.