| ObjectivePancreatic cancer is one of the digestive system cancers with high degree of malignancy, early metastasis and poor prognosis. The exploration of the mechanism of tumor invasion and metastasis is of great importance for controlling the progress and improving the prognosis of pancreatic cancer. Chronic pancreatitis is a risk factor of pancreatic cancer. Studies have indicated that epithelial mesenchymal transition (EMT) is closely related to the development of chronic inflammatory diseases and cancer. It has been reported that paired related homeobox 1 (Prrx1) gene is highly expressed in many solid tumors including lung, colon and thyroid carcinoma, along with a positive correlation with the invasion and metastasis of cancer cells. However, Prrxl is rarely studied in pancreatic cancer, with unclear molecular mechanisms. This study aims to investigate the role and molecular mechanisms of gene Prrxl in the development of pancreatic cancer through researches on clinical tissue samples and in vitro pancreatic cancer cells, thus providing new insights for developing novel molecular targets for the gene therapy of pancreatic cancer.MethodsStudies in clinical tissue samples:Tissue samples of pancreatic cancer, chronic pancreatitis and normal pancreas tissues were collected. Immunohistochemical method was employed for detecting the expression levels of Prrx1 and EMT related molecules, including E-cadherin and vimentin. The differences of the positive expression rates of Prrxl, E-cadherin and vimentin between different tissues were analyzed. The correlations of the positive expression rates of Prrxl, E-cadherin and vimentin with pathological characteristics of pancreatic cancer were investigated, respectively. Furthermore, the correlation of the positive expression rate of Prrxl with that of E-cadherin or vimentin in pancreatic cancer was explored.Studies in cells:(1)Human pancreatic duct epithelial cell HPDE6-C7 and 3 pancreatic cancer cell lines (AsPC-1, BxPC-3 and CFPAC-1) were cultured. The expression of Prrx1 protein in cells was detected with Western blot.(2)Based on the characteristics of Prrx1 mRNA,3 siRNAs were designed and synthesized, which were transfected into pancreatic cancer BxPC-3 cells. The siRNA with best knockout efficiency was screened by fluorescence quantitative real-time PCR.(3)The lentivirus vector with overexpression of Prrxl was transfected into human pancreatic cancer cell line BXPC-3 to obtain a cell line with stable and high expression of Prrx1 (Prrx1 overexpressing BXPC-3 cells). The expressions of Prrxl mRNA and protein were detected with fluorescence quantitative real-time PCR and Western blot, respectively. Cancer cell morphology was observed by microscope. By Western blot and immunofluorescence, the expressions of E-cadherin and Vimentin proteins were detected. The proliferation, migration and invasion of pancreatic cancer cells were evaluated with clone formation, scratch test and Transwell method, respectively.(4)The siRNA of Prrx1 was used to transfect Prrxl overexpressing cells. The expressions of Prrx1 mRNA and protein were detected with fluorescence quantitative real-time PCR and Western blot, respectively. Cancer cell morphology was observed by microscope. The expressions of E-cadherin and Vimentin proteins were detected by Western blot and immunofluorescence. The proliferation, migration and invasion of pancreatic cancer cells were evaluated with clone formation, scratch test and Transwell method, respectively.(5)EMT involves a number of signaling pathways. To further understand the molecular mechanism of Prrx1 gene regulating biological behavior, Prrx1 overexpressing BXPC-3 cells were treated with PI3K-Akt pathway inhibitor LY294002, NF-κB pathway inhibitor PDTC and Wnt pathway inhibitor XAV939. Western blot was used to detect the expression of Prrx1 and EMT associated proteins, so as to explore the pathways through which Prrx1 induces EMT.ResultsStudies in clinical tissue samples:The positive expression rate of Prrxl protein in pancreatic cancer tissues was significantly higher than that in chronic pancreatitis and normal pancreas tissue samples (P< 0.05). Besides, the positive expression of Prrx1 protein in pancreatic cancer tissue was significantly related with age, tumor vascular and lymphatic invasion, lymph node metastasis, TNM stage and 5 year survival rate (P<0.05), but not significantly related with sex, tumor location, degree of differentiation and depth of invasion (P>0.05). Further analysis revealed that the positive expression of Prrxl was negatively correlated with the expression of E-cadherin (P<0.01, R=0.401). The positive expression of Prrx1 was positively correlated with the high expression of Vimentin (P<0.01 R= 0.361). Statistical analysis showed that the overexpression of Prrxl gene was associated with EMT process.Studies in cells:(1)Normal human pancreatic ductal epithelial cells and 3 cell lines of pancreatic cancer were cultured. Western blot was used to detect the Prrxl protein expression in cells. It was found that Prrxl exhibited a very low expression of protein in HPDE-C7 cells, but high expressions in AsPC-1 and BXPC-3 and CFPAC-1 cells. BxPC-1 was chosen for further research.(2Real-time quantitative PCR and Western blot analysis was used to screen the siRNA with best knockout efficiency. siRNA-b showed the best effect of downregulating Prrxl. Therefore, siRNA-b was chosen to transfect Prrx1 overexpressing cell lines for investigating its effect on the biological behavior and its molecular mechanism.(3)Prrxl overexpressing BXPC-3 cell line was established with a stable and high expression of Prrxl. By fluorescence quantitative real-time PCR and Western blot, the expressions of Prrxl at mRNA and protein levels in Prrxl overexpressing BXPC-3 cell line group were significantly upregulated compared with control group. The morphology of cells in the Prrxl overexpressing group transformed from pebble shape to spindle shape, which indicated EMT. The Western blot results showed that, compared with the blank control group, the expression of epithelial marker E-cadherin in the Prrxl overexpressing group was downregulated, while that of Vimentin was upregulated. Immunofluorescence further confirmed that the overexpression of Prrxl caused the downregulation of E-cadherin and upregulation of Vimentin. The above results suggest that Prrxl overexpression can promote the occurrence of EMT in pancreatic cancer cells.(4)The clone formation, scratch test and Transwell method revealed that compared with blank control group, the Prrx1 overexpressing group exhibited significantly increased cell proliferation, migration and invasion.(5)The fluorescence quantitative real-time PCR and Western blot were applied after siRNA-b transfected Prrxl overexpressing BXPC-3 cells. The expressions of Prrxl mRNA and protein in siRNA group were significantly downregulated, suggesting a successful transfection. The morphology of cells in the Prrxl overexpressing group transferred from spindle shape to pebble shape, indicating that the EMT was reversed. Western blot results showed that in siRNA group, the expression of epithelial cell marker E-Cadherin was increased and that of Vimentin was decreased, compared with Prrxl overexpressing group. Immunofluorescence further confirmed that siRNA transfection induced elevated E-cadherin and decreased Vimentin. The results showed that when the expression of Prrxl was downregulated by siRNA, the EMT process in Prrxl overexpressing cells was reversed.(6)The clone formation, scratch test and Transwell method revealed that compared with Prrxl overexpressing group, the siRNA group exhibited significantly decreased cell proliferation, migration and invasion.(7)The experiment on signaling pathways showed that Prrx1 can induce the EMT process in pancreatic cancer cells at least partially through NF-κB and Wnt pathways.Conclusions(1)Prrxl gene is closely related with the invasion and migration of pancreatic cancer cells, which may become a new transfer promoting factor for pancreatic cancer.(2)Prrxl enhances the proliferation, migration and invasion of pancreatic cancer cells through inducing EMT.(3)The activation of NF-κB and Wnt signaling pathways is involved in the EMT process induced by Prrxl in pancreatic cancer cells. |
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