| ObjectiveTo determine the expression of Wnt2 in pancreatic cancer tissue slices and preliminary research the mechanism of Wnt2 signaling pathway in proliferation and migration and invasion. To explore the pathway of Wnt2 protein on invasion and metastasis. To investigate the role played by Wnt2 protein in pancreatic stellate cells(PSCs) affecting pancreatic cancer cells.Methods1. Immunohistochemistry of 86 cases of pancreatic tissue slice was used to analyze the expression of Wnt2 in pancreatic cancer.2. We built Wnt2 overexpression plasmid and Wnt2 interference RNA, transfected human pancreatic cancer cellline(PANC-1, CFPAC-1), and use recombinant Wnt2 protein. Then cell growth curve, wound healing assays, transwell test were used to analyze changes of biogical behavior of processed cells.3. Detecting the expression of EMT related mRNA and protein of processed cells using real time quantitative PCR and Western Blot.4. Detecting Wnt2 protein expression of pancreatic stellate cells and supernatant and detecting function of pancreatic stellate cell supernatants on pancreatic cancer cell.Results1. Through 86 cases of pancreatic tissue slices of Wnt2 protein immunohistochemical staining, 55 cases(63.5%) of pancreatic cancer cells showing Wnt2 protein positive expression; 77 cases(89.5%) positive stromal cells Wnt2 expression; Logistic regression analysis showed that among pancreatic cancer stromal cells Wnt2 protein expression and survival time and disease-free survival(DFS) correlation(P=0.002, P =0.007); Survival analysis showed a significant difference(P =0.019) compared to patients with positive stromal cells Wnt2 protein between immunohistochemical staining score more than 10 points in the survival time of patients with a score less than 10 points.2. Biological behavior observations: Compared with the control group, pancreatic cancer cell lines overexpressing group, Wnt2 recombinant protein group were to accelerate the rate of pancreatic cancer cell proliferation(P<0.05); while the rate of cell proliferation reduction in interference group(P<0.05); Wnt2 recombinant protein enhanced pancreatic cancer cell migration(P <0.05) and invasion capacity(P <0.05).3. The key EMT’s gene m RNA expressing results of pancreatic cancer cells processed: Wnt2 recombinant proteins stimulate CFPAC-1 and PANC-1 cells after 6 hours, the relative expression of Vimentin m RNA of Wnt2 recombinant protein stimulated group were 2.06(P<0.05) and 1.96 times(P <0.05) compared with the control group, respectively; relative expression of E-cadherin m RNA was 0.47(P> 0.05) and 0.33 times(P<0.05) compared with control group respectively; Vimentin and E-cadherin mRNA expression of PANC-1 cell interference and over-expression group was no significant difference compared with the control group. The key EMT’s protein expressing results of pancreatic cancer cells processed: CFPAC-1 and PANC-1 cells were stimulated by Wnt2 recombinant protein after 6 hours, Vimentin protein levels increased; E-cadherin expression levels reduced only in CFPAC-1 cells stimulated by Wnt2 recombinant protein. PANC-1 cell interference Wnt2 group, Vimentin protein level were lower than the control group, and E-cadherin protein expression level elevated; PANC-1 cells overexpressing Wnt2 group, E-Cadherin, Vimentin protein expression levels were reduced.4. Wnt2 protein was detected in activated stellate cells and culture medium, pancreatic cancer cell cultured in PSC supernatant was faster in proliferation rate(P <0.05) and cell migration(P<0.05) and invasion capacity enhancement(P<0.05); nucleus increasement in the β-catenin.Conclusion1. Wnt2 protein positive expression in stromal cells was associated with pancreatic cancer patients prognosis.2. Overexpressing Wnt2, Wnt2 recombinant protein can stimulate PANC-1 and CFPAC-1 cell proliferation; contrary, reducing the expression of Wnt2 would inhibit cell proliferation; Wnt2 recombinant protein can enhance capacity of pancreatic cancer cell migration and invasion; interference and over-expression of Wnt2 but did not affect cell migration and invasion.3. Wnt2 recombinant protein caused Vimentin protein and m RNA expression levels increase, E-cadherin protein and m RNA expression levels down; the display raised Wnt2 protein induces cell EMT, so that the increase in cell invasion and migration.4. Pancreatic stellate cells and their supernatant showed Wnt2 positive expression; pancreatic cancer cells cultured by PSC medium, canonical Wnt signaling pathway is activated, proliferation and cell migration and invasion capacity enhancement. |
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