| Background:Pancreatic carcinoma (PC) is a malignant tumor in pancreatic tissue. WNT5A, which synthesized and secreted by pancreatic cancer cells (PCCs), plays an important role in the progression of malignancy. Up to now, statistical analysis of WNT5A expression in PC is rare. Although growing evidence has been reported WNT5A’s function on PCCs invasion and migration and the role of WNT5A in chemoresistance and angiogenesis remain to be studied.Objective:For a new strategy for PC diagnosis and treatment, the expression of WNT5A with clinical-pathological characteristic in PC was evaluated, and the role of WNT5A in gemcitabine resistance and tumor angiogenesis was studied.Methods:1. Resected specimens of68patients identified as PC were obtained from Cancer Institute and Hospital of Tianjin Medical University. The expression of WNT5A in PC and paracarcinoma was analyzed by using immunohistochemistry method. And the correlation between WNT5A’s expression and clinicopathological characteristics such as age, gender, TNM stage, tumor grade, location and volume was also evaluated.2. Small interference RNA (SiRNA) was used to detect the WNT5A’s functions on the resistance to gemcitabine in PANC-1, MiaPaCa2and Capan-2. Then the stable PANC-1cell lines with knockdown or over-expression of WNT5A were established. CCK8cytotoxic assay was used for the detection of half inhibiting concentration.3. After WNT5A was knocked down, the percentage of cells in different cell cycle was determined by flow cytometry. Real time PCR was used for the detection of Cyclins, and western blotting was used for testing the protein level of Cyclin D and p-AKT. Then the immunoprecipitaion study was used to observe the mechanism of WNT5A’s function on pRb-E2F complex.4. By using immunoblotting method, the expression of HIF-1α was checked. And the translocation of HIF-1α from cytoplasm to nucleus was detected by immunofluorescence. Transcriptional level of Vasuclar Endothelial Growth Factor (VEGF) was detected by Real time PCR, and the secreted VEGF protein was detected by ELISA kit.5. Transwell chamber was used to co-culture PCCs and Human Umbilical Vein Endothelial cells (HUVECs). Proliferation of HUVECs was checked by Cell counting and migration was checked by scratch test and transwell assay.6. Female BALB/c nude mice were contributed for xenograft. After subcutaneous injection of PANC-1-sh1RNA-WNT5A and PANC-1-sh1RNA-Ctl, tumor sizes were measured every5days by caliper and tumor volume was calculated.30days later, mice were sacrificed and the expression of HIF-1α was detected by immunohistochemistry and the Micro-vascular density (MVD) was calculated.Results:1. WNT5A was positive expression in PC and faint staining in paracarcinoma. WNT5A stain was correlated with TNM stages.2. PCCs showed sensitive to gemcitabine with knockdown of WNT5A in PANC-1, MiaPaCa2and Capan-2. The same result was obtained in stable interference or overexpression of WNT5A in PANC-1.3. WNT5A caused cell cycle G1phase arrest and decreased mRNA levels of Cyclin D. These changes were mediated by phosphorylation of AKT on Ser473. By activating AKT and promoting Cyclin D, WNT5A could dephosphorlate pRb and stabilized the pRb-E2F complex, which reinforced function of restriction in G1/S phase.4. WNT5A could stabilize the HIF-1α’s expression and promote the nucleus translocation, which activated the transcription and promoted protein secretion of VEGF.5. After co-culture of PCCs and HUVECs, latter were showed to be proliferation and migration. In vivo, knockdown of WNT5A could block the proliferation of pancreatic cancer and tumor angiogenesis.Conclusion:1. WNT5A was positive expression in PC and the expression was correlation with TNM stages.2. WNT5A increased the expression of Cyclin D via phosphorylated-AKT, and Cyclin D promoted pRb-E2F complex depolymerization, which makes the cell easily pass through the G1/S restriction point and contribute to gemcitabine resistance.3. WNT5A promote HIF-1α stabilization and nucleus translocation in PCCs, which activated the expression and secretion of VEGF. VEGF facilitated the proliferation and migration of HUVECs, and result in tumor angiogenesis. |
PDF Full Text Request