The Role Of Dual Specificity Phosphatase 1 In The Proliferation, Invasion And Angiogenesis Of Gallbladder Cancer

Part one Expression of Duspl in gallbladder cancer tissues and normal gallbladder tissuesObjectiveThis study was aimed to investigate the expression level of dual specificity phosphatase 1 in gallbladder cancer tissues and normal gallbladder tissues, and preliminarily investigated its role in the progression of gallbladder cancer.MethodsThe expression of Dusp1 was examined in 47 gallbladder cancer tissues and 25 normal gallbladder tissues by immunohistochemistry (IHC). The expression level of Dusp1 in gallbladder cancer tissues and normal gallbladder tissues, in high invasion capacity and low invasion capacity tumor tissues were analyzed and compared.ResultsIn the gallbladder cancer tissues, Duspl mainly showed weak or negative positive expression (51.1%), then the moderate positive expression (31.9%). Few of them were strong positive expression (18%). However, in normal tissues, Duspl mainly showed moderate positive expression (40%) and strong positive expression (32%), left of the total cases were weak or negative expression (28%). The expression of Dusp1 is lower in tumor tissues compared to normal gallbladder tissues. The expression of Duspl is lower in high invasion capacity tumor tissues compared to low invasion capacity ones.ConclusionCompared to normal tissues, the Dusp1 expression in gallbladder cancer was down regulated. The Duspl expression was also down regulated in high invasion capacity tumor tissues compared to low invasion capacity ones. This study suggested Dusp1 may play a suppression role in the progression of gallbladder cancer.Part two The role of Dusp1 in tumor proliferation and cloning formation of gallbladder cancerObjectiveThis study was aimed to investigate the role of Dusp1 in tumor proliferation and cloning formation of gallbladder cancer.MethodsIn this study, we used two gallbladder cancer cell lines SGC996 and GBC-SD for in vitro study. Establishing Duspl knocking down cells in GBC-SD cell line and Dusp1 over-expression cells in the two gallbladder cancer cell lines by lentivirus transfection. In both cell lines, cell proliferation assay and cloning formation assay were performed to investigate the role of Dusp1 in tumor proliferation and cloning formation capacity of gallbladder cancer. Meanwhile, a subcutaneous xenotransplanted tumor model of SGC996 cell lines was established in nude mice to exam the influence of Dusp1 on tumor proliferation of gallbladder cancer in vivo.ResultsSuccessfully, we had established Duspl knocking down cells in GBC-SD cell line and Duspl over-expression cells in the two gallbladder cancer cell lines by lentivirus transfection. Knocking down and over-expression efficiency were verified by Real-time PCR and Western Blot. MTS assay and cloning formation assay demonstrated that Duspl knocking down promoted proliferation and cloning formation in GBC-SD cell line and Duspl over-expression inhibited proliferation and doing formation in SGC996 and GBC-SD cell lines. Further mechanism dissection revealed the expression of pERK in Duspl knocking down cells was increased and the expression of pERK in Duspl over-expression cells was decreased which implies a negative regulation of pERK caused by Duspl. When Dusp1 over-expression SGC996 cells and their control cells were implanted subcutaneously in 6 nude mice respectively, subcutaneous tumors were found in all 12 nude mice within 10 days, a lower tumor volume or weight was observed in Duspl over-expression conditions compared with their control groups. IHC result of the subcutaneous tumors revealed a decreased expression level of pERK in Dusp1 over-expression conditions.ConclusionDusp1 inhibits the proliferation and cloning formation capacity in gallbladder cancer which probably by negatively regulating pERK.Part three The role of Duspl in tumor metastasis and agiogenesis of gallbladder cancerObjectiveThis study was aimed to investigate the role of Duspl in tumor metastasis and angiogenesis of gallbladder cancer.MethodsIn this study, we had established Duspl knocking down cells in GBC-SD cell line and Duspl over-expression cells in the two gallbladder cancer cell lines by lentivirus transfection. In both cell lines, cell scratch assay and cell migration/invasion assays were applied to test the influence of Duspl on tumor migration and invasion of gallbladder cancer. At the same time, we subcutaneously implated Duspl over-expression SGC996 cells and their control cells in 10 mice. The role of Duspl in tumor metastasis and tumor angiogenesis of gallbladder cancer was explored in vivo.ResultsSuccessfully, we had established Dusp1 knocking down cells in GBC-SD cell line and Duspl over-expression cells in the two gallbladder cancer cell lines, which were verified by Real-time PCR and Western Blot. Cell scratch assay and migration/invasion assays revealed the increased tumor migration/invasion ability in Duspl knocking down GBC-SD cells and decreased migration/invasion ability in Duspl over-expression SGC996 and GBC-SD cells. Further mechanism dissection revealed Duspl may inhibit the migration/invasion ability via the Dusp1-pERK-MMP2 pathway.Dusp1 over-expression SGC996 cells and their control cells were each implanted subcutaneously in 10 nude mice. After one month, mice were sacrificed and metastasis situation were observed. Tumor metastasis was observed in 1 mouse in control group and 3 mice in Duspl over-expression group. So Duspl was also testified to inhibit gallbladder cancer metastasis in vivo assay. The tumor vessel density was decreased in Dusp1 over-expression subcutaneous tumors which was also testified by CD31 IHC result. Further mechanism dissection revealed Duspl may inhibit the tumor angiogenesis ability via the Duspl-pERK-VEGFA pathway.ConclusionDuspl may inhibit the migration/invasion and tumor angiogenesis ability in gallbladder cancer. Meachanism dissection revealed it may depend on the Dusp1-pERK-MMP2 and Dusp1-pERK-VEGFA pathways.