| Objective: To gain insight into the relationship between CUL4 B expression level in gastric cancer tissues and clinical pathological characteristics and prognosis of patients,we detected the expression level of CUL4 B in gastric cancer tissues.We then monitored and analyzed the biological behavior changes of gastric cancer cells after using small interfering RNA to shut down expression of CUL4 B.Methods: 1.Adopted immunohistochemical staining method to detect CUL4 B expression in 82 cases of gastric cancer tissue specimens,which were surgically resected and diagnosed by pathological.Then analysed the relation of CUL4 B expression level with invasive phenotypes of gastiric cancer by SPSS 16.0 software.2.Used western blot assay to text the expression of CUL4 B in gastric cancer tissues and normal tissues.3.Analysed the relative expression level of CUL4 B in gastric cancer cell lines.4.Used small interfering RNA to down-regulate the expression level of CUL4 B in MGC803 cells and then divided the cells into 3 groups,which were negative contorl group,interfereing group(si RNA-958,si RNA-2247),respectively.Next,detected the level of CUL4 B protein by western blot assay and detected the level of CUL4 B m RNA by Real-time PCR to confirm the effect of small interfering RNA in CUL4 B down-regulated cells.5.Transwell assay was used to test the ability of migration and invasion of MGC803 cells before and after down-regulating the expression of CUL4 B.6.Plate clone formation assay was used to detect the ability of proliferation of MGC803 cells before and after transfected with small interfering RNA.7.Western blot assay was used to detect the changes of Wnt/ β-catenin downstream preteins expression levels after transfected MGC803 cells with si RNA.Results: 1.CUL4 B protein was found to be mainly distributed in the nucleus and was up-regulated in gastric cancer tissues.The immunohistochemical staining method was adopted to detect CUL4 B expression in gastric cancer tissue specimens,and the result showed that the up-regulated rate of CUL4 B in gastric cancer tissue specimens was 78.04%(64/82).The statistical analysis of clinical data showed that high expression of CUL4 B was related to invasive phenotypes of gastric cancer(high TNM staging,lymph node metastasis,vascular invasion)(p<0.05).2.CUL4 B protein was up-regulated in gastric cancer cells.Western blot assay discoverd that CUL4 B expression level in gastric cancer cells were higher than that in normal gastric mocosal cell and there were remarkable differences between them(p<0.05).3.Western blot assay proved that the expression level of CUL4 B in gastric cancer tissues was higher than that in normal gastric tissues,and p<0.05,and a statistically significant differences.4.Western blot assay discovered CUL4 B that expressed in gastric cancer cells were significantly down-regulated after transfected with small interfering RNA(p<0.05).5.Semi-quantitative RT-PCR method detected CUL4 B expression in gastric cancer cells were remarkably reduced after transfected with small interfering RNA(p<0.05).6.Transwell assay showed that small interfering RNA of CUL4 B significantly reduced the ability of migration and invasion of gastric cancer cells when compared with the negative control group(p<0.05).7.Plate clonality assay detected that the proliferative capability of gastric cancer cells were markedly inhibited after transfected with small interfering RNA as compared to those in the negative control group(p<0.05).8.The expression levels ofβ-catenin and Cyclin-D1 preteins of Wnt/β-catenin signaling in gastric cancer cell line MGC803 were obviously decreased after tranfected with si RNA.Conclusion: 1.CUL4 B protein was mainly expressed in the nucleus of gastric cancer cells,and its expression was up-regulated in gastric cancer tissues.High expression of CUL4B was related to invasive phenotype of gastric cancer.2.The expression level of CUL4 B in gastric cancer tissues was higher than that in normal gastric cancer and the difference was statistically significant.3.The expression of CUL4 B was generally increased in gastric cancer cell lines.4.Down-regulation of CUL4 B was associated with marked decrease in the ability of migration and invasion of gastric cancer cells.5.Wnt/β-catenin signaling proteins β-catenin and Cyclin-D1 might have an effect on the biological behaviour of gastric cancer cells.6.CUL4 B may participate in tumorgenesis and development of gastric cancer,which can provide new ideas for the diagnosis and treatment of gastric cancer. |
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