Study On The Efiect Of ZEB2on Invasion And Metastasis In Gastric Cancer And Its Mechanism

Part1Expression and significance of ZEB2and E-cadherin in human gastric carcinoma tissues and cell linesObjective:To study the expression and significance of ZEB2and E-cadherin in human gastric carcinoma tissues and cell lines.Methods:Immunohistochemistry was used to detect the expression of ZEB2and E-cadherin protein in gastric carcinoma tissues and nontumorous gastric tissues. The relationship between the clinicopathologic features and the expression of ZEB2and E-cadherin was investigated. Survival analyses were performed using the Kaplan-Meier method and Cox regression model. Expressions of ZEB2mRNA and E-cadherin mRNA in human normal gastric epithelium cell line (GES-1) and gastric carcinoma cell lines (SGC7901, HGC27) were detected respectively by real-time fluorescence quantitative PCR.Results:1. Positive expression rate of ZEB2in gastric carcinoma tissues was higher than that in nontumorous gastric tissues. The expression of ZEB2protein was correlated with depth of invasion, lymph node metastasis and tumor TNM stage (all P<0.05), but not with age, gender, tumor size and tumor differention degree (all P>0.05). Kaplan-Meier analysis indicated that the mean survival time for patients with positive and negative ZEB2 expression was37.603±3.909mo,52.258±4.046mo; The five-year cumulative survival rate for patients with positive ZEB2expression was lower than that for patients with negative ZEB2expression (P<0.05).2. Positive expression rate of E-cadherin in gastric carcinoma tissues was lower than that in nontumorous gastric tissues. The expression of E-cadherin protein was correlated with tumor differention degree, depth of invasion, lymph node metastasis and tumor TNM stage (all P<0.05), but not with age, gender and tumor size (all P>0.05). Kaplan-Meier analysis indicated that the mean survival time for patients with positive and negative E-cadherin expression was52.473±3.675mo,32.667±4.225mo; The five-year cumulative survival rate for patients with positive E-cadherin expression was higher than that for patients with negative E-cadherin expression (P<0.05).3. The ZEB2level was negatively related to the E-cadherin level in human gastric carcinoma tissues (r=-0.295, P=0.01).4. The level of ZEB2mRNA in gastric carcinoma cell lines was higher than that in human normal gastric epithelium cell line (GES-1), and HGC27cells expressed the highest levels of ZEB2mRNA.5. The level of E-cadherin mRNA in gastric carcinoma cell lines was lower than that in human normal gastric epithelium cell line(GES-l), and HGC27cells expressed the lowest levels of E-cadherin mRNA.Conclusion: 1. The high expression of ZEB2and low expression of E-cadherin existed both in gastric carcinoma tissues and cell lines, which may play an important role in the carcinogenesis and progression of human gastric carcinoma.2. The expression of ZEB2and E-cadherin protein was correlated with tumor depth of invasion, lymph node metastasis, tumor TNM stage and poor survival. The abnormal expression may play an important role in the invasion and metastasis of gastric carcinoma.3. Inverse correlation between ZEB2and E-cadherin expression in human gastric carcinoma tissues. ZEB2could promote the invasion and metastasis of gastric carcinoma by inhibiting the expression of E-cadherin protein.Part2Construction and identification of shRNA expression plasmids targeting the ZEB2geneObjective:To construct and identify eukaryotic expressing plasmids of short hairpin RNA (shRNA) targeting the ZEB2gene and to evaluate their inhibitory effect on ZEB2expression in human gastric cancer cell line HGC27.Methods:Three pairs of complementary shRNA oligonucleotides targeting the ZEB2gene were designed, synthesized, annealed and inserted into the pGPU6/GFP/Neo plasmid. The recombinant plasmids (pGPU6/GFP/Neo-ZEB2-shRNA1,2,3) were identified by restriction enzyme analysis and sequence analysis. Then recombinant plasmids were introduced into HGC27cells by lipofectamine-mediated transfection and then fluo-rescence photographs were taken. The gene silencing efficiency was measured by Real-time PCR and Western blot.Results:1. After restriction enzyme analysis and sequence analysis, three eukaryotic expression plasmids of shRNA targeting the ZEB2gene were successfully constructed.2. Recombinant plasmids were successfully transfected to HGC27cell line. The inhibition ratio of three recombinant plasmids (pGPU6/GFP/Neo-ZEB2-shRNA1,2,3) to the mRNA expression of ZEB2were81%,42%,74%by Real-time PCR analysis; The inhibition ratio of three recombinant plasmids to the protein expression of ZEB2were73%,33%,64%by Western blot analysis. It was most remarkable for the plasmid ZEB2-shRNA1to inhibit the expression of ZEB2, and it was significantly different compared with the blank control group (HGC27cells)(P<0.05).Conclusion:1. Eukaryotic expression plasmids of shRNA targeting the ZEB2gene are successfully constructed.2. The expression of ZEB2gene could be effectively reduced by transfection of three recombinant plasmids in HGC27cells. The silencing effect of ZEB2-shRNA1on the expression of ZEB2was most remarkable, which can be selected for follow-up research.Part3Effect of ZEB2gene silencing on the biological characteristics of human gastric carcinoma HGC27and its mechanismObjective:To study the effect of RNA interference (RNAi) targeting the ZEB2gene on proliferation, migration and invasion of human gastric carcinoma cell line HGC27in vitro and its mechanism.Methods:Experiments were divided into three groups, experimental group (HGC27cells were transfected with ZEB2-shRNA1plasmids), negative control group (HGC27cells were transfected with ZEB2-shRNA NC plasmids), and blank control group (untreated HGC27cells). Cell proliferation was examined by MTT assay; Migration and invasive capability were evaluated by in vitro cell migration and invasion assay; the expression of epithelial marker E-cadherin, mesenchymal markers fibronecin and vimentin, and MMPs was detected by Western blot analysis.Results:1. MTT assay indicated that the proliferation level of HGC27cells was not significantly different after transfection with ZEB2-shRNA1plasmids. 2. The detection of invasion by transwell indicated that the capacity of experimental group on invasion was extremely lower than that of the negative control group and blank control group (P<0.05).3. The detection of migration by transwell suggested that the capacity of experimental group on migration was extremely lower than that of the negative control group and blank control group (P<0.05).4. Western blot analysis showed that the expression level of E-cadherin protein in experimental group increased compared with that in the negative control group and blank control group, however, the expression level of vimentin, fibronectin, MMP2and MMP9protein decreased (P<0.05).Conclusion:1. Down-regulation of ZEB2could lead to a significant decrease in migration and invasive capability of HGC27cells (P<0.05), but no significant effect on HGC27cells proliferation was found. ZEB2may play an important role in the invasion and metastasis of gastric carcinoma.2. Down-regulation of ZEB2enhanced the recruitment of E-cadherin and decreased the expression of vimentin, fibronectin, MMP2and MMP9. ZEB2promotes the invasion and metastasis of gastric cancer at least partly via the regulation of EMT. The downregulation of ZEB2expression is able to block the occurrence of EMT.