Effect And Mechanism Of Gli2 Gene Silencing On Invasion And Metastasis In Hepatocellular Carcinoma Cell Line SMMC-7721

Objective:To explore the effect and mechanism of Gli2 on invasion and metastasis in the hepatocellular carcinoma cell line SMMC-7721.Methods:Three pairs of complementary shRNA oligonucleotides targeting the Gli2 gene were designed and inserted into the pLKD.CMV.GFP.U6 lentiviral vector. Then shRNA lentivirus(pLKD.CMV.GFP.U6 shRNA-Gli2-1,2,3) were transfected into SMMC-7721 cells and then fluorescence photographs were taken. Transfection efficienc of shRNA lentiviral were detected by Flow cytometry. The gene silencing efficiency was measured by qRT-PCR and Western blot. After the lentivirus with the best silencing effect was screened, it would be the only silencing one in following experiments;The experiment was divided into 3 groups:shRNA-Gli2 group(SMMC-7721 cells were transfected with shRNA-Gli2 lentivirus),shRNA-NC group(SMMC-7721 cells were transfected with shRNA-NC lentivirus) and control group(untreated SMMC-7721 cells).Adhesion assay was used to detect the rates of homogeneous and heterogeneous cells intercellular adhesion of each group;Migration and invasion ability were evaluated by transwell chambers assay;Endothelial tube formation assay was used to detect the ability of angiopoiesis;The expression of epithelial marker E-cadherin, mesenchymal marker N-cadherin, MMP-2 and VEGF-A were detected by qRT-PCR and Western blot.Results:1. Flow cytometry indicated that the transfection efficiency of shRNA lentivirus was about 95%.2. shRNA-Gli2-1 with the best silencing effect was selected, the relative mRNA expression of Gli2 compared with control group were 0.37 ±0.02, and the relative protein expression of Gli2 compared with control group were 0.43 ± 0.05.SMMC-7721 cells were transfected with shRNA-Gli2 -1lentivirus was named shRNA-Gli2 group.3. Adhesion assay indicated that the rates of homogeneous cells intercellular adhesion in shRNA-Gli2 group increased compared with that in shRNA-NC group and control group(13.71% ± 1.87 vs 6.35% ± 1.33&5.53%±2.01)(P<0.05),however, the rates of heterogeneous cells intercellular adhesion were decreased(20.57%± 1.44 vs 35.64% ±2.58 &33.34%±1.40)(p<0.05).4. The detection of migration by transwell suggested that the capacity of shRNA-Gli2 group on migration was significantly lower than that of the shRNA-NC group and control group(40.00 ± 1.46 vs 162.33 ± 12.81&176.67±7.02)(p<0.05).5. The detection of invasion by transwell indicated that the capacity of shRNA-Gli2 group on invasion was significantly lower than that of the shRNA-NC group and control group(18.00 ± 2.66 vs 100.33 ± 3.06&106.33±4.16)(p<0.05).6. The detection of angiopoiesis by endothelial tube formation assay indicated that the capacity of shRNA-Gli2 group on angiopoiesis was significantly lower than that of the shRNA-NC group and control group(8.67±2.08 vs 27.33±2.52&28.33±3.51)(p<0.05).7. qRT-PCR and Western blot analysis showed that the expression level of E-cadherin mRNA and protein in shRNA-Gli2 group compared with that in shRNA-NC group and control group was significantly higher(p<0.05). However, the expression level of N-cadherin,MMP-2 and VEGF-A mRNA and protein declined(p<0.05).Conclusion:Silencing Gli2 gene could suppress invasion and metastasis ability on hepatocellular carcinoma cell line SMMC-7721.its mechanism may be related to the up-regulation of E-cadherin and the down-regulation of N-cadherin,MMP-2 and VEGF-A.