Effects of Gli1 on Twist1 expression and migration of human hepatoma cells

GAN Luhui; LYU Muhan; DENG Mingming

doi:10.3969/j.issn.1002-266X.2018.09.001

Abstract：Objective To explore the effects of the Hedgehog signal pathway crucial protein Gli1 Hedgehog signal pathway on the expression of Twist1 protein and migration of human hepatoma cells.Methods The cell lines with highly expressed Gli1 were screened out from the httman hepatoma cell lines Huh7,hepG2,PLC,SMMC-7221,and Bel-7404 by Western blotting.According to the biological information of GenBank database,three Gli-siRNA were transfected into the screened human hepatoma cells with highly expressed Gli1 via plasmids:pGCsi-U6-GLi1 siRNA-1 (siRNA-1 group),pGC-si-U6-GLi1siRNA-2 (siRNA-2 group),and pGCsi-U6-GLi1siRNA-3 (siRNA-3 group).The cells in the blank control group (group N) were not transfected with any siRNA sequence,and the cells in the negative control group (NC group) were transfected with the negative control sequence.The mRNA and protein expression of Gli after 48-hour transfection was assessed by qRT-PCR and Western blotting to screen out the most optimal siRNA sequence.The migration ability and viability of the cell lines with the most optimal siRNA sequence were detected by Scratch test.The protein expression of Twist1 and E-cadherin of cells was measured by Western blotting.Results Western blotting showed that compared with the other four human hepatoma cell lines,the protein expression of Gli1 in the Bel-7404 cells was significantly up-regulated (all P ＜0.05);compared with the other groups,the expression of Gli1 in the siRNA-2 group was significantly the lowest (all P ＜0.05);compared with the group N,the protein expression of Twist11 was down-regulated while the expression of E-cadherin was up-regulated,the expression of N-Cadherin and Vimentin was down-regulated,and the migration ability significantly decreased in the siRNA-2 group (all P ＜0.05).Conclusion Silencing the expression of the Gli1 with thc siRNA can upregulate the expression of Twistl in the Bel-7404 cells,and promote the transformation and migration of epithelial-mesenchymal cells.