The Influence Of Smad4 On The Migration Of Hepatocarcinoma Cell By Regulating Fascin And Cortactin

Objective:1. To construct a lentiviral vector of RNA interference of Smad4 gene, and down-regulate the expression of Smad4 in hepatocarcinoma cells SMMC-7721.2. To investigate the influence of Smad4 on the migration of hepatocarcinoma cells SMMC-7721.3. To investigate the influence of Smad4 on the expressions of fascin and cortactin in hepatocarcinoma cells SMMC-7721, and explore the underlying mechanism via which Smad4 influences the migration of hepatocarcinoma cells.Methods:1. 16 RNA interference sequences targeting Smad4 gene were designed. Then the complementary DNA containing both sense and antisense Oligonucleotides were designed, synthesized and cloned into the pMAGic1.0 vector to construct a lentiviral vector which expressed short hairpin RNA. The sequences were identified by PCR and DNA sequencing analysis.2. 293T cells were transfected with the lentiviral vector plasmid and lentiviral packaging system. After lentiviral packaging and concentration, the titer of virus was detected.3. Hepatocarcinoma cells SMMC-7721 were infected with the lentivirus, and then the efficiency of RNA interference was detected by Western blot. According to the efficiency, two of them were selected to follow up. Uninfected hepatocarcinoma cells SMMC-7721 and SMMC-7721 cells infected with empty lentiviral vector (RNAi-NC) were used as control.4. The healing of interference cells and control cells were detected, at 0 h, 12 h, 24 h and 48 h by wound healing experiment, respectively.5. The morphology of interference cells and control cells were detected in 3D culture system, respectively.6. The expressions of fascin and cortactin in interference cells and control cells were detected by Western blot, respectively.Results:1. PCR identification suggested that the segment of positive clones with double-stranded DNA is 343bp; the segment without double-stranded DNA is 306bp. The DNA suqencing exhibits that the sequence is identical with the construction.2. 293T cells were transfected with the lentiviral vector and lentiviral packaging system. After lentiviral concentration, the virus titer was 2×108 TU/mL to confirm the success of lentiviral package.3. Hepatocarcinoma cells SMMC-7721 were infected with the lentivirus, and then the efficiency of RNA interference was detected by Western blot. According to the efficiency, two of them were selected, which were named RNAi-Smad4-2 and RNAi-Smad4-12. Compared with SMMC-7721 cells and RNAi-NC cells, the expression of Smad4 was decreased in RNAi-Smad4-2 cells and RNAi-Smad4-12 cells.4. The wound-healing assay revealed that healing of RNAi-Smad4-2 cells and RNAi-Smad4-12 cells was faster than SMMC-7721 cells and RNAi-NC cells.5. Morphological aspect of RNAi-Smad4-2 cells and RNAi-Smad4-12 cells took on the irregular borders, penetrating through the surrounding gel, in 3D culture system. SMMC-7721 cells and RNAi-NC cells exhibit the normal structure with the well borders.6. The expressions of fascin and cortactin were increased in RNAi-Smad4-2 cells and RNAi-Smad4-12 cells comparing with SMMC-7721 cells and RNAi-NC cells, respectively.Conclusion:Smad4 can influence the migration of hepatocarcinoma cells SMMC-7721, and the contribution of Smad4 to the migration may be related with fascin and cortactin, which will provide a theoretical basis for the prediction of early metastasis and targe drug therapy.