Hypoglycosylated E-Cadherin And Proliferation, Invasion And Mobility Of TCA8113Human Tongue Carcinoma Cell Line

OBJECTIVE:To enhance the stability of tumor cell adherens junctions and tight junctions to inhibit tumor cell proliferation, invasion and mobility by changing the E-cadherin N-glycosylation may be a new method of gene therapy of cancer. The purpose of this experiment was the study of the effects of hypoglycosylated E-cadherin gene transfected Tca8113cells on the tongue cancer cell’s proliferation, invasion and mobility.METHODS:1. We use Tca8113tongue carcinoma cell lines as samples. Wild-type E-cadherin protein gene and hypoglycosylated E-cadherin (V13) gene were transfected into Tca8113cell. G418will be used to screen the successfully transfected cells. The stable expression of the gene transfected will be detected by western blot.2. Proliferation of the different group of cells will be measured by MTT assay.3. Cell cycle in each group will be detected by FACS analysis, in order to understand the activity of cell proliferation.4. The BrdUrd incorporation is presented to determine the ratio of BrdU positive cells to total cell number in each group, and the BrdUrd incorporation is presented as the percentage of positive nuclei relative to total cell number. An analysis of the above three experiment results will show us the influence to cell proliferation of Tca8113cell after hypoglycosylated E-cadherin gene was transfected.5. The Matrigel invasion assay will be used to observe the invasion ability differences among hypoglycosylated E-cadherin (V13) gene transfected Tca8113cells, wild-type E-cadherin gene transfected Tca8113cells and Tca8113cell.6. Wound healing assay will be employed to observe the mobility differences among hypoglycosylated E-cadherin (V13) gene transfected Tca8113cells, wild-type E-cadherin gene transfected Tca8113cells and Tca8113cell.RESULTS:1-Western blotting experiments:After the hypoglycosylated E-cadherin (V13) gene and wild-type E-cadherin gene transfected into Tca8113cells were stably expressed, we used anti-flag antibogy in the western blot assay. The resault showed that molecular weight of hypoglycosylated E-cadherin was smaller than wild-type E-cadherin. The molecular weight of hypoglycosylated E-cadherin was120KD, and the molecular weight of wild-type E-cadherin was150KD, while the control group had no flag-marked protein expression.2. MTT assay showed that:Relative to the the Tca8113blank control cells, the proliferation of hypoglycosylated E-cadherin (V13) gene transfected group had significant decrease. Since after48hours, the OD ratio of the V13transfected cells was significantly lower than the control cells, while the proliferative activity of wild-type E-cadherin gene transfected Tca8113cells had no obvious change.3. FACS results showed that:Relative to the the Tca8113blank control cells, the S-phase ratio of hypoglycosylated E-cadherin (V13) gene transfected group had significant decrease(18.42%reduced), while the S-phase ratio of the wild-type E-cadherin transfected Tca8113cell had no significant change.4. BrdU incorporation assay results showed that:The ratio of the BrdU positive cells in Tca8113control cells group was the highest. The ratio of the BrdU positive cells of the wild-type E-cadherin transfected Tca8113cell was lesser but not much(P>0.05). Transfection of hypoglycosylated E-cadherin (V13) gene caused BrdUrd-positive cells to drop obviously(P<0.01). That means cell DNA synthesis was significantly weakened in hypoglycosylated E-cadherin (V13) gene transfected Tca8113cells;5. Matrigel in vitro invasion assay results showed that:Relative to the the Tca8113blank control cells, the invasion ability of the hypoglycosylated E-cadherin (V13) gene transfected Tca8113cell was significantly decreased, but it had no obvious change in the wild-type E-cadherin gene transfected Tca8113cell.6. Wound healing assay results showed that:Relative to the the Tca8113blank control cells, the mobility of the the hypoglycosylated E-cadherin (V13) gene transfected Tca8113cell was significantly decreased, but it had no obvious change in the wild-type E-cadherin gene transfected Tca8113cell.CONCLUSION:Hypoglycosylated E-cadherin protein (V13) gene transfection can inhibit cell proliferation, invasion and mobility of Tca8113cell more significantly than the wild-type E-cadherin gene transfection. This is a new reasonable and workable idea to inhibit tumor cell proliferation, invasion and mobility, and the study laid an experimental and theoretical basis to inhibit tumor proliferation, invasion and mobility by hypoglycosylated E-cadherin gene.