The Effects Of LIMK1 That Over-expression On Inhibiting Proliferation,migration And Invasion Of MGC803 With Rosiglitazone

Objective Through the construction of human gastric carcinoma MGC803 cells with high expression of LIMK1, we observed that the influence and the mechanism of action of the expression of LIMK1 in the Rosiglitazone(Rosiglitazone, ROS) on the inhibition of proliferation, migration and invasion of gastric cancer cells.Methods To construct The eukaryotic expression vector cells of EX-C0763-M98-5 containing high LIMK1 expression and to transfect it into MGC803 cells. To test and verify the expressions of LIMK1 and protein in cells treated with ROS blot authentication by using RT-PCR and Western blot. And test and verify Flow cytometry, CCK-8 assay, plate clone formation assay, Transwell migration, invasion of ROS processing chamber before and after the experimental detection, overexpression of effect of LIMK1 on MGC803 cell cycle, cell proliferation, migration and invasion ability.Results 1. The eukaryotic expression vector cells of EX-C0763-M98-5 which successfully constructed expression of LIMK1. The sequencing result showed that the eukaryotic overexpression vector cells of EX-C0763-M98-5 which successfully constructed expression of LIMK1. Detecting fluorescent display that plasmid was successfully transfected MGC803 cells after the plasmid was transfected into MGC803 cells. RT-PCR and Western blot detection display that LIMK1 protein expression was significantly increased(P<0.05) in The eukaryotic expression vector cells of EX-C0763-M98-5. The expression of MGC803 cells, m RNA of empty vector EX-NEG-M9 and protein had no obvious change. 2.The ROS Effect on the expression of MGC803 cells of the high expression of LIMK1 and expression protein expression.The results of RT-PCR and Western blot suggest, before ROS treatment, cells with high expression of LIMK1 m RNA was significantly higher than that in control group and empty vector group(P<0.05). After ROS treatment, the control group and empty vector group of m RNA and protein expression were significantly decreased(P<0.05). Before and after ROS treatment, the m RNA and protain didn’t differ obviously in cells MGC803 overexpressing LIMK1. 3. Effect of MGC803 on cell proliferation of ROS and the high expression of LIMK1. CCK-8 test showed that respective treatment by ROS 1, 2 and 3 days later, MGC803 cell proliferation inhibition rates were 34.84%,75.81% and 80.16%(P<0.05), while that rate of cell viability comparing group of MGC803 with the high expression of LIMK1 were 66.59%,136.43% and 245.52%(P<0.05); and before and after ROS treatment, the differences of cell viability of MGC803 with over expression of LIMK1 was not significant(P=0.36). The experiment of Plate clone formation showed, before ROS treatment, cells relative colony forming rate with the high expression of LIMK1 is 37.3%, which is significantly higher than the control group 25.3% and empty vector group 24%(P<0.05). After ROS treatment, the colony formation rate of the control group and empty vector group were decreased to 15.3% and 14.7%(P<0.05). However, before and after ROS treatment, relative colony formation rate of MGC803 cells with overexpression of LIMK1 were not significantly different(P=0.14). 4. Effect of ROS on cycle of MGC803 cell with the high expression of LIMK1. Flow cytometry displayed, before ROS treatment, the G2/M phase of MGC803 cells with high expression of LIMK1 was25.27±2.53%, significantly higher than the control group 14.6±1.47% and empty vector group14.18±2.21%(P<0.05). After ROS treatment, the percentage of G0/G1 phase of the control group and empty vector group were 64.6±3.27% and 65.7±1.60%, and there are obvious G0/G1 arrest(P<0.05). The high expression group of ROS was not significantly different before and after treatment(P>0.05). 5. Effect of ROS on the migration, invasion of MGC803 cell with the high expression of LIMK1. Transwell migration experiment showed that 24 hours after the migration of MGC803cells,before ROS treatment, the number of Transmembrane cells in high expression group of LIMK1 was 196 ± 7, which was significantly higher than that of MGC803 group 158±3 and empty vector group 160±4(P<0.05). After ROS treatment, the number of Transmembrane cells of MGC803 group and empty vector group obviously reduced, were 69±1 and 69±3(P<0.05). LIMK1 with high expression(imported exogenous LIMK1), the migration ability of MGC803 cell treated by ROS recover 193±9. Invasive experiments showed, 48 h after invasion of MGC803 cells, before ROS treatment, the number of Transmembrane cells in high expression group of LIMK1 112±1 and was significantly higher than that of MGC803 group 73±2and empty vector group 76±2(P<0.05). After ROS treatment, the number of Transmembrane cells of MGC803 group and empty vector group were significantly reduced, which were 47±1 and 46±4(P<0.05). But LIMK1 with high expression(imported exogenous LIMK1), the invasion ability of MGC803 cells treated by ROS recovers 110±2.Conclusion 1.Overexpression of LIMK1 may promote the proliferation, migration and invasion in MGC803 cells. 2. Rosiglitazone may inhibit the proliferation, migration and invasion in MGC803 cells. 3.LIMK1 may be a potential target of the inhibition effects of rosiglitazone on the proliferation, migration and invasion in MGC803 cells.