Rosiglitazone Inhibits Human Gastric Cancer MGC803 Cell Proliferation,Migration And Invasion After LIMK1 Silencing

Objective: We used lentivirus-mediated RNAi technique to make LIMK1 silencing, and then observed the role of ROS inhibits human gastric cancer MGC803 cell proliferation, migration and invasion after LIMK1 silencing.Methods : We designed and composed three sh RNA which could down-regulate LIMK1 expression, and packed lentivirus. MOI value was confirmed by transfection efficiency of previous study. LIMK1 silence was detected by q RT-PCR and western blotting in m RNA and protein level, and one highest inhibition efficiency of sh RNA was screened. ROS treated human gastric cancer MGC803 cells and LIMK1 silenced MGC803 cells, and then LIMK1 expression was detected in m RNA and protein level. The CCK-8, plate clone formation, flow cytometry, transwell migration and invasion assays were used to observe inhibition of ROS in human gastric cancer MGC803 cells and LIMK1-silenced MGC803 cells.Results:1. Successfully constructed and packed three LIMK1-sh RNA lentivirus.LV-LIMK1-RNAi(37154-1),LV-LIMK1-RNAi(37155-1), LV-LIMK1-RNAi(37156-1) and a negative control sequence were designed and packed lentivirus. MOI50 value was confirmed by transfection efficiency of previous study.2.Successfully screened the highest LIMK1-RNAi sequence(37156-1).The inhibition efficiency of LV-LIMK1-RNAi(37156-1),(37155-1) and(37154-1) were 91.9%, 75.9% and 90.7% in m RNA level, and in protein level were 72.4%?29.4%?63.7%. Whereas it has no statistical difference between negative virus group and MGC803 control groups(P>0.05). Therefore, the inhibition efficiency of LV-LIMK1-RNAi(37156-1) was the highest.3. ROS inhibits LIMK1 expression in m RNA and protein level.Results showed that ROS inhibits LIMK1 m RNA and protein level expression in MGC803 cells(P<0.05); ROS further inhibits LIMK1 expression in LIMK1-silenced MGC803 cells(P<0.05).4.LIMKI-silenced enhances the effect of ROS on gastric cancer MGC803 cell proliferation,migration and invasion.4.1 The CCK-8 assay showed that the cell vitality of ROS-treated group was obviously lower than untreated group(P<0.05) in MGC803 cells; The silence of LIMK1 could reduce cell proliferation(P<0.05); The cell vitality of LIMK1-silenced group was significantly lower than unsilenced group after ROS treated(P<0.05).4.2 The plate clone forming assay showed that the colony forming units of ROS-treated group was obviously lower than untreated group(P<0.05) in MGC803 cells; The silence of LIMK1 could reduce the colony forming units(P<0.05); The colony forming units of LIMK1-silenced group was significantly lower than unsilenced group after ROS treated(P<0.05).4.3 The flow cytometry assay showed that ROS induced the cell cycle G1 phase block, and corresponding the number of S phase cells were significantly reduced(P<0.05); The silence of LIMK1 could increase the number of G1 phase cells(P<0.05); The number of G1 phase cells of LIMK1-silenced group was significantly more than unsilenced group after ROS treated(P<0.05).4.4 Transwell migration assay showed that the transfered cell number of ROS-treated group was obviously reduce than untreated group(P<0.05) in MGC803 cells; The silence of LIMK1 could reduce the transfered cell number(P<0.05); The transfered cell number of LIMK1-silenced group was significantly reduce than unsilenced group after ROS treated(P<0.05).4.5 Transwell invasion assay showed that the transmembrane cell number of ROS-treated group was obviously reduce than untreated group(P<0.05) in MGC803 cells; The silence of LIMK1 could reduce the transmembrane cell number(P<0.05); The transmembrane cell number of LIMK1-silenced group was significantly reduce than unsilenced group after ROS treated(P<0.05).Conclusions:1.ROS could inhibit LIMK1 expression in human gastric MGC803 cells, and LIMK1 silenced could enhance ROS inhibition.2. LIMKI-silenced enhances the effect of ROS on gastric cancer MGC803 cell proliferation,migration and invasion.