磷脂酰肌醇3激酶/蛋白激酶B/基质金属蛋白酶-9信号通路在芬太尼抑制人胃癌细胞侵袭和迁移中的作用

The role of phosphatidylinositol 3 kinase/protein kinase B/matrix metalloproteinase-9 signaling pathway in fentanyl-induced inhibition of invasion and migration ability of human gastric cancer cells

目的观察磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/基质金属蛋白酶(MMP)-9信号通路在芬太尼抑制人胃癌MGC-803细胞侵袭和迁移中的作用。方法将人胃癌MGC-803细胞随机分为空白对照组(C组)、芬太尼组(F组),LY294002组(LY组),SB-3CT组(SB组),LY294002+芬太尼组(FLY组)和SB-3CT+芬太尼组(FSB组)。Transwell实验和划痕实验检测细胞侵袭迁移能力,实时荧光聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)分别检测MMP-9和Akt的mRNA和蛋白质的表达,使用方差分析和独立样本t检验分析组间差异。结果F组胃癌细胞侵袭数低于C组(35.91±3.36比48.46±4.86,t=3.679,P<0.05)、迁移数低于C组(74.75±3.76比96.00±5.20,t=5.739,P<0.05)细胞迁移率(%)低于C组(14.29±4.29比22.43±3.37,t=3.591,P<0.05),Akt mRNA表达低于C组(0.77±0.02比1.00,t=8.110,P<0.05)、MMP-9 mRNA表达低于C组(0.87±0.08比1.00,t=2.967,P<0.05)、Akt-1蛋白表达低于C组(0.76±0.04比0.90±0.05,t=3.940,P<0.05)和MMP-9蛋白表达低于C组(0.76±0.05比0.94±0.04,t=4.774,P<0.05),差异均有统计学意义。FLY组胃癌细胞侵袭数低于LY组(18.45±4.86比33.62±4.13,t=4.999,P<0.05)、细胞迁移数低于LY组(42.79±3.56比71.79±7.60,t=5.986,P<0.05)和细胞迁移率(%)低于LY组(7.52±1.30比11.02±1.73,t=2.811,P<0.05),FLY组Akt mRNA表达低于LY组(0.60±0.05比0.76±0.17,t=3.468,P<0.05)、MMP-9 mRNA表达低于LY组(0.75±0.06比0.88±0.03,t=3.288,P<0.05)、Akt-1蛋白表达低于LY组(0.60±0.02比0.75±0.05,t=4.783,P<0.05)和MMP-9蛋白表达低于LY组(0.48±0.03比0.74±0.06,t=7.266,P<0.05),差异均有统计学意义。FSB组胃癌细胞侵袭数低于SB组(18.67±2.15比32.33±6.20,t=3.607,P<0.05)、细胞迁移数低于SB组(45.67±4.63比75.04±4.47,t=7.898,P<0.05)和细胞迁移率(%)低于SB组(7.09±1.69比14.39±2.06,t=4.745,P<0.05),FSB组Akt mRNA表达低于SB组(0.53±0.11比0.76±0.07,t=2.950,P<0.05)、MMP-9 mRNA低于SB组表达(0.63±0.07比0.84±0.01,t=3.687,P<0.05)、Akt-1蛋白表达低于SB组(0.61±0.03比0.75±0.04,t=4.573,P<0.05)和MMP-9蛋白表达低于SB组(0.53±0.07比0.72±0.05,t=3.730,P<0.05),差异均有统计学意义。芬太尼可降低细胞侵袭和迁移能力,MMP-9和p-Akt表达减少,且芬太尼联合LY294002或SB-3CT较单独用药时进一步加强了抑制效果。结论芬太尼抑制人胃癌MGC-803细胞侵袭和迁移的机制与抑制PI3K/Akt/MMP-9信号通路有关。

Objective To investigate the role of phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)/matrix metalloproteinase(MMP)-9 signaling pathway in the inhibition of invasion and migration of human gastric cancer MGC-803 cells by fentanyl.Methods MGC-803 cells were randomly divided into blank control group(group C),fentanyl group(group F),PI3K inhibitor LY294002 group(group LY),MMP-9 inhibitor SB-3CT group(group SB),and LY294002+fentanyl group(group FLY)and SB-3CT+fentanyl group(group FSB).Transwell test and scratch test were used to detect the invasion and migration ability of the cells.Real time fluorescent polymerase chain reaction(RT-PCR)and Western blotting were used to detect the mRNA and protein expression of MMP-9 and Akt.Results Compared with group C,the number of invasive cells(35.91±3.36 vs.48.46±4.86),t=3.679,P<0.05),number of migrating cells(74.75±3.76 vs.96.00±5.20,t=5.739,P<0.05)and cell migration rate[(14.29±4.29)%vs.(22.43±3.37)%,t=3.591,P<0.05]in group F were significantly decreased.The expression of Akt mRNA(0.77±0.02 vs.1.00,t=8.110,P<0.05)and MMP-9(0.87±0.08 vs.1.00,t=2.967,P<0.05)were significantly decreased,the expression of Akt-1 protein(0.76±0.04 vs.0.90±0.05,t=3.940,P<0.05)and MMP-9(0.76±0.05 vs.0.94±0.04,t=4.774,P<0.05)was significantly decreased.As compared with group LY,number of invasive cells(18.45±4.86 vs.33.62±4.13,t=4.999,P<0.05),number of migrating cells(42.79±3.56 vs.71.79±7.60,t=5.986,P<0.05),and cell migration rate[(7.52±1.30)vs.(11.02±1.73)%,t=2.811,P<0.05]in group FLY were significantly decreased;the expression of Akt mRNA(0.60±0.05 vs.0.76±0.17,t=3.468,P<0.05)and MMP-9 mRNA(0.75±0.06 vs.0.88±0.03,t=3.288,P<0.05),the expression of Akt-1 protein(0.60±0.02 vs.0.75±0.05,t=4.783,P<0.05)and MMP-9 protein(0.48±0.03 vs.0.74±0.06,t=7.266,P<0.05)in group FLY were significantly decreased.As compared with group SB,the number of invasive cells(18.67±2.15 vs.32.33±6.20,t=3.607,P<0.05),the number of migrating cells(45.67±4.63 vs.75.04±4.47,t=7.898,P<0.05)and cell migration rate[(7.09±1.69)%vs.(14.39±2.06)%,t=4.745,P<0.05]in FSB group were significantly decreased;the expression of Akt(0.53±0.11 vs.0.76±0.07,t=2.950,P<0.05)and MMP-9(0.63±0.07 vs.0.84±0.01,t=3.687,P<0.05)in group FSB was significantly decreased;the expression of Akt-1 protein(0.61±0.03 vs.0.75±0.04,t=4.573,P<0.05)and MMP-9 protein(0.53±0.07 vs.0.72±0.05,t=3.730,P<0.05)in group FSB was significantly decreased.Fentanyl can inhibit the invasion and migration of cells,and the expression of MMP-9 and p-Akt is decreased.Fentanyl combined with LY294002 or SB-3CT can further enhance the inhibitory effect.Conclusion Fentanyl inhibits the invasion and migration of MGC-803 cells by inhibiting PI3K/Akt/MMP-9 signaling pathway.