抑制血清淀粉样蛋白A对肺癌细胞增殖、迁移、侵袭及MAPK信号通路的影响

Influence of inhibition of SAA on proliferation,migration,invasion and MAPK pathway of lung cancer cells

目的探讨抑制血清淀粉样蛋白A(SAA)的表达对人肺癌细胞A549增殖、迁移、侵袭能力以及丝裂原活化蛋白激酶(MAPK)信号通路的影响。方法体外培养A549细胞,并特异性转染SAA-短发夹RNA(shRNA),另外以非特异性小干扰RNA(siRNA)转染A549细胞作为阴性对照,同时设置Blank组。采用MTT法和Edu细胞荧光染色法检测细胞增殖活力,Transwell小室法检测细胞侵袭活力,划痕实验检测细胞迁移活力。另采用Western blot法检测MAPK信号通路相关蛋白的表达。结果经G418筛选及培养后,置于荧光显微镜下观察细胞形态呈圆梭形,而Blank组A549细胞则呈长梭形。转染phU6/GFP/Neo-SAA-1序列的A549细胞SAA蛋白表达量最低,可用于后续实验。MTT法检测,SAA-shRNA组A549细胞培养24、48、72、96 h后490 nm波长处吸光度值分别为0.383±0.079、0.572±0.094、0.785±0.092、0.891±0.104,Edu细胞荧光染色法检测Blank组、shRNA对照组、SAA-shRNA组A549细胞相对增殖率分别为(100.00±5.70)%、(96.54±6.28)%、(48.96±5.33)%,均显示SAA-shRNA组细胞增殖活力低于shRNA对照组和Blank组(P<0.05)。Transwell小室法检测SAA-shRNA组A549细胞转移至下层小室中的细胞数为265±48,划痕实验检测SAA-shRNA组A549细胞24 h愈合率为(24.73±6.14)%,二者同样低于shRNA对照组和Blank组(P<0.05)。Western blot法检测,SAA-shRNA组A549细胞磷酸化p38(p-p38)、磷酸化细胞外调节蛋白激酶1/2(p-ERK1/2)、磷酸化c-Jun氨基末端激酶(p-JNK)蛋白相对表达水平(以β-actin作为内参)分别为0.45±0.13、0.85±0.16、0.47±0.11,三者明显低于Blank组和shRNA对照组(P<0.05)。结论抑制SAA表达可降低人肺癌细胞株A549体外增殖、迁移、侵袭活性,而MAPK信号通路在其中可能发挥着重要作用。

Objective To investigate the effects of inhibiting the expression of serum amyloid A(SAA) on the proliferation,migration,invasion ability and mitogen-activated protein kinase(MAPK) signaling pathway of human lung cancer cell A549.Methods A549 cells were cultured in vitro and specifically transfected with SAA-shRNA.A549 cells were transfected with non-specific siRNA as negative control,and blank group was set up.Cell proliferation activity was measured by MTT and Edu cell fluorescence staining.Cell invasion activity was detected by Transwell chamber method.Cell migration activity was detected by scratch test.The protein expression of MAPK signal pathway was detected by Western blot.Results After being screened and cultured by G418,the cell morphology was round fusiform under a fluorescence microscope,while A549 cells in blank group was long fusiform.Transfection of phU6/GFP/Neo-SAA-1 sequence showed the lowest expression of SAA protein in A549 cells,which could be used for subsequent experiments.According to the MTT method,the OD 490 nm values of A549 cells in the SAA-shRNA group after 24,48,72 and 96 h of culture were 0.383±0.079,0.572±0.094,0.785±0.092 and 0.891±0.104,respectively.The relative proliferation rates of A549 cells in the Blank group,the shRNA control group,and the SAA-shRNA group were(100.0±5.7)%,(96.54±6.28)% and(48.96±5.33)%,the cell proliferation activity of SAA-shRNA group was lower than that of shRNA control group and blank group(P<0.05).Transwell chamber method showed the number of A549 cells transferred to the lower compartment in SAA-shRNA group was 265±48,scratch test showed the 24-hour healing rate of A549 cells in SAA-shRNA group was(24.73±6.14)%,both of which were lower than those in shRNA control group and blank group(P<0.05).Western blot showed that the relative expression levels of p-p38,phosphorylated extracellular regulatory protein kinase 1/2(p-ERK1/2),and phosphorylated c-Jun amino terminal kinase(p-JNK) protein of A549 cells in SAA-shRNA group(β-actin as an inner consult) were 0.45±0.13,0.85±0.16 and 0.47±0.11,respectively,which were lower than those in blank group and shRNA control group(P<0.05).Conclusions Inhibition of SAA expression can reduce the proliferation,migration and invasion of human lung cancer cell line A549 in vitro,and the MAPK signaling pathway may play an important role in it.