LMTK3对人肺癌A549细胞生物学功能的影响

摘要

Objective To investigate the effects of down-regulation of lemur tyrosine kinase 3 (LMTK3) on the cytobiological behaviors of human lung cancer A549 cells.Methods The expression of LMTK3 in A549 cells was interfered with small hair RNA (shR-NA),and the expression level of LMTK3 was determined by RT-PCR.The effects of LMTK3 on the proliferation,migration,cell cycle and apoptosis of A549 cells were determined by the CCK-8 assay,scratch assay,Transwell assay and flow cytometry,respectively.Results The shLMTK3 cells with stably low expression of LMTK3 and control cells (Scramble) with normal expression of LMTK3 were successfully obtained.The relative proliferation rates of shLMTK3 cells cultured for 24,48 and 72 hours were significantly lower than those in the control cells (0.305 ±0.018 vs 0.354 ±0.011,t =5.24,P＜0.01;0.461 ±0.044 vs 0.551 ±0.027,t =3.91,P ＜0.01;0.74 ± 0.029 vs 0.881 ± 0.028,t =7.70,P ＜ 0.01).The relative migration rate of shLMTK3 cells 24 hours after scratching was significantly lower than that of control cells (0.51 ±0.096 vs 1.00 ± 0.029,t =4.81,P ＜ 0.01).Transwell assay showed that the number of migration cells in shLMTK3 group was significantly less than that in Scramble group (161 ±9.29 vs 308.66 ± 17.60,t =7.42,P ＜ 0.05).The results of flow cytometry showed that shLMTK3 cells were blocked at G1 phase (t =4.35,P ＜ 0.05),and that the inhibition of LMTK3 had no influence on the apoptosis of A549 cells.Conclusion Down-regulation of LMTK3 expression in human lung cancer A549 cells may inhibit the proliferation and migration of A549 cells significantly,indicating that the abnormal expression of LMTK3 in lung carcinoma cells may regulate the biological behaviors and progression of tumors.%目的 探讨Lemur酪氨酸激酶3(LMTK3)在人肺腺癌细胞系A549中表达下调后对细胞生物学功能的影响.方法 采用shRNA(small hairpin RNA)技术干扰A549细胞中LMTK3的表达并用RT-PCR对其进行鉴定,采用细胞增殖实验、划痕实验、Transwell实验及流式细胞术检测LMTK3对肺癌细胞增殖、迁移、细胞周期及凋亡等生物学行为的影响.结果 通过shRNA技术成功构建LMTK3表达下调的肺癌细胞系shLMTK3及对照组Scramble.细胞增殖实验结果显示,在培养24、48、72h后,shLMTK3组细胞的增殖水平(0.305±0.018,0.461±0.044,0.74±0.029)明显低于Scramble组(0.354±0.011,0.551±0.027,0.881±0.028),差异均有统计学意义(t分别为5.24,3.91,7.70,P均＜0.01);划痕试验结果表明,划痕24h后的shLMTK3组细胞相对倍数(0.51±0.096)明显低于Scramble组(1.00±0.029),差异有统计学意义(t=4.81,P＜0.01);Tran-swell迁移实验结果表明,shLMTK3组迁移到膜下的细胞数[(161±9.29)个]明显低于Scramble组[(308.66±17.60)个],差异有统计学意义(t=7.42,P＜0.01);细胞周期检测结果显示,抑制LMTK3的表达可将细胞阻滞于G1期(t=4.35,P＜0.05);细胞凋亡试验显示,抑制LMTK3的表达对A549细胞的凋亡率无影响.结论 下调A549细胞中LMTK3的表达可明显抑制细胞的增殖及迁移能力,提示肺癌细胞异常表达LMTK3参与调节其肿瘤生物学行为.