保罗样激酶1表达抑制导致胃癌MKN45细胞有丝分裂停滞

Mitosis arrest caused by inhibition of PLK1 expression in gastric cancer MKN45 cells

目的探讨保罗样激酶1(PLK1)基因在胃癌MKN45细胞有丝分裂中的作用。方法应用RNA干扰(RNAi)技术阻断MKN45细胞PLK1基因的表达;real time定量PCR和Westernblot检测干扰前后PLK1mRNA及蛋白质表达的变化;免疫荧光及激光共聚焦显微镜观察MKN45细胞微管及有丝分裂表型的改变;倒置显微镜观察MKN45细胞形态的变化;流式细胞仪检测细胞周期的变化。结果经靶向PLK1的小型干拢RNA(siRNA)作用后,MKN45细胞的PLK1mRNA及蛋白质表达明显下降;细胞微管结构变得模糊,失去完整性;PLK1细胞有丝分裂表型发生明显变化,有丝分裂开始时,较高比例(46.3%)的细胞处于Ⅰ亚期,较低比例的细胞处于Ⅱ亚期(1.2%)及Ⅲ亚期(1.7%),有较高比例的带哑铃状细胞核的MKN45细胞(32.8%)及细胞间连接有胞质桥的MKN45细胞(8.4%),与对照siRNA组比较,差异有统计学意义(P<0.05);较多MKN45细胞呈圆形改变,并呈现G2期细胞的DNA含量,siRNA作用24、48及72h后,PLK1-组G2期DNA含量细胞分别为43.7%、41.0%和58.5%,与对照siRNA组在3个时间点差异均有统计学意义(P<0.05)。结论PLK1基因在MKN45细胞有丝分裂过程中起着关键作用,抑制其表达可导致MKN45细胞有丝分裂停滞。

Objective To observe the effect of polo-like kinase 1 （ PLK1 ） gene depletion on mitosis phenotype and elucidate its vital role in gastric cancer cell line （MKN45） mitosis. Methods The PLK1 expression in MKN45 cells was blocked by RNA interference （ RNAi）, the expression level of PLK1 mRNA and protein were measured by real-time quantitative PCR and Western blot, respectively. The morphological change of microtubules and mitosis phenotype in MKN45 cells were observed by immunofluorescence staining and laser confocal microscopy, the morphological changes of cells were observed by reverse microscopy, the variation of cell cycle distribution was detected by flow-cytometry. Results After RNAi targeting PLK1, PLK1 mRNA and protein level decreased obviously, the cell microtubules became obscure and lost cohesiveness, the mitosis phenotype also varied substantially （ P 〈 0.05 ）, more gastric cancer ceils became rounded and showed G2 phase cell DNA content （P 〈 0.05 ）. Conclusion PLK1 gene plays a key role in mitosis and its inhibition can lead to mitosis arrest in MKN45 ceils.