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目的观察抑制保罗样激酶基因(pololikekinase1,plk1)表达对胃癌细胞——MKN45凋亡的诱导,探讨plk1基因对胃癌细胞增殖及生存力的作用。方法化学合成小片断干扰RNA(siRNA)阻断MKN45细胞plk1基因的表达;实时定量PCR及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细胞微管改变,流式细胞仪检测MKN45细胞周期及凋亡的变化;Westernblot检测pro-caspase3水平的变化。结果经靶向plk1的siRNA作用后,plk1siRNAmRNA及蛋白质表达明显下降;MKN45细胞纺锤体结构变得模糊,失去完整性;较多MKN45细胞呈现G2期细胞DNA含量(P<0.05);MKN45细胞在48、72h凋亡率较对照组明显上升(P<0.05);伴随pro-caspase3水平在72h出现下降。结论抑制plk1基因表达可导致MKN45细胞凋亡,凋亡机制可能通过caspase3途径;plk1基因对胃癌细胞增殖及存活起着至关重要的作用。
Objective To investigate the effect of inhibiting the expression of pololikekinase1 (plk1) on the apoptosis of gastric cancer cell line - MKN45 and the effect of plk1 gene on the proliferation and viability of gastric cancer cells. Methods The small interfering RNA (siRNA) was chemically synthesized to block the expression of plk1 gene in MKN45 cells. The expression of plk1 mRNA and protein was detected by real-time PCR and Western blot. The changes of microtubules in MKN45 cells were observed by immunofluorescence and confocal microscopy. Cytokines were used to detect the cell cycle and apoptosis of MKN45 cells. The level of pro-caspase 3 was detected by Western blot. Results The expression of plk1 siRNA mRNA and protein was significantly decreased after siRNA targeting plk1. The spindle structure of MKN45 cells became vague and loss of integrity. The expression of DNA in G2 phase cells was higher in MKN45 cells (P <0.05) , 72h apoptosis rate was significantly higher than the control group (P <0.05); accompanied by pro-caspase3 levels in 72h decreased. Conclusion Inhibition of plk1 gene expression can lead to apoptosis of MKN45 cells. The mechanism of apoptosis may be through the caspase3 pathway. Plk1 gene plays a crucial role in the proliferation and survival of gastric cancer cells.