黏着斑激酶siRNA抑制结肠癌细胞株侵袭的机制研究

Effect of siRNA Targetting Focal Adhesion Kinase on Invasion of Colon Cancer Cells

目的研究胃泌素-胆囊收缩素2受体(CCK2R)-黏着斑激酶(FAK)信号通路在人结肠癌细胞侵袭过程中的作用,探讨通过FAK siRNA降低FAK表达抑制癌细胞侵袭的可行性。方法使用CCK2R的真核表达载体pCR3.1/CCK2R,转染人结肠癌细胞株Colo320,使用RT-PCR检测细胞CCK2R表达情况。设计并合成针对FAK的siRNA,转染细胞,抑制FAK表达;使用胃泌素刺激结肠癌细胞,免疫沉淀和蛋白质印迹法检测不同情况下FAK第397位酪氨酸(tyr-397)磷酸化表达情况;Boyden小室法检测相应细胞侵袭力变化。结果真核表达载体pCR3.1/CCK2R可以有效增加CCK2R表达,增加CCK2R可以增强胃泌素促FAK tyr-397磷酸化和细胞侵袭力作用。使用RNA干扰技术可以明显降低细胞内FAK tyr-397磷酸化,并抑制胃泌素促结肠癌细胞侵袭作用。结论胃泌素-CCK2R-FAK信号通路在结肠癌细胞侵袭和转移过程中发挥关键作用,阻断FAK表达可以有效抑制胃泌素促结肠癌细胞侵袭作用。

Objective The study was designed to examine the effect of focal adhesion kinase（FAK） on invasion of human colon cancer cell,Colo320 cells induced by gastrin and cholecystokinin2 receptor（CCK2R）,and to investigate the feasibility of inhibiting colon cancer cell invasion by decreasing expression of FAK with RNA interference. Methods： Vector pCR3.1/CCK2R which expressed cholecystokinin 2 receptor（CCK2R） was stably into Colo320 with lipofectamine 2000 stably,RT-PCR was used to detect the expression of CCK2R after the transfection. FAK siRNA was designed, synthesized and transfected into Colo320 cell, after the cells were stimulated by gastrin, expression of FAK tyr-397 was detected by immunoprecipitation and western-blotting. Modified Boyden Chamber assay was used to examine the cell invasiveness before and after gastrin stimulation respectively. Results： Expression of GCK2R in Colo320 cells increased obviously after transfection of pCR3.1/CCK2R. Meanwhile, peR3.1/ CCK2R promoted FAK tyr-397 phosphorylation and cell invasiveness induced by gastrin. FAK siRNA decreased FAK tyr-397 phosphorylation and inhibited cell invasiveness induced by gastrin（P〈0. 01） simulta- neously. Conclusion： Gastrin-CCK2R-FAK signaling pathway plays an important role in the invasion and metastasis of colon cancer cells. Blockade of FAK expression is effective to inhibit colon cancer invasion induced by gastrin.