反义局部粘着斑激酶脱氧寡核苷酸对肝癌细胞侵袭性生长的抑制作用

Inhibitory effects of antisense focal adhesion kinase oligodeoxynucleotides on the invasion of Bel 7402 hepatocellular carcinoma cells

目的 观察反义局部粘着斑激酶脱氧寡核苷酸(FAK ODN)转染对肝癌细胞侵袭性生长的影响,并探讨其作用机制。 方法 以LipofecTAMINE介导的反义FAK ODN转染Bel 7402肝癌细胞株,测定Bel 7402肝癌细胞株体外生长曲线、细胞活力,测定不同时间点该细胞体外黏附能力变化,以Transwell小室测定细胞的体外侵袭能力,同时行FAK表达与细胞DNA含量的双参数流式细胞仪检测及细胞凋亡的流式细胞仪检测。 结果 p125FAK表达在反义转染组(6.49％±0.10％)显著低于正义转染组(14.33％±1.88％)与对照组(16.68％±1.62％),F=7.66,P<0.01;反义FAK ODN转染显著抑制Bel 7402肝癌细胞株的生长,其细胞活力显著下降,肿瘤细胞抑制率在30％-60％之间;细胞体外黏附能力受到显著抑制,黏附抑制率在25％-55％间;细胞的体外侵袭能力显著下降,侵袭抑制率在15％-25％之间;细胞凋亡显著增加;细胞周期分析显示S期细胞比率显著降低,细胞生长主要阻滞在G2/M期。 结论 FAK在Bel 7402肝癌细胞的黏附与迁移运动中发挥重要作用,其表达阻断显著抑制肝癌细胞的体外黏附与侵袭活性。FAK表达阻断显著抑制肝癌细胞的体外增殖,促进细胞凋亡。

Objectives To study the inhibitory effects of antisense focal adhesion kinase (FAK) oligodeoxynucleotides (ODN) on the invasion of Bel 7402 cells, and investigate the mechanisms. Methods LipofecTAMINE-mediated antisense FAK ODN was transfected into Bel 7402 cells. Cell number and viability were evaluated every 24 hours by trypan blue dye exclusion. Cell attachment assay was carried out at intended time points in a microculture well pre-coated with fibronectin (FN). The invasive activity of tumor cells was assayed in a transwell cell culture chamber. Cell cycle and cell apoptosis analysis were performed with flow cytometry (FCM). Results The expression of p125FAK in the group treated with antisense FAK ODN (6.49%±0.10%) significantly decreased, compared with those in the group treated with sense FAK ODN (14.33%±1.88%) and control group (16.68%±1.62%), F=7.66, P<0.01. Antisense FAK ODN significantly inhibited the growth of Bel 7402 cells by 30%-60%, the attachment by 25%-55%, and the invasion, 15%-25%. The decreased expression of FAK in Bel 7402 cells caused a G2/M cell cycle arrest, and the cells at S phase decreased significantly. The occurrence of apoptosis detected by FCM increased significantly in the group treated with antisense FAK ODN. Conclusions Inhibition of FAK expression significantly decreases the attachment between ECM and Bel 7402 cells, and the ability of Bel 7402 cells to invade the reconstituted basement membrane. In addition, FAK suppression significantly inhibits the proliferation of Bel 7402 cells in vitro, and increases their apoptosis.