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目的研究抑制人肝癌细胞 MHCC97-H 中黏着斑激酶(FAK)表达对细胞黏附、侵袭和骨架重塑的影响。方法利用脂质体转染 FAK siRNA 至 MHCC97-H 细胞内。Western blot 法检测RNA 干扰前后 MHCC97-H 细胞中 FAK 蛋白的表达水平。黏附试验和侵袭试验分别检测 MHCC97-H细胞黏附和侵袭能力在干扰前后的变化。明胶酶谱试验检测 MHCC97-H 细胞分泌 MMPs 的变化。用激光共聚焦显微镜观察免疫荧光标记细胞的骨架重塑的变化。结果特异性 FAK siRNA 明显抑制 MHCC97-H 细胞中 FAK 表达。干扰后 MHCC97-H 细胞与细胞外基质纤维连接蛋白的黏附率为35.8%,低于对照组的57.3%(P<0.05)。干扰后 MHCC97-H 细胞穿透 Tanswell 小室的数目由对照组的(31.3±2.6)个减少至(14.5±3.1)个(P<0.05)。干扰后 MHCC97-H 细胞分泌 MMPs 的量明显减少。FAK 表达抑制影响骨架蛋白 Vinculin 在 PDGF-BB 诱导细胞骨架重塑中分布,阻碍片状伪足形成,延迟黏着斑形成时间。结论 FAK 表达受到抑制后,通过减少 MMPs 分泌和影响细胞骨架重塑可降低 MHCC97-H 细胞黏附、侵袭能力。
Objective To study the effect of inhibiting the expression of focal adhesion kinase (FAK) in human hepatoma cell line MHCC97-H on cell adhesion, invasion and remodeling. Methods Lipofectamine was used to transfect FAK siRNA into MHCC97-H cells. Western blot was used to detect the expression of FAK protein in MHCC97-H cells before and after RNA interference. Adhesion test and invasion assay were used to detect the changes of adhesion and invasion ability of MHCC97-H cells before and after interference. Gelatin zymography assay to detect the changes of MMPs secreted by MHCC97-H cells. Changes in scaffold remodeling of immunofluorescently labeled cells were observed by laser confocal microscopy. Results Specific FAK siRNA significantly inhibited FAK expression in MHCC97-H cells. The adhesion rate of MHCC97-H cells to extracellular matrix fibronectin was 35.8% after interference, which was lower than 57.3% of the control group (P <0.05). The number of MHCC97-H cells penetrating Tanswell cells after interference decreased from (31.3 ± 2.6) to (14.5 ± 3.1) (P <0.05) in the control group. The amount of MMPs secreted by MHCC97-H cells after interference was significantly reduced. Inhibition of FAK expression affects the distribution of Vinculin, a cytoskeletal protein involved in PDGF-BB-induced cytoskeletal remodeling, hindering the formation of lamellipodia and delays the formation of focal adhesions. Conclusion After FAK expression is inhibited, the adhesion and invasion ability of MHCC97-H cells can be decreased by reducing the secretion of MMPs and affecting cytoskeletal remodeling.