热休克蛋白90α在人类肝癌细胞外的表达及其对侵袭、迁移能力的影响

Expression of extracellular heat shock protein 90α in human hepatocellular carcinoma cells and its effect on cells migratory and invasive capability

目的探讨细胞外热休克蛋白90α（HSP90α参与肝癌细胞转移的可能分子机制。方法采用免疫印迹法检测HSP90α在低、高转移性肝癌MHCC97-L和MHCC97-H细胞外的表达。选取高转移潜能MHCC97-H细胞，应用不穿透细胞膜的小分子抑制剂抑制细胞外HSP90α表达，体外侵袭实验观察细胞侵袭、迁移能力的变化，明胶酶谱法分析基质金属蛋白酶2（MMP-2）活性的改变。免疫印迹和免疫共沉淀法检测细胞外辅助分子伴侣HSP70和MMP-2的表达及其与HSP90d的相互作用。利用RNA干扰技术下调细胞HSP70表达，观察细胞外HSP90仅与MMP-2相互作用及其细胞转移潜能的变化。结果HSP90d在不同转移潜能肝癌细胞内外均表达，且与肝癌细胞转移潜能呈正相关。MHCC97-H细胞经特异性HSP90抑制剂二甲氨基乙氨基-17-去甲氧基格尔德霉素-N-氧化物（DMAG—N—oxide）作用24h后，穿过上室基底膜的细胞数较未处理组和空白对照组明显下降（P〈0．01），分别为（28．11±3．56）、（80．12±4．16）、（82．24±4．12）个。体外侵袭实验显示，穿过Matrigel人工基底膜的细胞数低于未处理组和空白对照组，分别为（36．54±4．12）、（95．12±3．48）、（101．11±3．36）个，差异有统计学意义（P=0．017），并伴有细胞外MMP-2活性减低。HSP70和MMP-2均可在MHCC97-H细胞外表达，且能与HSP90α相互结合。小分子干扰RNA（siRNA）能有效抑制细胞HSP70表达，MHCC97-H细胞外HSP90d和MMP-2相互作用减弱，MMP-2活性下降，细胞侵袭、迁移能力受抑制。同时联合应用MMP-2抑制剂，具有协同抑制效应。结论细胞外HSP90饯可能通过与HSP70形成伴侣复合体介导MMP-2成熟活化，增强肝癌细胞侵袭、迁移运动，提示其可能是潜在的对肝癌转移实施防治的靶点。

Objective To explore the expression of heat shock protein （HSP） 90α in outside of different metastatic hepatoeellular carcinoma （HCC） cell lines and its role in the cells migration and invasion. Methods The expression of HSP90α was detected by Western blot analysis in conditioned media of MHCC97L and MHCC97H with low and high metastatic HCC cell lines. A small molecule eell-impermeant HSP90 inhibitor DMAG-N-oxide was used to inhibit extracellular HSP90α. Changes of the cells migratory and invasive capability were assessed by in vitro motility and invasion assay. The endogenous matrix metalloproteinase 2 （MMP-2） was demonstrated by Zymography. The expression of extraeellular co-chaperone HSP70 and MMP-2 were tested by Western blots and the association between HSP90α, HSP70 and MMP-2 was analyzed by immunoprecipitation. The effects of HSP70 knockdown by siRNA, with or without MMP-2 inhibitor Batimastat, on the level of active MMP-2 and cell migration and invasion were also evaluated. Results HSP90α can express both inside and outside of different metastatic HCC cell lines, and the level of expression was consistent with metastasis potentials. After MHCC97-H cells were treated with a special HSP90α inhibitor DMAG-N-oxide for 24 h, the average migratory cell numbers （28.11 ±3.56） had a significantly reduction, compared with those without treatment group （80.12±4.16） and empty control group（82.24±4.12）, respectively （P 〈 0.01）. In vitro invasion assay showed the average invaded cell numbers in treatment group （36.54±4.12） were more fewer than without treatment group （95.12±3.48） and empty control group （101.11±3.36）, respectively （P = 0.017）, and accompanying with decreasing of the extracellular MMP-2 activity. HSP70 and MMP-2 could express outside of MHCC97-H cells and interact with HSP90α. Small molecular interfere RNA （siRNA） dramatically inhibited HSP70 expression and reduced the interaction HSP90α with MMP-2 and MMP-2 activity outside MHCC97-H cells, and also suppressed MHCC97-H cells migration and invasion. In addition, combining MMP-2 inhibitor had additive inhibition effects. Conclusion Extracellular HSP90α and HSP70 form chaperone complex to assist in MMP-2 activation and increases HCC cells migration and invasion, which maybe a novel therapeutic target against metastatic HCC.