CAJ | 학술논문

目的探讨细胞外热休克蛋白90α(HSP90d)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HSP90 α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HSP90α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 α与MMP-2相互作用及其细胞转移潜能的变化.结果 HSP90α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24 h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HSP90α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HSP90α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.
목적 탐토세포외열휴극단백90α(HSP90d)삼여간암세포전이적가능분자궤제.방법 채용면역인적법검측HSP90 α재저、고전이성간암MHCC97-L화MHCC97-H세포외적표체.선취고전이잠능MHCC97-H세포,응용불천투세포막적소분자억제제억제세포외HSP90α표체,체외침습실험관찰세포침습、천이능력적변화,명효매보법분석기질금속단백매2(MMP-2)활성적개변.면역인적화면역공침정법검측세포외보조분자반려HSP70화MMP-2적표체급기여HSP90α적상호작용.이용RNA간우기술하조세포HSP70표체,관찰세포외HSP90 α여MMP-2상호작용급기세포전이잠능적변화.결과 HSP90α재불동전이잠능간암세포내외균표체,차여간암세포전이잠능정정상관.MHCC97-H세포경특이성HSP90억제제이갑안기을안기-17-거갑양기격이덕매소-N-양화물(DMAG-N-oxide)작용24 h후,천과상실기저막적세포수교미처리조화공백대조조명현하강(P＜0.01),분별위(28.11±3.56)、(80.12±4.16)、(82.24±4.12)개.체외침습실험현시,천과Matrigel인공기저막적세포수저우미처리조화공백대조조,분별위(36.54±4.12)、(95.12±3.48)、(101.11±3.36)개,차이유통계학의의(P=0.017),병반유세포외MMP-2활성감저.HSP70화MMP-2균가재MHCC97-H세포외표체,차능여HSP90α상호결합.소분자간우RNA (siRNA)능유효억제세포HSP70표체,MHCC97-H세포외HSP90α화MMP-2상호작용감약,MMP-2활성하강,세포침습、천이능력수억제.동시연합응용MMP-2억제제,구유협동억제효응.결론 세포외HSP90α가능통과여HSP70형성반려복합체개도MMP-2성숙활화,증강간암세포침습、천이운동,제시기가능시잠재적대간암전이실시방치적파점.
Objective To explore the expression of heat shock protein (HSP) 90α in outside of different metastatic hepatocellular 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 cell-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 extracellular 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 (101.1 1±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.