热休克蛋白27在转移潜能不同人肝癌细胞系中的表达及机制研究  

Expression and Biological Mechanism Exploration of HSP27 in Different Metastatic HCC Cell Lines

作　　者：郭坤[1] 刘银坤[1] 周海君[1] 代智[1] 陈洁[1] 孙瑞霞[1] 孙强玲[1] 卢雯静[1] 康晓楠[1] 

机构地区：[1]复旦大学附属中山医院肝癌研究所,上海200032

出　　处：《生物化学与生物物理进展》2007年第7期738-745,共8页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展计划(973)(2004CB520802);国家自然科学基金(3017416)资助项目.~~

摘　　要：利用不同转移潜能肝癌细胞系探讨热休克蛋白27(HSP27)参与肝癌细胞转移潜能形成的可能分子机制.细胞免疫荧光技术、RT-PCR和免疫印迹技术显示,HSP27在转移潜能不同的肝癌细胞Hep3B、MHCC97L和MHCC97H中定位于细胞浆,亦可见于细胞核,HSP27 mRNA和蛋白质的表达水平与肝癌细胞转移潜能呈正相关.高转移潜能肝癌细胞系MHCC97H的HSP27 RNA干扰试验结果显示,HSP27 RNA干扰后MHCC97H的侵袭(MHCC97H组:21.36±2.92;对照RNAi组:19.88±2.23;RNAi组:11.40±2.05)、运动能力(MHCC97H组:26.35±3.29;对照RNAi组:24.43±3.17;RNAi组:10.92±2.27)明显减弱,细胞凋亡显著增加(MHCC97H组:15.12%;对照RNAi组:17.56%;RNAi组:27.64%).同时进行信号转导基因芯片检测发现,HSP27干扰后核因子κB(NF-κB)通路抑制,并且免疫印迹显示细胞核内活化的NF-κBp65减少,细胞内磷酸化IκBα降低.另外,免疫共沉淀检测发现,在肝癌细胞内HSP27可与IKKβ、IκBα共沉淀,且在HSP27RNA干扰后IKKβ与IKKα结合能力下降.这些结果提示,HSP27可能通过参与细胞内NF-κB通路的激活,影响细胞凋亡和细胞运动,在肝癌细胞侵袭转移过程中发挥作用.To reveal molecular mechanisms responsible for that over-expression of heat shock protein 27 （HSP27） is associated with poor prognosis in liver cancer, expression of HSP27 was analyzed by RT-PCR and Western blot in Hep3B, MHCC97L and MHCC97H, which had higher metastasis potential than Hep3B, and after HSP27 RNA interference, MHCC97H migratory and invasive capability were evaluated by in vitro migration and invasion assay, apoptosis ratio was detected by flow cytometry（FCM） and TUNEL method, prominent altered signal pathway was analyzed through a Human Q Series Signal Transduction in Cancer Gene Array. The results showed that the up-regulation of HSP27 expression in HCC cell lines was consistent with metastasis potentials. Using RNA interference technique, mRNA and protein level of HSP27 in MHCC97H cell line were specifically reduced by up to 80%. In vitro migration and invasion assay showed the average migratory and invaded cell numbers per field were 21.36 ± 2.92, 19.88 ± 2.23, 11.40 ± 2.05 and 26.35±3.29, 24.43± 3.17, 10.92 ± 2.27 respectively in MHCC97H, control RNAi, RNAi groups. Moreover, MHCC97H cell apoptosis ration was 15.12%, 17.56%, 27.64% （MHCC97H, control RNAi, RNAi） respectively by FCM analysis, which was reconfirmed by TUNEL morphological assessment. Furthermore cDNA microarray analysis revealed that NF-κB pathway activation was inhibited after HSP27 RNAi. Immunoblotting analysis showed that the decreasing of nuclear activated NF-κB p65 and phosphorylated IKBα, meanwhile reducing of the association between IKKβ and IKKα in MHCC97H cells after HSP27 RNAi, further communoprecipitation assay showed interaction of IKKβ, IκBβ with HSP27 in three HCC cell lines. Altogether, these findings revealed possible roles of HSP27 in being of HCC cell lines metastatic potentials through involving in cellular NF-κB pathway activation.

关 键 词：肝细胞性肝癌细胞 热休克蛋白27 信号转导 核因子κB(NF-κB) 肿瘤转移 凋亡 RNA干扰 

分 类 号：R735.7[医药卫生—肿瘤]