Wang Jian-Wei, Peng Shu-You, Li Jiang-Tao, Wang Yong, Zhang Zhi-Ping, Cheng Yan, Cheng De-Qing, Weng Wei-Hong, Wu Xiang-Song, Fei Xiao-Zhou, Quan Zhi-Wei, Li Ji-Yu, Li Song-Gang, Liu Ying-Bin
Department of Surgery, Second Affiliated Hospital, Zhejiang University, Hangzhou, China.
Cancer Lett. 2009 Aug 18;281(1):71-81. doi: 10.1016/j.canlet.2009.02.020. Epub 2009 Mar 18.
Advanced gallbladder cancer has an extremely poor prognosis because of metastasis. Identification of metastasis-related biomarkers is essential to improve patient survival. In the present study, metastasis-associated proteins were identified by comparative proteomic analysis and the metastasis-related function of the candidate protein, chloride intracellular channel 1 (CLIC1), was further elucidated. Two cell lines with high or low metastatic potential (termed GBC-SD18H and GBC-SD18L, respectively), originating from the same parental gallbladder carcinoma GBC-SD cell line, were identified by spontaneous metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised of two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between GBC-SD18L and GBC-SD18H. Twenty-six proteins were identified and further verified by one-dimensional Western blotting and semiquantitative reverse transcriptase polymerase chain reaction analysis. It was determined that CLIC1, ezrin, vimentin, annexin A3, WD repeat domain 1, triosephosphate isomerase, C1-tetrahydrofolate synthase, Rho GDP-dissociation inhibitor 1, T-complex protein 1, heterogeneous nuclear ribonucleoprotein K, glutamate dehydrogenase 1, proteasome activator complex subunit 3 and Rab GDP-dissociation inhibitor beta were significantly up-regulated in the highly metastatic GBC-SD18H cell line compared to the poorly metastatic GBC-SD18L cell line. However, phosphoglycerate kinase 1 and programmed cell death protein 8 were significantly down-regulated in the highly metastatic GBC-SD18H cell line compared to GBC-SD18L. Considering that CLIC1 was profuse in highly metastatic GBC-SD18H but scarce in poorly metastatic GBC-SD18L, the association of CLIC1 with metastasis was further elucidated by the overexpression and RNA interference of CLIC1 in GBC-SD18L cells and GBC-SD18H cells, respectively. The results demonstrated that the overexpression of CLIC1 promoted cell motility and invasion of GBC-SD18L in vitro, while RNA interference of CLIC1 remarkably decreased cell motility and invasive potency of GBC-SD18H in vitro, indicating that CLIC1 might play an important role in metastasis of gallbladder carcinoma.
晚期胆囊癌由于发生转移，预后极差。鉴定与转移相关的生物标志物对于提高患者生存率至关重要。在本研究中，通过比较蛋白质组学分析鉴定了转移相关蛋白，并进一步阐明了候选蛋白氯离子细胞内通道1（CLIC1）的转移相关功能。通过体内自发转移鉴定了源自同一亲本胆囊癌GBC-SD细胞系的两种具有高转移潜能或低转移潜能的细胞系（分别称为GBC-SD18H和GBC-SD18L），并通过体外转移表型分析对其进行了表征。随后，采用二维凝胶电泳分析和质谱组成的蛋白质组学方法来鉴定和比较GBC-SD18L和GBC-SD18H之间的蛋白质表达模式。鉴定出26种蛋白质，并通过一维蛋白质印迹和半定量逆转录聚合酶链反应分析进一步验证。结果表明，与低转移潜能的GBC-SD18L细胞系相比，高转移潜能的GBC-SD18H细胞系中CLIC1、埃兹蛋白、波形蛋白、膜联蛋白A3、WD重复结构域1、磷酸丙糖异构酶、C1-四氢叶酸合酶、Rho GDP解离抑制剂1、T复合蛋白1、不均一核核糖核蛋白K、谷氨酸脱氢酶1、蛋白酶体激活复合物亚基3和Rab GDP解离抑制剂β显著上调。然而，与GBC-SD18L相比，高转移潜能的GBC-SD18H细胞系中磷酸甘油酸激酶1和程序性细胞死亡蛋白8显著下调。鉴于CLIC1在高转移潜能的GBC-SD18H中丰富而在低转移潜能的GBC-SD18L中稀少，分别通过在GBC-SD18L细胞和GBC-SD18H细胞中过表达和RNA干扰CLIC1，进一步阐明了CLIC1与转移的关联。结果表明，CLIC1的过表达促进了GBC-SD18L在体外的细胞运动性和侵袭能力，而CLIC1的RNA干扰显著降低了GBC-SD18H在体外的细胞运动性和侵袭能力，表明CLIC1可能在胆囊癌转移中起重要作用。
