Identification of metastasis-associated proteins involved in gallbladder carcinoma metastasis by proteomic analysis and functional exploration of chloride intracellular channel 1.

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.