AIM:To identify potential biomarkers of primary gallbladder cancer(PGC).METHODS:Fresh PGC,cholecystitis and normal gallbladder tissue specimens collected from 10 patients,respectively,were subjected to comparative proteomic analysis.The proteomic patterns of PGC were compared with those of cholecystitis and normal gallbladder tissues using two-dimensional gel electrophoresis(2-DE).The differentially expressed proteins were then identified using a MALDI-TOF mass spectrometer(MS)and database searches.To further validate these proteins,20 samples of PGC tissues and normal tumoradjacent tissues were collected for Western blot,quantitative real-time PCR,and immunohistochemical staining assay.RESULTS:Seven differentially expressed protein spots were detected by 2-ED analysis by comparing the average maps of PGC,cholecystitis and normal gallbladder tissues.Six of the seven differentially expressed proteins were identified using MALDI-TOF MS,with three overexpressed and three underexpressed in PGC tissue.Protein levels of annexin A4(ANXA4)were significantly elevated,and heat shock protein90-beta(Hsp90β)and dynein cytoplasmic 1 heavy chain 1(Dync1h1)were decreased in PGC tissues relative to the normal tumor-adjacent tissues as shown by Western blot analysis.However,levels of actin,aortic smooth muscle and gamma-actin were unchanged.In addition,the mRNA levels of all 5 proteins showed similar changes to those of the protein levels(P<0.01).Further validation by immunohistochemical analysis showed the upregulated expression of ANXA4 and decreased expression of Hsp90βand Dync1h1 in the cytoplasm of PGC tissues relative to the normal tumoradjacent tissues.CONCLUSION:Three proteins are identified as potential biomarkers of PGC using proteomic analysis.The functions of these proteins in the carcinogenesis of PGC remain to be studied. AIM: To identify potential biomarkers of primary gallbladder cancer (PGC). METHODS: Fresh PGC, cholecystitis and normal gallbladder tissue materials collected from 10 patients, respectively, were subjected to comparative proteomic analysis. The proteomic patterns of PGC were compared with those of cholecystitis and normal gallbladder tissues using two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were then identified using a MALDI-TOF mass spectrometer (MS) and database searches. further validate these proteins, 20 samples of PGC tissues and normal tumoradjacent tissues were collected for Western blot, quantitative real-time PCR, and immunohistochemical staining assay. RESULTS: Seven differentially expressed protein spots were detected by 2-ED analysis by comparing the average maps of PGC, cholecystitis and normal gallbladder tissues. Six of the seven differentially expressed proteins were identified using MALDI-TOF MS, with three overexpressed and three underexpressed in PGC tissue. Protein levels of annexin A4 (ANXA4) were significantly elevated, and heat shock protein90-beta (Hsp90β) and dynein cytoplasmic 1 heavy chain 1 (Dync1h1) were decreased in PGC tissues relative to the normal tumor-adjacent tissues as shown by Western blot analysis. Of the levels of actin, aortic smooth muscle and gamma-actin were unchanged. In addition, the mRNA levels of all 5 proteins showed similar changes to those of the protein levels (P <0.01). Further validation by immunohistochemical analysis showed the upregulated expression of ANXA4 and decreased expression of Hsp90β and Dync1h1 in the cytoplasm of PGC tissues relative to the normal tumoradjacent tissues. CONCLUSION: Three proteins are identified as potential biomarkers of PGC using proteomic analysis. These functions of these proteins in the carcinogenesis of PGC remain to be studied