Proteomic Analysis Of Human Gallbladder Cancer And Study Of Biological Effects Of AnnexinA3-miRNA In Gallbladder Cancer Cells

Part I Proteome analysis of human gallbladder cancer serum and normal human serumObjective To compare the serum proteome difference of gallbladder carcinoma patients and healthy subjects, and to provide experimental evidence for finding serum biomarkers of gallbladder cancer. Methods Proteomes of 6 pairs of clinical gallbladder cancer serum samples and normal human serum samples were obtained by two-dimensional gel electrophoresis(2-DE). Comprehensive analyses of proteins were focused on total protein spots exhibiting statistical alternations between the two groups. Protein identification was done by peptide mass fingerprinting with tandem mass spectrometry(MS). Western blot and immunohistochemistry(IHC) were used to detect the expression levels of differential protein S100A10 and haptoglobin in an independent series of serum and tissue samples. Results A total of 12 protein spot-features were found to be significantly increased and 12 significantly decreased in gallbladder cancer serum, including: Splicing factor 3B subunit 5, Cystatin-B, S100A10 Protein, Histone H2B type 2-E, Profilin-1, Eukaryotic translation initiation factor 1A, Isoform 1 of Eukaryotic translation initiation factor 5A-1, FERM domain containing 3, haptoglobin protein, Glyceraldehyde-3-phosphate dehydrogenase, Serum amyloid P-component precursor, harmonin isoform b3, Apolipoprotein A-I precursor, Myosin regulatory light chain 2, Isoform 1 of Protein canopy homolog 2 precursor, Proteasome subunit beta type-2, ARHGDIA, Superoxide dismutase [Mn], Isoform 1 of Ficolin-3 precursor, zinc alpha-2-glycoprotein 1, HP haptoglobin isoform 2 preproprotein, CD5 antigen-like precursor, CLU clusterin isoform, ITIH4 71kDa protein. The increased levels of S100A10 and haptoglobin protein found to be associated with gallbladder cancer was further confirmed by Western blot and immunohistochemistry analysis.Conclusion These results suggest that the combination of 2-DE with MS provides an effective strategy to discover differentially expressed proteins in gallbladder cancer which may be molecular markers for diagnosis or therapeutic targets.Part II Proteome analysis of human glabbladder cancer tissue and benign gallbladder tissueObjective To find out potential molecular targets for gallbladder carcinoma diagnosis and treatment by analyzing and comparing the proteins expressed in human gallbladder carcinoma tissue and benign gallbladder tissue.Methods Proteomes of 6 pairs of glabbladder cancer tissue samples and benign gallbladder tissues were analyzed by two-dimensional gel electrophoresis(2-DE). Comprehensive analyses of proteins were focused on total protein spots exhibiting statistical alternations between the two groups. Protein identification was done by peptide mass fingerprinting with mass spectrometry(MS). In addition, Western blotting and immunohistochemistry (IHC) were performed to examine the expression of certain candidate proteins in an independent series of samples. .Results Protein extracts of individual sample in each type of tissues were separated on two-dimensional gels. Seventeen proteins were successfully identified by MS, in which nine proteins were overexpressed in tumors and the other eight proteins were underexpressed. Western blotting and IHC further validated up-regulated expressions of two candidate protein in tumorous tissues: AnnexinA3 and PEBP1.Conclusion These results suggest that the combination of 2-DE with MS provides an effective strategy to discover differentially expressed proteins in gallbladder cancer which may be molecular markers for diagnosis or therapeutic targets. Part III Study of Biological Effects of AnnexinA3-miRNA in Gallbladder Cancer CellsObjectives To construct recombinant interfering RNA(miRNA) plasmid vector targeting annexinA3, and observe its impact on the growth and apoptosis of human gallbladder cancer cell line SGC-996 in vitro. Methods An annexinA3-miRNA targeting human annexinA3 mRNA common sequence was synthesized and it was inserted into pcDNA?6.2-GW/EmGFPmiR vector. The recombinant plasmid was transfected into SGC-996 cell by Lipofectin. The proliferation of SGC-996 cell was assessed by cell counting in experimental and control groups. The cell cycle and apoptosis of SGC-996 cells was observed by flow cytometry(FCM). The migratory and invasive ability of these cells was detected by scratch-wound healing assay and Transwell.Results The recombinant plasmids containing annexinA3 miRNA were successfully constructed. The expressions of annexinA3 mRNA and protein in SGC-996 cells of experimental groups were significantly decreased,compared to controls(p<0.05). The proliferation of SGC-996 cells in experimental groups was inhibited, compared to controls(p<0.05). FCM analysis showed that the cell cycle had no change, but annexinA3-miRNA can promote apoptosis(p<0.05). AnnexinA3 knockdown cells (pcDNA6.2-miR) exhibited significant decrease in migration. The number of migrated SGC-996 cells in experimental group was far less compared with controls(p<0.01). Conclusions It indicated that miRNA eukaryotic expression vector for annexinA3 would be successfully established. The administration of annexinA3-miRNA in SGC-996 cells can down-regulated annexinA3 expression, inhibit proliferation, induced apoptosis, and also inhibit the migratory and invasive ability. It suggested that miRNA-based targeting annexinA3 strategy might build the experimental foundation for the research of gene therapy in gallbladder cancer.