Glycoproteomics Of Human Immortalized Non-tumor Liver Cell Line And High Metastatic Liver Cancer Cell Line And The Up-regulation Of Prohibitin 1 Is Involved In The Proliferation And Migration Of Liver Cancer Cells

Part one Glycoproteomics of human immortalized non-tumor liver cell lineIn this article, we presented here an effective technique for the large-scale separation andidentification of N-linked glycoproteins from Chang liver cells, which were human normalliver cells. To separate N-linked glycoproteins high-through from the whole cells lysis, wefirstly perform a procedure containing the lysis of human liver cells, the solubilization of totalproteins, lectin affinity chromatography including concanavalin A (Con A) and wheat germagglutinin (WGA). Then the captured N-linked glycoproteins were separated bytwo-dimensional electrophoresis (2-DE), and identified by mass spectrometry and databasesearching. After analyzing image data with ImageMaster^TM 2-D Platinum software, there were323±5 (n=3) Con A binding spots detected on the Chang liver cells gels, and there were 256±5 (n=3) WGA binding spots. The spots matched Con A binding spots with WGA bindingspots were 124±5 (n=3), and there were totally 454 protein spots after eliminating theoverlap spots from two gels. For the glycoproteins captured with Con A and WGA, theselected spots were identified by MALDI-TOF MS. Finally we identified 84 positive proteinspots. Among these spots, 47 positive protein identifications from Con A binding spotsrepresented 40 unique protein, and 37 from WGA binding spots representing 34 uniqueproteins. After eliminating the overlap proteins captured by Con A and WGA, 70 uniqueproteins were obtained. Our results showed that we have established a high-throughputproteomic analysis for separating N-linked glycoproteins from human liver cells. Our workwas part of the HUPO HUMAN LIVER PROTEOME PROJECT (HLPP) studies and wassupported by CHINA HUPO. The establishment of the technique will contribute to theresearch of the N-glycosylation of human liver cells.Part two Comparative glycoproteomics of human immortalized non-tumor liver cell line and high metastatic liver cancer cell lineApplying the method in part one, we compared the Con A and WGA capturedglycoprotein profiles of Chang liver cells and MHCC97-H cells, which was high metastatichuman liver cancer cell line. Combining software analysis with artificial correction, wefocused on 2 spots from Con A captured proteins and 5 spots from WGA captured proteinsthat showed statistically significant up-regulation (p＜0.05) in MHCC97-H cells. After MSidentification, there were carboxylesterase, heat shock protein gp96 precursor, prohibitin 1, glutamate-cysteine ligase modifier subunit, hypothetical protein, pre-B cell enhancing factorprecursor and dihydrolipoamide dehydrogenase. Carboxylesterase and heat shock proteingp96 precursor were captured by Con A, others captured by WGA. These glycoproteins werereported to be associated with the occurrence and development of cancer.Part three The up-regulation of prohibitin 1 is involved in the proliferation and migration of livercancer cellsIn part two, we found that prohibitin 1 (PHB) was up regulated in MHCC97-H cells.Although PHB has been reported as a potential tumor suppressor protein for many years,there are few studies about the association of PHB with hepatocellular carcinoma and theglycosylation of PHB. So we selected PHB for the further study. Firsyly, western blot andreverse transcription-PCR experiments confirmed that PHB was up regulated about 2-fold inMHCC97-H cells comparing to chang liver cells. At the same time, total proteins from 4 pairsof matched tissues including normal liver tissue, primary hepatocarcinoma tissue and adjacentnormal liver tissue were detected by Western blot (pair test p＜0.01). The expression of PHBdisplayed significant higher in two of primary hepatocarcinoma tissues than their matchednormal and adjacent normal liver tissues at p＜0.05. Secondly, PHB was not significantly upregulated in other cancer cell lines including SMMC-7721, BEL-7404, HUH-7, Lmb,MHCC97-L cell lines. MHCC97-H and MHCC97-L cell lines had the same geneticbackground but with different metastatic potential. So we further study the association of theup-regulation of PHB with the migration and proliferation of liver cancer cells. When PHBwas over-expressed in MHCC97-L cells, the cell proliferation was inhibited 35% and themigration was increased about 2.1±0.1 (p＜0.05) folds. Furthermore, SWISS-PROT databaseprediction, immunoprecipitation and lectin L-PHA blot validated that PHB was a glycoprotein.Our results firstly showed that PHB was an N-linked glycoprotein and was associated with theproliferation and migration of liver cancer cells.In conclusion, the experiment established a high-through method of glycoproteomics withlectin affintity and proteomics to separate and identify the N-linked glyproteins. Then weapplied the method to perform the comparative glycoproteomics analysis and found 7glycoproteins which were remarkably up regulated in MHCC97-H cells comparing to Changliver cells. Among the 7 glycoproteins, we preliminarily investigated on the functions of PHBon tumor cells and the glycosylation of PHB.