Influence Of HepaCAM On Gene Expression Profile Of Human Bladder Transitional Cell Carcinoma And HepaCAM Can Suppress The Proliferation Of Bladder Cancer Cells Through AMPK/mTOR Pathway

PART ONEINFLUENCE OF HEPACAM ON GENE EXPRESSION PROFILEOF HUMAN BLADDER TRANSITIONAL CELL CARCINOMAObjective: To investigate the changes of gene expression profile oftransitional cell carcinoma of bladder after hepaCAM was overexpressed, inorder to explore the underlying molecular machanism.Methods：Affymetrix Human Genome U133Plus2.0Array was usedto investigate the changes of gene expression profile betweenAd-GFP-hepaCAM group and Ad-GFP group in transitional cell carcinomaof bladder EJ cells. Significant Analysis of Microarray (SAM) methods wasused to screen the differentially expressed genes, DAVID software was usedto conduct Go(Gene Ontology) analysis and wikiPathway analysis based onthe differentially expressed genes. RT-PCR and western blot were applied to verify microarray data.Results：Compare with the Ad-GFP group, a total of2469genes wereup-regulated or down-regulated by more than2times in theAd-GFP-hepaCAM group. Among these genes,1602genes wereup-regulated and867genes were down-regulated, a large proportion of the2469differentially expressed genes were involved in the function of cellproliferation and cell cycle regulation. The mRNA expression of NBN,LKB1and cyclinD1detected by RT-PCR in three different bladder cancercell lines were consistent with the microarray data, the protein expression ofNBN and LKB1in the three cell lines measured by western blot wereaccordance with the mRNA expression.Conclusion: HepaCAM significantly alter the gene expression profileof bladder cancer EJ cells. The well-known anti-tumor effect of hepaCAMmay mediated by regulating the gene expression profiles of multiplepathways. PART TWOCORRELATION OF THE PROTEIN EXPRESSION OF HEPACAMAND P-MTOR IN BLADDER TRANSITIONAL CELLCARCINOMA.Objective: To explore the correlation of protein expression ofhepaCAM and p-mTOR in bladder transitional cell carcinoma and therelationship between hepaCAM or p-mTOR and variousclinicopathological parameters.Methods: Western blot was used to measure the protein level ofhepaCAM and p-mTOR in25men and5women of bladder transitionalcell carcinoma patients, disease was Ta-T1in7patients, T2-T4in23, G1in13, G2in9, G3in8, primary in13and recurrent in17.Results: HepaCAM protein was significantly lower, and p-mTORprotein was remarkably higher in bladder transitional cell carcinomacompared to adjacent tissues(P<0.001, P<0.001). Spearman correlationanalysis showed the decrease of hepaCAM level was associated with theincrease of p-mTOR level (r=-0.533P=0.002). Neither hepaCAM orp-mTOR was correlated with any of the clinicopathological parameters.Conclusion: The low expression of hepaCAM is closely correlatedwith high expression of p-mTOR, which laid a foundation for further studythe mechanism of hepaCAM involving in inhibiting the proliferation of bladder transitional cell carcinoma. PART THREEHEPACAM INHIBITS PROLIFERATION OF BLADDERTRANSITIONAL CELL CARCINOMA CELLS THROUGHAMPK/MTOR PATHWAYObjective: To study the molecular mechanisms of hepaCAM ininhibiting the proliferation of bladder transitional cell carcinoma in vitro.Methods: WST-8assay was used to study the effect of hepaCAM oncell proliferation ability. The phosphorylation of AMP-activated proteinkinase(AMPK) and mTOR, total AMPK, total mTOR, c-Myc and cyclin D1were determined by western blot.Results: HepaCAM inhibited proliferation of T24, BIU-87and EJ cells.Over-expression of hepaCAM activated AMPK phosphorylation,down-regulated mTOR phosphorylation and its targets c-myc and cyclin D1.Treatment with AMPK inhibitor (Compound C) prevented the growthinhibitory effect of hepaCAM, down-regulated AMPK activation, increasedmTOR phosphorylation and its corresponding targets c-Myc and cyclin D1.Conclusion: HepaCAM can inhibit the proliferation of bladder transitional cell carcinoma cells through AMPK/mTOR pathway.