Expression Of HEPACAM And VEGF In Transitional Cell Carcinoma Of The Bladder And The Effect Of The HEPACAM Gene On The Expression Of VEGF In Bladder Cancer Cell Lines

PART ONE The correlation of the expression of hepaCAM and VEGF in TCCBObjective: To investigate the expression of hepatocyte cell adhesion molecule(hepaCAM)and vascular endothelial growth factor (VEGF) mRNA in transitional cell carcinoma of the bladder(TCCB). The potential relationship between hepaCAM, VEGF and TCCB was analyzed.Methods: Expression of hepaCAM and VEGF gene was determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) in 55 paired TCCB specimens.Results: RT-PCR showed that hepaCAM expression level was significantly lower and VEGF was significantly higher in TCCB tissues than peripheral noncancerous tissues(P<0.05). HepaCAM and VEGF was strongly correlated with tumor stage(P<0.05),but no correlation with gender, age and grades. Spearman correlation analysis showed lower hepaCAM level were associated with higher VEGF level(r=-0.277,P<0.05).Conclusion: The downexpression of hepCAM is associated with the high expression of VEGF. It established a foundation for further study the mechanism of hepaCAM participating in the invasion and metastasis in TCCB.PART TWO Construction of TCCB cell lines expressing hepaCAM geneObjective: To construct T24, BIU-87 cells stably expressing hepaCAM. To construct T24 and BIU-87 cells stably expresseing Green Fluorescent Protein.Methods: T24, BIU-87 cells were transfected with hepaCAM plasmid (hepaCAM-pEGFPN2) or empty vector (pEGFPN2) using Lipofectamine 2000 according to the manufacturer's instructions. Forty-eight hours after transfection, the location of fluorescent protein in cells was observed by fluorenscene microscope,the transfected cells were selected by G418.The expression of hepaCAM in different groups was detected by RT-PCR.Results: The green fluorenscene of T24/pEGFPN2 and BIU-87/pEGFPN2 was on the all parts of the cells, but the green fluorenscene of T24/pEGFPN2-hepaCAM and BIU-87/pEGFPN2-hepaCAM were only on the perinuclear, cytoplasm and the tip of the cells.RT-PCR showed that there was no hepaCAM mRNA expression in T24/pEGFPN2 and BIU-87/pEGFPN2 cells, but there was an obviously increased hepaCAM mRNA expression in T24/pEGFPN2-hepaCAM and BIU-87/pEGFPN2-hepaCAM.Conclusion: The stablely transfected cells with hepaCAM-pEGFPN2 or pEGFPN2 were constructed successfully, which lays the experimental foundation for the further study about the relationship between hepaCAM and the invasion, metastasis of TCCB.PART THREE The correlation of expressing hepaCAM and VEGF in TCCB cell linesObjective: To investigate the effect of hepaCAM on the expression of VEGF in bladder cancer cell lines in vitro.Methods: MTT was performed to evaluate cell proliferation activity of different cells (cells transfected with hepaCAM, cells transfected with GFP,untreated cells); the expression of VEGF mRNA and protein in different cells were detected by RT-PCR and Western blot respectively.Results: MTT showed that expressing hepaCAM resulted in a significant inhibition of T24 and BIU-87 cells proliferation (P <0.05).RT-PCR showed that the expression of VEGF mRNA was significantly decreased in T24 and BIU-87 cells after expressing hepaCAM gene (P<0.05). Western blot showed that VEGF protein was significantly decreased in T24 and BIU-87 cells after expressing hepaCAM gene (P<0.05).Conclusion: The proliferation activity of T24, BIU-87 cells and the expression of VEGF mRNA and protein were decreased by expressing hepaCAM gene which suggests that hepaCAM may participate in the invasion and metastasis of bladder cancer by influencing the expression of VEGF.