Objective To investigate the effect of 5-aza-CdR on expression of hepaCAM mRNA and the inhibition of T24 and BIU-87cell lines.Methods The effects of 5-aza-CdR on cell proliferation of T24 and BIU-87 cell lines were measured by MTT;Flow cytometry was used to examine apoptosis of T24 and BIU-87 cells;RT-PCR was used to detect the expression of hepaCAM in T24 and BIU-87 cell lines with or without 5-aza-CdR treatment.ResultsThe inhibition ratios of 3.0 and 10.0 μmol/L 5-aza-CdR were significantly higher than that of 0.3 and 1.0 μmol/L 5-aza-CdR in T24 cell(P0.05).The inhibition ratio of 5.0 μmol/L 5-aza-CdR was higher than that of 0.1 and 0.5 μmol/L 5-aza-CdR in BIU-87 cell(P0.05).Cell apoptotic rate increased in cells with 5-aza-CdR treatment(P0.05).The expression of hepaCAM mRNA was reversed in T24 and BIU-87 cells after treatment.Conclusions 5-aza-CdR can reverse the expression of hepaCAM mRNA and therefore inhibit the cell growth through inducing apoptosis in bladder cancer tissue.

Objective To investigate the mechanism of vascular endothelial growth factor(VEGF) in influencing the angiogenesis of tumors through phospholipase C epsilon(PLCe)-NF-κB signal pathway.Methods Plasmid pGenesil-PLC for expression of PLC e-shRNA was transfected to human renal cell carcinoma 786-0 cell strain to silence the expression of PLCe gene.The expressions of PLC e,NF-κB and VEGF in transfected cells at mRNA level were determined by RT-PCR,while those at protein level by Western blot.The cells were treated with BAY11-7082,a specific inhibitor of NF-κB,and determined for inhibiting effect on cell proliferation by MTT method,then for expressions of VEGF mRNA and protein by RT-PCR and Western blot respectively.Results Recombinant plasmid pGenesil-PLCe inhibited the expressions of PLCe at mRNA and protein levels significantly,of which the inhibiting rates were 71.43% and 50.01% respectively.The plasmid also down-regulated the expressions of NF-κB and VEGF mRNAs and proteins significantly.BAY11-7082 showed significantly dose-and time-dependent inhibitory effect on the proliferation of 786-0 cells.Both the expressions of VEGF mRNA and protein in the cells after treatment with BAY11-7082 were inhibited significantly.Conclusion PLCe might inhibited the expression of NF-κB-dependent VEGF gene by inhibiting the expression of NF-κB gene and influence the angiogenesis of renal cell carcinoma.

It was to investigate the expression of hepaCAM and VEGF mRNA in renal cancer cells 786-0,RC-2,renal cell carcinomas(RCC)and their relation to invasion and metastasis of renal cell carcinomas(RCC).The expression of hepaCAM and VEGF mRNA was determined in 786-0,RC-2,normal renal tissue,the expression of hepaCAM mRNA was determined in 73 cases of RCC and corresponding peripheral noncancerous tissue and the expression of VEGF mRNA was determined in 43 cases of RCC and corresponding peripheral noncancerous tissue by reverse transcription polymerase chain reaction(RT-PCR).The difference and corelation between each group were compared and analyzed.Results indicate that hepaCAM mRNA's expression was lower in 786-0,RC-2 than in the normal tissue(P<0.05),VEGF mRNA's expression was higher in 786-0,RC-2 than that of in the normal tissue(P<0.05).In RCC tissues hepaCAM mRNA's expression was significantly lower than corresponding peripheral noncancerous tissues(P<0.05),VEGF mRNA's expression was significantly higher than corresponding peripheral noncancerous tissues.There are significance between stage Ⅰ+Ⅱgroup and stageⅢ+Ⅳ group as far as VEGF mRNA expression are concerned(P<0.05)and hepaCAM mRNA expression level was negatively correlated with the VEGF mRNA in RCC(r =-0.329,P<0.05).Therefore,the down regulation of hepaCAM contributes to the invasiveness and metastasis of RCC,and the mechanism may be associated with the change of VEGF expression.hepaCAM is expected to become a new molecular in the gene therapy of RCC.

It was to investigate the relationship between the expression of hepaCAM and methylation of hepaCAM exon 2 in renal carcinoma.Methylation-specific restriction-PCR-assay and reverse transcription-polymerse chain reaction(RT-PCR)were used to examine the methylation status of hepaCAM exon 2 and mRNA in renal carcinoma cell lines(786-0,RC-2)and 43 cases of renal carcinoma tissues and paired adjacent tissues.The expression of hepaCAM mRNA was down-regulated in 786-0 renal cell lines as compared with RC-2 renal cell lines(P<0.05);786-0 cell lines showed hepaCAM exon 2 methylation but RC-2 didn't.The expression of hepaCAM mRNA in renal carcinomas was significant lower than that in the adjacent renal tissues(P<0.001).The methylation rate of hepaCAM exon 2 was significant higher in renal carcinomas than in adjacent renal tissues(34.9% vs 2.33%,P<0.001).But,there was no statistically significant difference in clinical pathology parameters(P>0.05).The expression of hepaCAM mRNA was significant lower in hepaCAM exon 2 methylation renal carcinoma than that in unmethylated renal carcinoma(P<0.05).Therefore,the methylation of hepaCAM exon 2 seems to be one of reasons inducing the down-regulation or deletion of hepaCAM expression.The study of hepaCAM methylation provides a new theoretical evidence for the mechanism of the occurrence and development of renal carcinoma.

Investigate the expression of hepatocyte cell adhesion molecule (hepaCAM) and vascular endothelial growth factor (VEGF) mRNA in 55 cases of urothelial carcinoma to examine the potential relationship between hepaCAM and VEGF in urothelial carcinoma.

Objective To investigate the effects of transfection of wild type hepatocyte cell adhesion molecule(hepaCAM) gene on the growth and metastasis of bladder cancer BIU-87 cells.Methods Plasmids pEGFPN2-hepaCAM and pEGFPN2 were respectively transfected into BIU-87 cells with Lipofectamine2000 to establish BIU-87 cells with stable over-expression of hepaCAM.MTT assay was used to observe the proliferation of hepaCAM transfection cells,pEGFPN2 transfection cells and untransfected cells.The mRNA expressions of hepaCAM and VEGF before and after the stable transfection were detected by reverse transcriptase polymerase chain reaction(RT-PCR) .The protein level of VEGF in the above cell groups was analyzed by Western blot analysis.Results MTT assay showed that the proliferation of BIU-87 cells transfected with wild type of hepaCAM was markedly inhibited compared with that of control groups(P<0.05) .There was no expression of hepaCAM mRNA in BIU-87 cells,but the expression of hepaCAM mRNA of BIU-87 cells transfected with hepaCAM gene was markedly increased(P<0.05) .The expression of VEGF mRNA(0.527±0.031) in BIU-87 cells transfected with hepaCAM was markedly inhibited as compared with that of transfected with empty vector cells(0.750±0.040) and untransfected cells(0.803±0.042,P<0.05) .The results of Western blotting showed the protein expression of VEGF in BIU-87 cells transfected with hepaCAM(0.057±0.015) was markedly inhibited as compared with transfected with empty vector cells(0.280±0.036) and untransfected cells(0.263±0.032,P<0.05) .Conclusion Transfection of hepaCAM gene significantly inhibits the cell proliferation,and VEGF expressions at mRNA and protein levels in BIU-87 cells.hepaCAM gene might play an important role in the proliferation,invasion and metastasis of bladder cancer.

Aim: We aimed to investigate the mechanisms of hepaCAM inactivation in transitional cell carcinoma of the bladder through the analysis of hepaCAM exon 2 methylation. Methods: The methylation of hepaCAM exon 2 and the expression of hepaCAM were determined by methylation-specific restriction PCR assay and RT-PCR in bladder cancer cells (T24, BIU-87) as well as in 55 paired bladder cancer specimens. The methylated bladder cancer cells were treated with 5-Aza-2'-deoxycytidine (5-Aza-CdR), a demethylating agent. MTT was used to detect the proliferation of T24 and BIU-87 cells. Results: The proliferation of T24 and BIU-87 cells was suppressed by treatment with different concentrations of 5-Aza-CdR; the expression of hepaCAM was absent in T24 and BIU-87 cells, and we found that exon 2 of hepaCAM was methylated in the 2 cells. hepaCAM mRNA was re-expressed and the methylation status of hepaCAM exon 2 was reversed after treatment with 5-Aza-CdR. The expression of hepaCAM mRNA in bladder cancer tissues was significantly lower than that in adjacent tissues. The methylation rate of hepaCAM exon 2 was significantly higher in bladder cancer tissues than in adjacent tissues. The methylation of hepaCAM exon 2 was related to hepaCAM expression in bladder cancer tissues. Conclusions: Downregulation of hepaCAM expression plays an important role in the tumorigenesis and development of bladder cancer. DNA methylation may be important for downregulation of hepaCAM expression in bladder cancer. Copyright (C) 2010 S. Karger AG, Basel

Objective:hepaCAM (hepatocyte cell adhesion molecule)is a kind of immunoglobulin superfamily member which was discovered as a tumor suppressor. This study was to investigate the effects of hepaCAM transfection on proliferation and invasion of human renal cancer cell line 786-0. Methods:An eukaryotic expression plasmid containing hepaCAM,hepaCAM-pEGFPN2 was transfected into 786-0 cells,then positive cell clones were selected with G418 and amplified. pEGFPN2 transfected cells and untreated cells were set as controls.The expression of hepaCAM was detected by reverse transcription-polymerase chain reaction (RT-PCR). Proliferative ability of 786-0 cells was measured by MTT assay and the invasive ability in vitro was determined by transwell chambers method. Results: Plasmid pEGFPN2,hepaCAM-pEGFPN2 were successfully constructed and stably transfected into 786-0 cells.After transfection,the inhibitory rates of cell growth at the fourth、 fifth and sixth day were 26.5%,38.1% and 35.7%,respectively. The penetrating cells were significantly less in 786-0/ hepaCAM-pEGFPN2 group than in 786-0/pEGFPN2 and 786-0 groups (24.6±5.3vs35.5±6.2 and 37.1±7.0,P0.01). Conclusion:Transfection with hepaCAM might suppress proliferative and invasive ability of renal cancer 786-0 cells.

Objective:This study was to evaluate the effects of hepaCAM (hepatocyte cell adhesion molecule) on invasiveness of human renal cell carcinoma (RCC) cell line 786-0 and explore the underlying mechanism. Methods:A recombinant plasmid containing hepaCAM cDNA (hepaCAM-pEGFPN2) was transfected into 786-0 cells, then the positive cell clones were selected with G418 and amplified. The 786-0 cells transfected with pEGFPN2 and untreated 786-0 cells were as controls. The mRNA expressions of hepaCAM and matrix metalloproteinase (MMP)-2 and MMP-9 before and after stable transfection are detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time fluorescence PCR. The activities of MMP-2 and MMP-9 were analyzed by gelatin zymography. The invasiveness of 786-0 cells was determined by Transwell migration assay in vitro. Results: The mRNA transcription of hepaCAM was significantly increased in 786-0 cells after stable transfection of hepaCAM (P0.01). The mRNA expressions and acti-vities of MMP-2 and MMP-9 in 786-0 cells were markedly inhibited by hepaCAM (P0.05). The transmembrane migration capacity of cells was significantly reduced after stable transfection of hepaCAM (P0.01). Conclusion:HepaCAM inhibits the activities of gelatinase by down-regulating the expressions of MMP-2 and MMP-9, thereby preventing the invasiveness of renal cell carcinoma 786-0 cells.