| PART ONE: THE EXPRESSION OF HEPACAM IN TCCBObjective: To explore the expression of hepaCAM (hepatocyte cell adhesion molecule) mRNA in TCCB(transitional carcinomal of the bladder)and the relationship between hepaCAM mRNA and the clinical pathology parameters.Methods: To detect expression of hepaCAM mRNA by RT-PCR in bladder cancer cell lines (T24,BIU-87), 55 TCCB tissues and corresponding adjacent tissues and analyze the relationship between hepaCAM mRNA and the clinical pathology parameters.Results: The expression of hepaCAM mRNA was deficient in bladder cancer cell lines(T24,BIU-87). the expression level of hepaCAM in TCCB tissues was lower than that in adjacent tissues (P<0.001). No significant difference of hepaCAM mRNA was detected in the pathological grade, gender and age, but there was statistically significant differences in clinical stage (P<0.05).Conclusion: The expression of hepaCAM mRNA is obviously decrease or deficient in TCCB tissues and bladder cancer cell lines (T24,BIU-87). HepaCAM may be a putative tumor suppressor gene in TCCB.PART TWO THE ANALYSIS OF THE MEHYLATION STATUS OF HEPACAM EXON2Objective: To detect the methylation status of hepaCAM exon 2 in bladder cancer cell lines(T24,BIU-87) and TCCB tissues and explore the potential mechanism of the down-regulation of hepaCAM expression in TCCB.Methods: We use the methylation-specific restriction-PCR-assay to detect the methylation status of hepaCAM exon 2 in TCCB and bladder caner cell lines(T24,BIU-87), analyze the relationship between the methylation of hepaCAM exon 2 and the clinical pathology parameters and explore the relationship between the methylation of hepaCAM exon 2 and expression of hepaCAM mRNA from the perspective of epigenetics.Results: The methylation status of hepaCAM exon 2 was observed in bladder caner cell lines(T24,BIU-87) and 29 of 55 primary cancers, whereas in the adjacent tissues by methylation-specific restriction-PCR-assay , only 3 of 55 cases exhibited hepaCAM exon 2 methylation, and the methylation rate of hepaCAM exon 2 was significant higher in TCCB than that in adjacent renal tissues(52.7% vs. 5.5%,P<0.001). the methylation of hepaCAM exon 2 was negatively correlated to the expression of hepaCAM(r=-0.403,P<0.05).Conclusion: The methylation of hepaCAM exon 2 may be one important reasion for down-regulation of hepaCAM expression in TCCB. The study of hepaCAM methylation provides a new theoretical evidence for the mechanism of the occurrence and development of TCCB.PART THREE THE MECHANISM OF DNA METHYLATION REFULATION HEPACM EXPRESSION IN BLADDER CANCER CELLSObjective: The study is to explore the transcription regulation 5-Aza-2-deoxycytidine (5-Aza-CdR) on hepaCAM tumor suppressor gene in bladder cancer cell lines(T24,BIU-87)and to investigate its effect on the growth of T24 and BIU-87 cells and to further explore the mechanism of hepaCAM exon2 resulting in down-regulation of hepaCAM expression in TCCB.Methods: T24 and BIU-87 cell lines were separately treated with different dose of 5-Aza-CdR.MTT was used to detect the effect of 5-Aza-CdR on the growth of T24 and BIU-87 cells. The status of hepaCAM exon 2 methylation was analyzed using methylation-specific restriction-PCR-assay. RT-PCR was used to examine the expression of hepaCAM gene in before and after treated T24 and BIU-87 cells.Results: The growth of the T24 and BIU-87 cells was suppressed by treatment with 5-Aza-CdR.Before 5-Aza-CdR treatment, hypermethylation of hepaCAM exon 2 exised and hepaCAM mRNA was not expressed in T24 and BIU-87 cells. HepaCAM mRNA was re-expressed and the methylation status of hepaCAM exon 2 was obviously reversed after treatment with 5-Aza-CdR.Conclusion: 5-Aza-CdR may effectively cause the demethyaltion of hepaCAM exon 2, induce the expression of hepaCAM, and inhibit the growth of tumor cells.The result suggest that the methylation of hepaCAM exon2 is a new mechanism of resulting in down-regulation of hepaCAM expression in TCCB.This provides new ideas for the treatment of TCCB. |
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