Identification Of Hypermethylation In Hepacam Gene Promoter Region In Bladder Carcinoma

PART ONE Effect of exogenous hepaCAM gene on cell cycle distribution in bladder carcinoma cell linesObjective:To investigate the effect of hepaCAM on cell cycle distribution, exogenous hepaCAM gene was imported into bladder cancer cell lines (T24and BIU-87).Methods:T24and BIU-87cell lines were infected with adenovirus vectors (pAdH5or pAdH5-hepaCAM). Cells divided into three groups: blank group, pAdH5group and pAdH5-hepaCAM group. After48h, RT-PCR、Quantity-RT-PCR and Western-blot detected the expression of hepaCAM mRNA and protein. FCM measured the cell cycle distribution in three group cells. Western-blot examined the expression of cyclinDl protein.Results:Results of PCR and Western-blot revealed that hepaCAM gene successfully expressed in pAdH5-hepaCAM group cells. It didn’t express in blank and pAdH5groups (p<0.05). Results of FCM showed that the cell cycle was arrested in G0/G1stage in pAdH5-hepaCAM groups. Compared with blank and pAdH5groups, pAdH5-hepaCAM groups were more statistically significant difference (p<0.05). Western-blot demonstrated that the expression of cyclinDl in pAdH5-hepaCAM groups was significantly lower than other groups (p<0.05).Conclusion:Exogenous hepaCAM gene can arrest cell cycle at G0/G1stage, which explained its inhibition ability of tumor growth through regulating the cell cycle distribution. PART TWO Methylation status of hepaCAM gene promoter region in bladder carcinoma cell lines and the demethylation of5-aza-CdRObjective:To examine the methylation and demethylation of hepaCAM gene promoter in vitroMethods:T24and BIU-87cell lines were treated with different concentrations of5-aza-CdR for different times and divided into three groups:DMSO group, blank group and5-aza-CdR group; MSP determined the methylation status of hepaCAM gene promoter in these groups; Western-blot and Immunofluorescence measured the re-expression of hepaCAM protein in5-aza-CdR group cells; FCM examined the distribution of cell cycle in T24and BIU-87cell lines with drug treatment.Results:The result of MSP revealed that hepaCAM gene promoter was methylated in T24and BIU-87cell lines;5-aza-CdR can apparently reverse the methylation of hepaCAM. The best time and concentration of 5-aza-CdR used in experiment was:3uM for72h in T24and5uM for72h win BIU-87cell. The cell population of G0/G1in cells with5-aza-CdRtreatment was significantly higher than cells without5-aza-CdR treatment (p<0.05). The results of western-blot and IF showed that5-aza-CdR can reverse the expression of hepaCAM protein. And hepaCAM protein was expressed at cell membrane and around the Cell nucleus.Conclusion:HepaCAM gene promoter was hyper-methylated in T24and BIU-87cell lines, and its hypermethylation can be reversed by5-aza-CdR. HepaCAM gene was re-expressed to regulate cell cycle distribution in bladder carcinoma. PART THREE The correlation between hypermethylation of hepaCAM gene promoter region and the expression of hepaCAM protein in bladder carcinomaObjective:To study the correlation between the methylation status of hepaCAM promoter and its expression in bladder carcinoma.Methods:MSP detected the methylation status of hepaCAM promoter region in30paired bladder carcinoma specimens and corresponding adjacent tissues. Western-blot measured the expression of hepaCAM protein in30paired patients.Results:The result of MSP demonstrated that hypermethylation of hepaCAM promoter region in83.3%(25of30) bladder carcinoma samples was higher than in33.3%(10of30) corresponding adjacent samples (p<0.05). HepaCAM protein was expressed at mostly adjacent normal tissues, and only a few tumor tissues. Spearman analysis showed that the correlation between the methylation of hepaCAM promoter and the expression of its protein in bladder carcinoma was negative (r=0.-894, p=0.000). Chi square test revealed that there was no correlation between the methylation of hepaCAM promoter and the Clinical pathological parameters.Conclusion:The hypermethylation of hepaCAM promoter region may be due to the silencing of hepaCAM gene in bladder carcinoma. And that may be a new research hot spot for gene therapy.