5-azacytidine Inhibite Proliferation Of Bladder Cancer Through Reverse Hypermethylation Of Hepacam Gene Promoter

Part I Construction And Identification Of hepaCAM GeneAdenovirusObjective：To construct hepaCAM gene recombinant adenovirus andidentify its expression in bladder cancer BIU-87cells.Method：HepaCAM gene fragment was cloned into the shuttle plasmidpAdtrack-CMV in order to construct the recombinant plasmidpAdtrack-CMV–hepaCAM, which homologously recombinated with theadenoviral backbone vector pAdeasy-1in BJ5183bacterial cells forrecombinant adenoviral plasmids pAdEasyI-hepaCAM. Then,transfect therecombinant adenoviral DNA into HEK-293cells for generating hepaCAMadenoviruses which was used for infect bladder cancer BIU-87cells.Subsequently the expression of hepaCAM in infected bladder cancerBIU-87cells was tested by real-time quantitative PCR and western blot.Results：HepaCAM gene sucessfully cloned and reconbinant adnoviralplasmid constructed wich conformed by restriction endonuclease and PCR. Bladder cancer cells expressed hepaCAM gene obviously after infection.Conclusion：HepaCAM gene adenovirus constructed successfully andthe expression of hepaCAM gene in bladder cancer BIU-87cells wasconfirmed. Part II5-azacytidine Inhibite Proliferation Of Bladder CancerThrough Reverse Hypermethylation of hepaCAM Gene PromoterObjective：To detect methylation status of hepaCAM gene in bladdercancer cells and investigate the effectiveness of azac on hepaCAMmethylation and proliferation of bladder cancer cells.Methods：We predicted a CpG island existing in hepaCAM promoterthrough an online database and design methprimer to detect mythylationstatus of hepaCAM gene in bladder cancer cells; RT-PCR and Western blotdetected the expression of hepaCAM and dnmt3a/3b after azac treatment;Immunofluorescence measured the expression of dnmt3a/3b protein;CCK-8assay and colony formation determined the proliferation of bladdercancer cells; FCM examined distribution of cancer cells treated with azac.Results：MSP showed that hepaCAM gene was hypermethylated inthree bladder cancer cell lines. Its hypermethylation can be reversed aftermethylation inhibitor azac treatment. Furthermore, azac can re-express hepaCAM and downregulate dnmt3a/3b in bladder cancercells.Re-expression of hepaCAM can inhibite bladder cancer cells growth.Conclusion: Our results suggest that absence or loss of hepaCAM genein human bladder cancer is associated with abnormal methylation of itspromoter CpG island. DNA methylation is a common mechanism ininactivating the hepaCAM tumor suppressor gene in human bladder cancercells. Part III Effectiveness Of5-azacytidine On Bladder Cancer Growth InVivoObjective: To study the effect of azac on human bladder cancerxenografts growth and expression of hepaCAM and dnmt3a/3b.Method:3x106viable EJ cells resuspend with PBS were injected intoright flanks of ten male nude mice.2weeks later, mice were randomized intotwo groups: Group A (blank group); group B (azac group). Azac wasinjected into mice in grop B and DMSO in group A every three days. Tumorsize and weight measured everyday, then mice were sacrificed3weeks laterand tumors were weighted after necropsy. Histopathology analysis usedH&E staining and expression of hepaCAM and dnmt3a/3b detected by IHC.Results: Compared with blank group, tumor in azac group were smaller and lighter, they were considered significant that p value was lessthan0.05. IHC results showed that expression of hepaCAM is upregulatedand dnmt3a/3b are downregulated in azac group.Conclusion: In vivo, azac also can inhibite bladder cancer growth andre-express hepaCAM and downregulate dnmt3a/3b. The results wereaccordanced with in vitro.