Aberrent Methylation Of RASSF1A Gene And Regulation Of MicroRNA In Bladder Cancer Recurrence Research

Part OneAnalysis of Risk Factors of Bladder Cancer RecurrenceObjectives:To evaluate the recurrence rate of bladder cancer patients after bladder-sparing surgery and to find the risk factors.Materials and Methods:452patients underwent bladder-sparing surgery (partial cystectomy or transurethral resection) and were confirmed as urothelial carcinoma pathologically. All cases were followed up for2years and the clinical pathological characteristics were collected. All cases were divided to three groups according to the recurrence frequency:no-recurrence group(NR), low-recurrence group(LR) which means recurrent only once during first2postoperative years, high-recurrence group(HR) which means recurrent more than twice. The clinical pathological datas were compared among the three groups. All the recurrent low-grade cases were divided into2groups:stable group (tumor grades remained low during first2postoperative years) and progress group(tumor grades elevated). The clinical pathological datas of the2groups were compared.Results:Total recurrence rate was45.1%(204/452). According to the recurrence frequency, NR group248cases, LR group127cases and HR group77cases. Significant differences were found among these three groups in smoking history, tumour grading, tumour staging, tumor quantity and regular intravesicle chemotherapy, while patient age, gender, intravenous chemotherapy, radiation therapy and BCG therapy were not different significantly. Concerning the110recurrent low grade cases,19were in stable group and31were in progress group. Significant difference were found in smoking history and tumor staging.Conclusions:Smoking history, tumor grading, staging, tumour quantity and regular intravesicle chemotherapy are risk factors of bladder cancer recurrence. Smoking and tumour staging are risk factors of tumour grade elevation. Part TwoRASSF1A gene methylation in human bladder cancer cell linesObjectives:To evaluate RASSF1A gene methylation status in T24human bladder cancer cell lines and its relationship of tumor cell proliferation and invasion.Materials and Methods:T24cell lines were treated with DNA methyltransferase inhibitor5-aza-dc of different concentration (0.1,2.5,12.5u mol/1, defined as low-dose group, medium-dose group and high-dose group). The cell proliferation inhibitor rates were detected by MTT assay, apoptosis rate and cell cycle changes were tested by flow cytometry and cell invasion rates were detected by transwell assay. RT-PCR and Western-blot were used to detect RASSF1A expression. Methylation specific PCR(MSP) was performed to detect the methylation status of RASSF1A gene in each group.Result:Cell proliferation inhibitor rate of low, medium and high dose group were7.621,8.297and8.478respectively. Apoptosis rate of null group, control group, low, medium and high dose group were2.5%,2.9%,4.2%,5.7%and13.8%respectively. No significant changes were found in cell cycle distribution. Cells treated with5-aza-dc showed lower invasive power than control or null group and these changes were dose-related. Using MSP assay, we found RASSF1A gene completely methylated in null and control group, partial methylated in low-dose group and unmethylated in medium and high dose group. Treated with5-aza-dc, RASSf1A gene expression were uploaded and the changes were also dose-related.Conclusion:RASSF1A gene is completely methylated in T24bladder cancer cell lines. DNA methyltransferase inhibitor5-aza-dc can inhibit the methylation of this gene and lead to gene expression uploaded. As a result the cell proliferation and invasion are suppressed and apoptosis are increased. Part ThreeCell proliferation and invasion in microRNA transfection cells and its relationship of RASSF1A methylationObjective:To evaluate the relationship among bladder cancer cell proliferation and invasion, microRNA-143and29a, and RASSFIA methylation.Materials and methods:MiR-143and miR-29a mimics were transfected to T24cell lines. Transfection efficiency was confirmed by RT-PCR. All cell lines were divided into four groups:WT group(wild T24cell lines without any treatment), NC group(transfected by negative control mimics), M1group(transfected by miR-143mimics) and M2group(transfected by miR-29a mimics). Apoptosis rates, cell cycle, cell invasive inhibitor rates, RASSF1A gene expression and methylation were detected.Results:Transfection was successful confirmed by RT-PCR. Apoptosis rates of WT, NC, Ml and M2group were2.5%,3.4%,12.4%and13.3%respectively. S-phase cells in M1and M2group increased significantly. Cell invasive inhibitor rates of NC, Ml and M2group were0.020,0.165and0.132respectively which showed the rates in M1and M2group were higher than NC group(P<0.05). RASSFIA gene expression was upgraded in M2group and no changes were found in M1group. What’s more, MSP results showed that RASSFIA gene was partial methylated in M2group while it was completely methylated in other groups. Conclusion:T24bladder cancer cell proliferation and invasion decreased after miR-143and miR-29a transfected. miR-29a transfection may inhibit RASSFIA gene methylation and lead to gene expression uploaded.Part FourRASSFIA methylation and microRNA expression in human bladder cancer tissues of different recurrence frequencyObjectives:To evaluate RASSFIA methylation status and miR-143and29a expression in human bladder cancer tissues and its relationship of tumor recurrence.Materials and methods:30patients underwent partial cystectomy in our centre from 2008to2009. All patients were devided into three groups according to different recurrence frequency(refer to part1). RASSF1A methylation status of each group was tested by MSP assay and miR-3p-143, miR-3p-29a and miR-5p-29a were tested by RT-PCR.Results:No significant difference was found in gender, age, tumor quantity and staging, while more high grade tumors were found in high recurrence group. The rates of complete or partial methylation of RASSF1A in NR, LR and HR group were40%,80%and100%. MiR-3p-143of each group were122.68,3.49and0.77. MiR-3p-29a of each group were246.99,65.60and62.25respectively. MiR-5p-29a of each group were11.30,3.65and2.20. Statistical analysis showed that miR-3p-143was different significantly between every two groups. MiR-3p-29a and MiR-5p-29a were higher in NR group than other two groups, while no significant difference was found between other two groups. In the group of non-methylation but recurrent and methylation but no-recurrent, miR-3p-143were99.98+44.90vs5.57+1.73respectively and difference was significant statistically; however, miR-3p-29a and miR-5p-29a were not significantly different between the above two groups.Conclusions:RASSF1A gene methylation status and miR-143, miR-29a expression have strong relationship with bladder tumor recurrence. Combined testing RASSF1A methylation status and miR-143might increase the accurancy of predicting bladder tumor recurrence.