Research On The Transcriptional Expression And Promoter Methylation Of RASSF1A In Bladder Cancer

PrefaceTransitional cell carcinoma of bladder is known as the most common urological malignant tumor in China, with high incidence of recurrence of superficial bladder cancer and poor prognosis of invasive or end-stage bladder cancer. Despite the progressing surgical techniques and chemotherapy, the therapeutic effect of bladder cancer remains unsatisfactory. Thus, improvement of prognosis of bladder cancer patients entails a deeper understanding of its etiology and mechanism of carcinogenisis. Gene expression alteration caused by DNA methylation is considered the main mechanism for carcinogenesis. Like gene deletion and mutation, DNA methylation is also a pathway for regulating gene expression. Methylation of promoter region can cause inactivation of tumor suppressor gene. Ras association domain family 1 Agene(RASSF1A) is a new candidate tumor suppressor gene cloned from 3p21.3 in 2000. Some studies have confirmed that RASSF1A is a common inactivated tumor suppressor gene. RASSF1A methylation occurs rarely in the normal tissue, but with a high frequency in cancer tissue more than 90%(liver cancer), the patient with precancer with RASSF1A methylation have been identified to deneloped into cancer, to characterize the alteration of RASSF1A methylation in the bladder cancer is of crucial theoretical and clinical application in understanding the mechanism of bladder cancer and establishing biomarker for high-risk subject screening and early diagnosis. In this study, we use semi-quantitative RT-PCR and methylation specific PCR to detect RASSF1A expression and promoter methylation in normal bladder tissue and bladder cancer, in the aim of determining the relationship between gene expression and biological behavior of bladder cancer. 5-Aza-CdR, a specific DNA methylation inhibitor, can reverse the expression of hyper-methylized tumor suppressor gene in CpG island. We treated BIU-87 cells with different concentration of 5-Aza-CdR and studied its effect on cellular apoptosis and RASSF1A expression, which may provide new clues for treating bladder cancer.Materials and methodsClinical materialsThe specimen included 30 samples (24 men, 6 women) of frozen bladder cancer tissue from cystecomy in urological department of sheng jing hospital from Jau, 2005 to August, 2006, and 10 samples of normal bladder tissue from patients who underwent prostatectomy. The age of patients ranged from 28 to 84 years (mean 55.6 years). The diagnosis was confirmed by pathological histology as transitional cell carcinoma of bladder. The stages of cancer were established on the criteria given in by UICC(2 cases of Ta, 10 cases of T1, 14 cases of T2, 2 cases of T3 and 2 cases of T4). In pathological grade, there were 8 cases of G1, 14 cases of G2 and 8 cases of G3. BIU-87 cell line was obtained from research institute of urology in Peking University.Methods1. RASSF 1AmRNA expression in bladder cancer: RNA extracted from 30 cases of bladder cancer tissue samples and 10 cases of normal bladder tissue samples. Semiquantitative RT-PCR was used to detect RASSF1AmRNA expression2. Methylation status of RASSF1A in bladder cancer: DNA was extracted from 30 cases of bladder cancer tissue samples and 10 cases of normal bladder tissue samples. Methylation specific PCR(MSP) was used to detect RASSF 1A promoter methylation in bladder cancer tissue and normal bladder tissue.3. BIU-87 cells were treated with different concentration of 5-Aza-CdR. AO fluorescent staining was used to observe morphology of cellular apoptosis. Cell proliferation was measured by MTT method. Effect of 5-Aza-CdR on BIU-87 cell apoptosis was measured by flow cytometry (AnnexinV-FITC-PI assay).4. BIU-87 cells were treated with different concentration of 5-Aza-CdR. We used semi-quantitative RT-PCR and methylation specific PCR(MSP) to determine the effect of 5-Aza-CdR on RASSF1A expression in BIU-87 cells. Protein expression of RASSF1A was measured by Western blot after being treated with different concentration of 5-Aza-CdR.Statistieeal analysisAll the data were processed by SPSS12.0 sol, ware. P -value of less than 0.05 was considered to be statistically significant.Results1. RASSF1A mRNA expression in bladder cancer: The expression of RASSF1A mRNA in normal bladder tissues was 9 out of 10, while that of transitional cell carcinoma was 13 out of 30. There was statistically significant difference between these two groups(P＜0.05). The expression of RASSF1A mRNA was not statistically associated with pathological stages(P＜0.05), but there was significant difference between superficial bladder cancer (12/12) and invasive bladder cancer(1 / 18) (P＜0.05).2. RASSF1A methylation in bladder cancer: The prevalence of methylation in normal bladder tissues was 1 out of 10, while that of transitional cell carcinoma was 21 out of 30. There was statistically significant difference between these two groups(P＜0.05). The prevalence of methylation of 5'CpG island in RASSF1A promoter was not statistically associated with pathological stages(p＞0.05), but there was significant difference between superficial bladder cancer (4/12) and invasive bladder cancer(17/18) (P＜0.05). The expression of RASSF1A mRNA was not statistically associated with pathological stages (p＞0.05), but there was significant difference between superficial bladder cancer and invasive bladder cancer(P＜0.05).3. After being treated with different concentration of 5-Aza-CdR, the proliferation of BIU87 cells was inhibited. Increased concentration result in increased inhibition, lower cellular survival rate and higher apoptosis rate. There was significant difference between treated group and control group (P＜0.05). Cellular apoptosis was dosagedependent on the concentration of 5-Aza-CdR(P＜0.05). After AO fluorescent staining, some cells in the 5-Aza-CdR treated group manifest characteristic morphological changes of apoptosis, with chromosome margination, karyopyknosis and breakage into numerous yellow-greenish strong-fluorescent granules4. Using GAPDH as the interior reference. Expression of RASSF1A mRNA was not detected by RT-PCR in the control group. After being treated with 0.5μmol/L, 1μmol/L, 5μmol/L 5-Aza-CdR respectively, RASSF1A mRNA was re-expressed, the relative amount being dosage-dependent on 5-Aza-CdR concentration, 0.73±0.05, 1.39±0.03, 1.76±0.02, respectively(P＜0.05). RASSF1A methylation of BIU-87 cells was also reversed after being treated with 5-Aza-CdR. After being treated with 0.5μmol/L,1μmol/L,5μmol/L 5-Aza-CdR respectively, the RASSF1A protein was reexpression in each group, while the control group was not expressed. RASSF1A protein was re-expressed, the relative amount being dosage-dependent on 5-Aza-CdR. concentration, 0.49±0.01, 0.99±0.05, 1.22±0.07, respectively(P＜0.05).ConclusionsAbnormal methylation of RASSF1A promoter result in the down-regulation of expression of RASSF1A mRNA. Inactivation of RASSF1A gene might be the main mechanism for carcinogenesis in bladder cancer. In vitro studies indicate that 5-AzaCdR could inhibit proliferation of tumor cells through the re-expressing RASSF1A gene, and thus induce apoptosis of BIU-87 cells in bladder cancer. Down-regulation of RASSF 1A mRNA in BIU-87 cells was caused by hyper-methylation of promoter region. RASSF1A gene can inhibit the proliferation of tumor cells, which might participate in the carcinogenesis of bladder cancer.