| [Backgroud]Bladder cancer is one of the most common urological tumors in the world.75% of patients present with superficial tumors (stage Tis, Ta, and T1) and 25% of patients present with invasive tumors (stage T2-T4) at the time of first diagnosis. The major treatments for superficial cases include complete transurethral resection and intravesical chemotherapy to prevent recurrence after complete transurethral resection. As for muscle-invasive bladder cancer, the primary treatment is the combined approach of radical cystectomy and adjuvant systemic chemotherapy. Although current doctors has owned more and more advanced therapeutic strategies, 50%-70% of superficial cases will suffer recurrent and patients with invasive tumors are at a high risk for metastases. The 5 year survival rate of patients with invasive tumors is just 14-30%. What is more, chemoresistance to traditional chemotherapy agents present limited treatment options for doctors. Therefore, it is necessary to develop novel therapeutic strategies to improve the treatment and reverse chemoresistance of bladder cancer.It is known that tumorigenesis is a multi-step process that involves accumulation and alteration of both genetic and epigenetic modifications. Epigenetic modification is inheritable, yet reversible change, referring to change gene phenotype where sequence of the genome is not altered. Epigenetic modification include DNA methylation, histone modification andchromatin remodeling. DNA methylation play an important role in multistage tumorigenesis, progression and chemoresistance. DNA methylation is catalyzed by DNA methyltransferases (DNMTs) that transfer the methyl group from the donor S-adenosyl methionine (SAM) to the fifth carbon of cytosine to form 5-methylcytosine (5mC). DNA methylation frequently occurs in CpG islands at 5’promoter region of gene. It is known that the promotor hypermethlation of tumor suppressor genes which lead to downregulation of tumor suppressor genes, play a crucial role in carcinogenesis. Recent studies have also established that promoter hypermethylation is closely related to drug resistance in tumor chemotherapy.DNA methylation is catalyzed by the family of DNMTs. The mammalian DNMT family includes three active members:DNMT1, DNMT3a and DNMT3b. DNMT1 plays an important role in the maintenance of methylation, whereas DNMT3a and DNMT3b are responsible for an initial setup of methylation patterns for developing genomes. DNMT3b play the leading role in de novo methylation. The overexpression levels of the DNMT3b have been reportedly elevated in various cancers including bladder cancer and were closely related to carcinogenesis and drug resistance in tumor chemotherapy. DNA methylation is different from genetic mutation, which causes suppression of gene achieved by inactivating the transcription. This kind of epigenetic change is reversible. Thus, it suggests that inhibition of DNMT3b may be a promising strategy for cancer therapy and reversal of drug resistance as it can restore the aberrant methylation status to normal. It has been demonstrated that DNMT3b knockdown inhibited tumor growth and reverse chemoresistance in breast and liver cancer. However, whether DNMT3b expression could affect tumor growth and chemosensitivity has yet to be evaluated.Thus, to investigate the potential role of DNMT3b knockdown in the tumor growth and chemoresistance of bladder cancer:1) we investigate DNMT3b knockdown in T24,5367 and BIU87 cell lines to exam whether epigenetic therapy can inhibit cell growth and induce cell apoptosis. Then the promoter methylation status and the expression level of cell growth and apoptosis related genes were assessed.2) We investigate DNMT3b knockdown in wild-type BIU-87 adriamycin-resistant BIU-87/ADM cell lines to exam whether epigenetic therapy can enhance chemosensitivity. Then the promoter methylation status and the expression level of chemoresistance related genes were assessed. The aim of our study is to develop a novel therapeutic strategy—DNMT3b, to improve the survival rate in bladder cancer.[Objective]1To investigate the effect of DNMT3b knockdown on the proliferation and apoptosis of T24、5637 and BIU87 bladder cancer cells. Then observe the relationships between DNMT3b knockdowns and the methylation status and expression of RASSF1A, DAPK, Bax and Bcl-2. And finally investige the effect of DNMT3b knockdown on bladder cancer cell lines biological behaviour change in the molecular biology mechanism.2 To investigate the effect of DNMT3b knockdown on Adriamycin resistance of bladder cancer wild cell line BIU-87 and BIU-87 Adriamycin resistance cell line (BIU-87/ADM). Then observe the relationships between DNMT3b knockdown and the methylation status and expression of RASSF1A and hMLHl. And finally investige the effect of DNMT3b knockdown on bladder cancer cell lines chemosensitivity change in the molecular biology mechanism.[Method]1 The effect of DNMT3b knockdown on cell proliferation and apoptosis of bladder cancer cell in vitro and in vivo1.1 Cell Culture and siRNA TransfectionThe bladder cancer T24,5637 and BIU-87 cell lines were routinely cultured in RPM1640 medium. The siRNA oligonucleotide sequence of DNMT3b (siRNA-DNMT3b) and negative control siRNA oligonucleotide (siRNA-NC) were designed. Then siRNA-DNMT3b and siRNA-NC were transfected into these bladder cancer cell lines.1.2 Quantitative RT-PCR AssaysQuantitative RT-PCR was performed to detect the expression of DNMT3b in transfected T24,5637 and BIU-87 cell lines and the expression of the genes DAPK, RASSF1 A, Bax and Bcl-2 in BIU-87 cell.1.3 Western Blot AnalysisWestern blot was performed to detect the expression of DNMT3b in transfected T24,5637 and BIU-87 cell lines and the expression of the genes DAPK, RASSF1A, Bax and Bcl-2 in BIU-87 cell.1.4 Methylation-specific PCR AssaysMethylation-specific PCR was performed to detect the expression of DNMT3b in BIU-87 cell lines and the expression of the genes DAPK, RASSF1A, Bax and Bcl-2 in BIU-87 cell.1.5 Cell Proliferation AssayThe proliferations of T24,5637, and BIU87 cells were measured using the MTT assay. The absorbance at 490 nm was then measured for each well by a Microplate Autoreader.1.6 Flow Cytometry Analysis of Cell ApoptosisAnnexin-V-FITC/PI double staining assay was used to detect cell apoptosis rates of transfected T24,5637 and BIU87 cells. The production of reaction was flow cytometrically analyzed.1.7 Lentivirus (LV)-Mediated Short Hairpin RNA (shRNA) Knockdown of DNMT3b ExpressionThe pGLV-H1-GFP+Puro LV particles targeting the human DNMT3b gene and nonsilencing pGLV-Hl-GFP+Puro control LV particles with viral titers of approximately 109 were designed. BIU87 cell was infected with LV-shRNA-DNMT3b or LV-shRNA-NC. The infection efficiency was determined through the measurement of the expression of green fluorescent protein with fluorescence microscopy 48 hours after infection. The stable cells (BIU-87-shRNA and BIU-87-shNC) were maintained in puromycin.1.8 In vivo experimentEach nude mouse was injected subcutaneously in the right flank with the BIU-87-shRNA and BIU-87-shNC cells. When the tumor became visible, the tumor volumes in each group were measured every 3-4 days with a micrometer caliper. Tumor growth was followed for 28 days from the first injection. Then the mice were killed and the tumors were excised to take photos.2 The effect of DNMT3b knockdown on Adriamycin resistance of bladder cancer cell in vitro and in vivo2.1 Cell Culture and siRNA TransfectionThe bladder cancer BIU-87 and BIU-87/ADM cell lines were routinely cultured in RPM1640 medium. Then siRNA-DNMT3b and siRNA-NC were transfected into these bladder cancer cell lines.2.2 Quantitative RT-PCR AssaysQuantitative RT-PCR was performed to detect the expression of DNMT3b, RASSF1A and hMLH1 in transfected BIU-87 and BIU-87/ADM cell lines.2.3 Western Blot AnalysisWestern blot was performed to detect the expression of DNMT3b> RASSF1A and hMLHl in transfected BIU-87 and BIU-87/ADM cell lines.2.4 Methylation-specific PCR AssaysMethylation-specific PCR was performed to detect the expression of RASSF1A and hMLHl in BIU-87 and BIU-87/ADM cell lines.2.5 Adriamycin Drug TreatmentTransfected wild-type BIU87and drug resistant BIU87/ADM cell lines were seeded in 96-well plates. Then these cells were treated with different concentrations of Adriamycin for 72h and cell viability was measured using MTT assay. IC50 values were taken to be the concentration of sample required to inhibit 50% of cell proliferation, and were calculated at both cell.2.6 Flow Cytometry Analysis of Cell Apoptosis induced by AdriamycinAnnexin-V-FITC/PI double staining assay was used to detect the apoptosis rates of transfected BIU-87 and BIU-87/ADM cells followed by treatment with different concentrations of Adriamycin for 24h. The production of reaction was flow cytometrically analyzed.2.7 Lentivirus (LV)-Mediated Short Hairpin RNA (shRNA) Knockdown of DNMT3b ExpressionBIU-87-shRNA cell and BIU-87-shNC cell were constructed and maintained in the part one of this study. BIU-87/ADM cell which express stably DNMT3b siRNA (BIU-87/ADM-shRNA) and the negative control cell (BIU-87/ADM-shNC) were constructed using the same method.2.8 In vivo experimentEach nude mouse was injected subcutaneously in the right flank with the BIU-87-shRNA, BIU-87-shNC, BIU-87/ADM-shRNA and BIU-87/ADM-shNC cells followed by treatment with Adriamycin (5mg/kg) injected in the abdominal cavity every three days. When the tumor became visible, the tumor volumes in each group were measured every 3-4 days with a micrometer caliper. Tumor growth was followed for 28 days from the first injection. Then the mice were killed and the tumors were excised to take photos.3 Statistical AnalysisThe results are expressed as the mean standard deviation. All statistical evaluations were performed with SPSS 18.0 (SPSS, Inc., Chicago, IL). Results were analyzed using Student’s t-test and One-way ANOVA to assess statistical significance, respectively, with values of P.05 considered statistically significant.[Results]1 The effect of DNMT3b knockdown on cell proliferation and apoptosis of bladder cancer cell in vitro and in vivo1.1 Effect of siRNA-DNMT3b on Expression of DNMT3b Gene in Bladder Cancer Cells linesThe mRNA and protein expression levels were determined quantitatively using RT-PCR and Western blot analyses, respectively. The expressions of DNMT3b mRNA and protein in T24,5637 and BIU-87 cells infected with siRNA-DNMT3b were significantly decreased, compared with cells infected with siRNA-NC (P<0.01). These results demonstrated that siRNA-DNMT3b specifically reduced DNMT3b mRNA as well as protein expression.1.2 DNMT3b knockdown suppress cell Proliferation of T24,5637 and BIU87 cellsThe cell activity at 0,24,48, and 72 hours after DNMT3b knockdown was determined using the MTT assay. The growth curves showed significant proliferation reduction in T24,5637 and BIU87 cells at 48 and 72h which had been transfected with siRNA-DNMT3b, compared with the siRNA-NC group. These results demonstrated that DNMT3b knockdown can suppress cell Proliferation of T24,5637 and BIU87 Cells.1.3 DNMT3b knockdown induce cell apoptosis of T24,5637 and BIU87 cellsThe cell apoptosis after DNMT3b knockdown was determined using the flow cytometry analysis. The apoptosis rates of T24-siRNA and T24-NC group were 28.5%±2.3% and 12.6%±1.9% respectively (P＜0.01). The apoptosis rates of 5637-siRNA and 5637-NC group were 24.6%±1.9% and 12.3%±1.8% respectively (P<0.01). The apoptosis rates of BIU-87-siRNA and BIU-87-NC group were 32.0%±2.2% and 12.7%±2.0% respectively (P<0.01). These results indicated that downregulation of DNMT3b can promote apoptosis at T24,5637 and BIU-87 cell line.1.4 Effect of DNMT3b knockdown on the Expression of relevant genesThe expression of the mRNA and protein of DAPK, Bax, and RASSFIA were found to be increased in BIU87 cells which had been transfected with siRNA-DNMT3b, compared with the siRNA-NC group. By contrast, the expression of the mRNA and protein of BCL2 was reduced in cells transfected with specific siRNA. These results demonstrated that DNMT3b knockdown can upregulation of DAPK, Bax, and RASSF1A and downregulation of Bcl-2.1.5 Effect of DNMT3b knockdown on the methylation status of relevant genesFollowing transfection of siRNA-DNMT3b in BIU-87 cell, DAPK, Bax, BCL2, and RASSF1A genes lost methylation marks, showing that the high-level methylation at these loci can in part be reversed. These results indicated that DNMT3b knockdown led to upregulation of RASSF1A, DAPK and Bax correlated with demethylation of these genes. However, the mechanism of downregulation of Bcl-2 remains to be further investigated in the future.1.6 Effect of DNMT3b stable knockdown to Reduce Tumor Growth in Human Bladder cancer Xenograft ModelsThe results showed that both of infected cells express fluorescent cells under a fluorescence microscope. The results of Real time PCR and western blot showed that DNMT3b mRNA and protein level were stable knockdown in BIU-87 cell.Stable cell lines were inoculated into mice. BIU-87 cells in which DNMT3b was silenced exhibited significantly reduced tumor growth in comparison with. LV-NC in day 20 to day 25 (P＜0.01). Tumors were removed and taken photographs in the end of the feeding examination. The tumors of BIU-87-shRNA group were significantly smaller than BIU-87-shNC group. These results suggest that DMNT3b stable inhibition can effectively induce tumor growth in an in vivo human bladder cancer environment.2 The effect of DNMT3b knockdown on Adriamycin resistance of bladder cancer cell in vitro and in vivo2.1 Effect of siRNA-DNMT3b on Expression of DNMT3b Gene in BIU-87 and BIU-87/ADM Cell linesThe mRNA and protein expression levels were determined quantitatively using RT-PCR and Western blot analyses, respectively. The expressions of DNMT3b mRNA and protein in BIU-87 and BIU-87/ADM cells infected with siRNA-DNMT3b were significantly decreased, compared with cells infected with siRNA-NC (P<0.01). These results demonstrated that siRNA-DN,MT3b specifically reduced DNMT3b mRNA as well as protein expression.2.2 Effect of DNMT3b knockdown on Drug resistance of bladder cancer cells to AdriamycinAfter treatment of different concentrations of adriamycin (0,0.0125,0.025, 0.05,0.1 and 0.2 mg/L) in transfected BIU87 cell for 72h, MTT assays were performed in these cells. Our results showed that significant inhibition of proliferation had been found in BIU-87 cells transfected with siRNA-DNMT3b versus those with siRNA-NC in the concentrations of adriamycin of 0.025,0.05, 0.1 mg/L. However, no differences were observed in the concentrations of adriamycin of 0.0125 and 0.2 mg/L.Similarly, MTT assays were performed in transfected BIU87/ADM cells after treatment of different concentrations of adriamycin (0,0.5,1,2,4 ' 8 mg/L). Our date showed that significant inhibition of proliferation had been found in BIU-87/ADM cells transfected with siRNA-DNMT3b versus those with siRNA-NC in the concentrations of adriamycin of 1,2,4 and 8 mg/L. However, no difference was observed in the concentrations of adriamycin of 0.5 mg/L.The IC50 of BIU87 cell was respectively 0.18 and 0.14 mg/L in negative control group and experimental transfected group. No difference was observed in the IC50 of BIU87 cell between negative control group and experimental transfected group. The IC50 of BIU87/ADM cell was respectively 13.96 and 7.10 mg/L in negative control group and experimental transfected group. The IC50 of BIU87/ADM cell was remarkably reduced after knockdown of DNMT3b, compared with the negative control group. These results indicated that downregulation of DNMT3b could reserve the drug resistance of BIU-87/ADM cell to Adriamycin in vitro.2.3 Effect of DNMT3b knockdown on adriamycin-induced Apoptosis of wild-type and adriamycin resistance Bladder Cancer cellsAfter BIU87 cell had been transfected with siRNA-DNMT3b or siRNA-NC, adriamycin (0.05mg/L) was administrated into BIU87 cell for 24h. Similarly, BIU87/ADM cell was transfected with siRNA-DNMT3b or siRNA-NC followed by adriamycin treatment (2 mg/L) for 24h. The apoptosis rates of BIU-87-siRNA and BIU-87-NC group were 27.84%±1.57% and 14.54%±2.12% respectively (P＜0.01). The apoptosis rates of BIU-87/ADM-siRNA and BIU-87/ADM-NC group were 25.68%±2.20% and 12.76%±1.39% respectively (P<0.01).These results indicated that combination therapy of siRNA-DNMT3b and adriamycin could promote apoptosis at BIU87 and BIU87/AMD cell lines.2.4 Effect of DNMT3b knockdown on the Expression of relevant mRNA and protein contentsDNMT3b knockdown increased the expression of RASSF1A in both BIU87 and BIU87/ADM cells, consistent with the results of cell proliferation and cell apoptosis detection. However, the expression level of hMLHl in BIU87/ADM cell was significantly higher than BIU87 after DNMT3b knockdown, coincident with the reversal of drug resistance. These results indicated that the increased expression level of hMLHl in BIU87/ADM cells might be related to reversal of chemoresistance to Adriamycin.2.5 Effect of DNMT3b knockdown on methylation status of relevant genes promotorFollowing transfection of siRNA-DNMT3b, RASSF1A gene in both BIU87 and BIU87/ADM cell and hMLHl gene in BIU87/ADM cell lost methylation marks, showing that the high-level methylation at these loci can in part be reversed. However, no significant difference was observed in the methylation status in hMLHl gene in BIU87 cell after DNMT3b knockdown. These results indicated that DNMT3b knockdown led to upregulation of RASSF1A in both BIU87 and BIU87/ADM cell and hMLHl gene in BIU87/ADM cell correlated with demethylation of these genes.2.6 Effect of combined treatment of DNMT3b stable knockdown and Adriamycin to Reduce Tumor Growth in Human Bladder cancer Xenograft ModelsThe results showed that both of infected cells express fluorescent cells under a fluorescence microscope. The results of Real time PCR and western blot showed that DNMT3b mRNA and protein level were stable knockdown in BIU-87 and BIU-87/ADM cells.Stable cell lines were inoculated into mice followed by treatment with Adriamycin (5mg/kg) injected in the abdominal cavity every three days. BIU-87 and BIU-87/ADM cells in which DNMT3b was silenced exhibited significantly reduced tumor growth in comparison with LV-NC in day 20 to day 25 (P<0.01).Tumors were removed and taken photographs in the end of the feeding examination. The tumors of BIU-87-shRNA and BIU-87/ADM-shRNA group were significantly smaller than BIU-87-shNC and BIU-87/ADM-shNC group. These results suggest that combined treatment of DNMT stable knockdown and Adriamycin can effectively induce tumor growth in an in vivo human bladder cancer environment.[Conclusion]1 DNMT3b knockdown can affect the methylation status of DAPK, Bax, Bcl-2 and RASSFIA to regulate their expression, which might be a possible mechanism for suppressed cell growth andenhanced apoptosis of T24,5637 and BIU-87 cells2 DNMT3b knockdown can reverse Adriamycin resistance of the bladder cancer cell line BIU-87/ADM via affecting the expression of hMLH1. |
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