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@#[摘 要] 目的：探讨EB病毒核抗原1（EBNA1） mRNA修饰的DC（EBNA1-DC）诱导的淋巴细胞联合甲基化抑制剂5-Aza-CdR对鼻咽癌C666-1细胞的杀伤作用。方法：以构建的EBNA1-pCDNA3.1质粒为模板，体外转录获得EBNA1 mRNA，通过脂质体转染至健康人外周血来源DC，构建EBNA1-DC疫苗。流式细胞术检测转染后DC表型及5-Aza-CdR处理后的C666-1细胞凋亡情况。实时无标记动态细胞分析技术检测EBNA1-DC疫苗诱导的淋巴细胞联合5-Aza-CdR的特异性抗肿瘤活性。结果：转染EBNA1 mRNA后EBNA1-DC表面EBNA1阳性率为（59.3±5.85）%，HLA-DR的表达与未转染DC相比显著升高[（84.9±5.5）% vs （68.0±5.8）%，P=0.026]，CD80的表达也显著升高[（88.2±3.9）% vs （61.1±4.4）%，P=0.015]。低剂量5-Aza-CdR处理后的C666-1细胞凋亡情况与未处理的细胞相比无显著差异。经低浓度5-Aza-CdR预处理的C666-1细胞中IRF7基因表达与未处理的细胞相比显著升高（P=0.000 1）。与空载的DC相比，EBNA1-DC诱导的淋巴细胞对EBV阳性表达的C666-1细胞具有更强的特异性杀伤活性（P=0.049）；经低浓度5-Aza-CdR预处理的C666-1细胞对EBNA1-DC诱导的特异性免疫杀伤更敏感（P=0.019）。结论：5-Aza-CdR与EBNA1-DC疫苗联合可显著增强对C666-1细胞的特异性免疫杀伤，本研究为开拓以mRNA为基础的DC疫苗及其在临床综合治疗中的应用转化提供前期研究基础。
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As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1-V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1-MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.
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Objective To investigate the relationship between the expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells,to observe the inhibitory effect of 5-Aza-CdR on the growth of gastric cancer cells,to observe the effect of demethylation on the expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells,and to explore the molecular mechanism of gastric cancer.Methods Real-time PCR was used to detect the expression of ASPP2 mRNA in two gastric cancer cells and normal gastric epithelial cells.BSP was used to detect the methylation of ASPP2 gene in two gastric cancer cells and normal gastric epithelial cells.CCK-8 was used to detect the growth inhibition rate of gastric cancer cells treated with 5-Aza-CdR of different concentrations,then they were used to detect expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells again after the demethylation.Results ① The expression of ASPP2 mRNA in MKN-45 cells was significantly lower than that in GES-1 cells(P＜0.01).The expression of ASPP2 mRNA in MGC-803 cells was significantly lower than that in GES-1 cells (P＜0.01).There was no significant difference in MGC-803 cells and MKN-45 cells(P＞0.05).② The methylation rate of ASPP2 in MKN-45 cells was significantly higher than that in GES-1 cells (P＜0.01).The methylation rate of ASPP2 in MGC-803 cells was not significantly different from that in GES-1 cells (P＞0.05).The methylation rate of ASPP2 in MKN-45 cells was significantly higher than that in MGC-803 cells (P＜0.01).③ At the same time,the growth inhibition rate of each 5-Aza-CdR concentration group increased as the drug concentration depended.4.The expression of ASPP2 mRNA in MKN-45 cells was significantly higher than that before treatment (P＜0.01),the expression of ASPP2 mRNA in MGC-803 cells was not significantly different from that before treatment(P＞0.05).The methylation rate of ASPP2 in MKN-45 cells was significantly lower than that before treatment (P＜0.01).The methylation rate of ASPP2 in MGC-803 cells was not significantly different from that before treatment (P＞0.05).Conclusion ① Abnormal hypermethylation of ASPP2 gene in MKN-45 cells may be a molecular mechanism of decreased ASPP2 mRNA expression.② 5-Aza-CdR can inhibit the growth of MKN-45 and MGC-803 cells,and it can enhance the expression of ASPP2 mRNA in MKN-45 cells.Reversal of methylation in the promoter region of ASPP2 gene is the possible mechanism.③ Abnormal hypermethylation of the promoter region of ASPP2 gene may lead to silencing of mRNA expression that may be associated with gastric cancer.
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Objective To investigate whether emodin combined with 5AzA-cdR can enhance the demethylation of tumor suppressor genes p16,RASSF1A and ppENK in nude mice with subcutaneously transplanted pancreatic cancer.Methods Pancreatic cancer cells Panc1 burdened subcutaneous xenograft nude mice model was established,which were randomly divided into control group,emodin group,5AzA-cdR group and emodin combined 5AzA-cdR group (combined group).The growth of transplanted tumors wasobserved in each group.Methylation specific PCR (MSP) was used to detect the methylation levels of p16,RASSF1A and ppENK in the xenograft tumor tissue among three groups.The mRNA and protein expression of three tumor suppressor genes were detected by FQ-PCR and Western blotting,respectively.Results The weight of xenografts in the control group,emodin group,5AzA-cdR group,and combination group were (0.28 ±0.01),(0.17 ± 0.01),(0.12 ± 0.02),(0.08 ± 0.01)g,respectively.The tumor volume was (517 ±0.02),(382 ± 0.01),(232 ± 0.03),(169 ± 0.01) mm3.The methylation levels of p16 were 1.00 ± 0.00,0.89 ± 0.02,0.63 ± 0.02,and 0.19 ± 0.01;the methylation levels of RASSF1A were 1.00 ± 0.00,0.88 ± 0.02,0.51 ± 0.01,and 0.32 ± 0.01;the methylation degree of ppENK was 1.00 ± 0.00,0.92 ± 0.02,0.77 ± 0.02 and 0.31 ± 0.01,respectively.The expression of p16 mRNA was 1.00 ± 0.00,1.71 ±0.02,2.67 ± 0.02,3.81 ± 0.01.The expression of RASSF1A mRNA was 1.00 ± 0.00,1.92 ±0.02,2.73 ± 0.03,3.77 ± 0.01.The expression of ppENK mRNA was 1.00 ± 0.00,1.69 ± 0.03,2.17 ± 0.02 and 4.28 ± 0.01.The expression of p16 protein was 1.00 ± 0.00,1.71 ± 0.02,2.67 ± 0.02,3.81 ± 0.01;the expression of RASSF1A protein was 1.00 ± 0.00,1.92 ± 0.02,2.73 ± 0.03.3.77 ± 0.01;ppENK protein expression levels were 1.00 ±0.00,1.69 ±0.03,2.17 ±0.02,4.28 ±0.01.The weight and volume of xenografts in the three treatment groups were significantly smaller than those in the control group.The methylation of three tumor suppressor genes was lower than that of the control group,and the expression of tumor suppressor mRNA and protein was all significantly higher than the control group,which the combination drug group was also significantly stronger than that in emodin group and 5AzA-cdR group,and the differences were statistically significant (P ＜ 0.05 or ＜ 0.01).Conclusions The combination of emodin and 5AzA-cdR can enhance the demethylation effect of 5A6A-cdR on the tumor suppressor genes p16,RASSF1A and ppENK in the tumor tissue of pancreatic cancer xenograft model.
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Objective To investigate the mechanism and cause of the inactivation of tumor suppressor gene RNF180 in prostate cancer cell line by observing the effect of 5-Aza-CdR on the RNF180 gene in prostate cancer cell line DU145. Methods MTT method was adopted to study the effect of 5-Aza-CdR(0,1,2,5,10,15 and 20μmoI/L)on the proliferation of prostate cancer cells. Western blotting,real-time PCR,and methyla-tion specific PCR(MSP)were separately used to detect the expression of RNF180 in prostate cancer cells before and after the treatment of the most suitable drug concentration(5μmoI/L). Results In a certain range,the effect of 5-Aza-CdR on the proliferation of prostate cancer cell line DU145 was increased with the increase of drug concentration and the time of drug treatment(P<0.05). After the treatment of the most suitable drug concentration,the protein and mRNA expression of RNF180 in prostate cancer cells was significantly increased(P<0.05),but the methyla-tion of the promoter region was obviously decreased. Conclusion 5-Aza-CdR can reverse the methylation status of RNF180 gene in DU145 pros-tate cancer cell line,and relieve the silencing status of RNF180gene expression.
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Objective To explore the function of 5-Aza-CdR in B-cell acute lymphocytic leukemia cell line NALM-6 and its influence on the expression of microRNA (miRNA) in the cells. Methods NALM-6 was treated with different concentrations of 5-Aza-CdR. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) test, and DNA methyltransferase (DNMT) mRNA expression level was detected by reverse transcription PCR (RT-PCR). The expression changes of miRNA were detected by miScript miRNA PCR Array chip in cells after methylation. Results NALM-6 cell growth was inhibited by different concentrations of 5-Aza-CdR processing time, reaching to the maximum inhibitory rate was (74.163 ±0.381) %. 5-Aza-CdR affected concentrations was inversely proportional with expression level of DNMT mRNA. After 1 000 μmol/L of 5-Aza-CdR was dealed with cell 72 h, the relative expression of DNMT-1 was reduced to 0.453 ±0.021, DNMT-3L was 0.003±0.001, DNMT-3B was 0.395±0.019. MiScript miRNA PCR array sieved out 3 miRNA (miR-184, miR-23a-3p, miR-34a-5p) associated with DNA methylation. Conclusions 5-Aza-CdR down regulates the expression of DNMT gene in NALM-6 cells, and inhibits the proliferation of cells. MiR-184, miR-23a-3p and miR-34a-5p are related to DNA methylation in the occurrence and development of B-cell acute lymphocytic leukemia.
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Objective To explore the effect of p21Waf1/Cip1 methylation changes on the process of cellular senescence .Methods Bisulfite sequencing was used to analyze the methylation changes of p21Waf1/Cip1 in the process of cellular senescence;p21Waf1/Cip1 ex‐pression was detected by RT‐PCR and Western‐blot ;Middle‐aged 2BS cells was treated by 5‐aza‐CdR and cellular senescence was detected by MTT and SA‐β‐Gal staining .Results Bisulfite sequencing analysis of p21Waf1/Cip1 promoter showed that CpGs were methylated by 1 .25% in the young 2BS cells ,by 27 .27% in the middle‐aged 2BS cells ,while only by 0 .64% in the senescent cells . The expression of p21Waf1/Cip1 was low in the young(28 PD) 2BS cells ,it increased first(35 PD) but decreased later in the middle‐aged(42 PD) cells .In the senescent 2BS cells ,p21Waf1/Cip1 expression was further increased .5‐aza‐CdR treatment resulted in de‐creased growth rate but increasedβ‐Gal staining of middle‐aged 2BS cells .Conclusion The process of cellular senescence is regula‐ted by the status of p21Waf1/Cip1 methylation ,and p21Waf1/Cip1 demethylation accelerates cellular senescence .
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Objective To discuss the effect of 5-Aza-CdR on pancreatic carcinoma Capan-2 cell proliferation and PCDH8 gene expression.Methods Pancreatic carcinoma Capan-2 cells were treated with different doses of 5-Aza-CdR with or without gemcitabine,negative control group without drug,0.08μmol/L group with 0.08μmol/L 5-Aza-CdR,0.40 μmol/L group with 0.40 μmol/L 5-Aza-CdR,2.00 μmol/L group with 2.00 μmol/L 5-Aza-CdR,10 μmol/L group with 10 μmol/L 5-Aza-CdR,50 μmol/L group with 50 μmol/L 5-Aza-CdR. The inhibition ratio of Capan-2 cell proliferation were observed by MTT assay and PCDH8 gene and protein expression were detected by RT-PCR and Western blot. Results The inhibition ratio was increased with 5-Aza-CdR dose increasing(P<0.01),but decreased apparently with times extending(P<0.01). Inhibition ratio in 5-Aza-CdR and gemcitabine group was higher than those with only 5-Aza-CdR or gemcitabine groups(P<0.05 or P<0.01).The levels of PCDH8 gene and protein expression were increased significantly in 5-Aza-CdR treatment groups,with dose dependent (P <0.01 ).Conclusion 5-Aza-CdR can inhibit the proliferation of pancreatic carcinoma Capan-2 cell proliferation,and increase PCDH8 gene expression. The inhibition effect is strong when combined with gemcitabine.
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Aim To investigate the mechanism of demethylation on adenosine (ADO )and homocysteine (HCY)-induced apoptosis in human hepatoma HepG2 cells .Methods HepG 2 cells were treated with differ-ent concentrations of ADO (1.0、2.0、4.0 mol · L-1 ) alone or in combination with HCY for 6h,12h and 24h,5-aza-2-deoxycytidine (5-Aza-CdR)as a positive control.Cell viabilities were assessed by CCK8 assay. Cell apoptosis was observed by AnnexinV-FITC/PI staining.The mitochondrial membrane potentials(ΔΨ) were measured by flow cytometry.The mRNA and pro-tein expressions of caspase-3,caspase-8,caspase-9, MDM-2,p53,Cytochrome C,DNMT1,DNMT3a,DN-MT3 b were detected by RT-qPCR and Western blot re-spectively.Results ADO alone or in combination with HCY significantly reduced the viability of HepG2 cells in a dose and time-dependent manner.The apoptotic rates of HepG2 cells after combination treatment with ADO and HCY at 1 .0,2.0,4.0 mol · L-1 for 24 h were (1 8.63 ± 1.25 )%,(29.42 ±2.37 )% and (42.47 ±3.09 )%,compared with the control group (1.30 ±0.82 )%,P <0.01;and the mitochondrial membrane potentials were decreased from 674.15 ± 82.8%(black control group)to (428.38 ±54.5)%, (297.78 ±30.5)%,(74.45 ±5.73)%,P<0.01, respectively.The expressions of caspase-3,caspase-8, caspase-9,MDM-2,p53,Cytochrome C were up-regula-ted and MDM-2 were down-regulated after combination treatment of ADO and HCY.The mRNA expressions of DNMT1 ,DNMT3 a and DNMT3 b were down-regulated after combination treatment with ADO and HCY or 5-Aza-CdR alone.Conclusion Combination treatment of ADO and HCY can cause cellular methylation chan-ges.The effects of demethylation of ADO and HCY may activate p53 gene and mitochondrial pathway, which at last leads to HepG2 cell apoptosis.
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ObjectiveTo investigate the effects of 5-Aza-CdR on the transcriptional regulation through methylation of the DNA promoter protocadherin 8(PCDHg) gene in pancreatic cancer cell line Capan-2.The Capan-2 retardation in growth rate and apoptosis were assessed in when administered 5-Aza-CdR and the chemotherapy agent,gemcitabine.MethodsMTT and flow cytometry were used to analyze the cell growth inhibition and apoptosis when treated with 5-Aza-CdR or in combination with gemcitabine.Methylation-specific PCR,RT-PCR and western blot were performed to detect methylation state,mRNA and protein respectively of PCDH8 gene in 5-Aza-CdR-treated Capan-2cells.Results Capan-2 cells treated with 5-Aza-CdR showed a slower growth rate,and a significant growth inhibition when given both 5-Aza-CdR in combination with gemcitabine.Compared with single drug administration and control,5-Aza-CdR together with gemcitabine can induce a stronger apoptosis signal.Different concentrations 5-Aza-CdR of were able to reverse methylation,restore mRNA and protein levels of PCDH8 in Capan-2.Conclusion5 Aza-CdR may demethylate the PCDH8 gene,which would effectively remove the gene silencing caused by high methylation,and thus induce gene mRNA transcription and protein expression to inhibit cell growth and have collaborative antitumor functions with gemcitabine.
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DNA methylation is crucial for mammalian development, and DNA methylation is always in the dynamic status during preimplantation mouse embryos development. The effects of 5-AZA-CdR on the development of preimplantation mouse embryos were evaluated. Preimplantation mouse embryos created by in vitro fertilization were cultured continuously in 5-AZA-CdR (0.2, 1.0, or 5.0 μmol/L). Fertilized oocytes exposed to CZB containing 5-AZA-CdR at the pronuclear stage were unable to form morulae (0.2 and 1.0 μmol/L) or 4-cell embryos (5.0 μmol/L), while 2-cell stage embryos exposed to 5-AZA-CdR developed into uncompacted 8-cell (0.2 and 1.0 μmol/L) or 3/4-cell (5.0 μmol/L) stage embryos. The rate of morula formation was significantly lower in 4-cell embryos cultured in 5-AZA-CdR (1.0 or 5.0 μmol/L) than that in control embryos (P < 0.05). These data indicate that 5-AZA-CdR inhibits the development of mouse preimplantation embryos. Apoptosis, DNA methylation, and transcriptional activity were analyzed to determine the reason for these developmental defects. An aunexin V-PI assay revealed that high doses of 5-AZA-CdR led to apoptosis. Compared to the controls, DNA methylation was significantly reduced in uncompacted 8-cell embryos and morulae (p < 0.05) in a dose- dependent manner, whereas no significant change was detected in 2-or 4-cell embryos (P > 0.05). The observed changes in transcriptional activity, determined by measuring the incorporation of BrUTP, were similar to the observed alterations in DNA methylation. Therefore, the developmental defects induced by 5-AZA-CdR appear to bc mediated by alterations in DNA methylation and transcriptional activity in preimplantation mouse embryos.
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Objective To observe the expression of RAR-β gene in SiHa, HeLa,C33A and CasKi cell lines of cervical carcinoma and to investigate the role of methylated RAR-β in its expressive defection. Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of RAR-β gene. Immunohistochemistry and Western Blot were used to analyze the protein expression of RAR-β gene in four cervical cancer cell lines as well as the influence of 5-Aza-cdR on gene expressive defection. Methylation specific PCR (MSP) was used to detect whether there was the methylation in RAR-β gene in four cell lines. The change of RAR-β gene methylation state was also observed by MSP. The cell proliferation rate influenced by the 5-Aza-cdR was observed by MTT assay. Results The expression of RAR-β mRNA and protein in SiHa, HeLa and CasKi cell lines of cervical cancer was silent or decreased, whereas its expression was detected in C33A cell line. By using MSP method, it was found that there was RAR-β gene methylation in those three cell lines, whereas there was no RAR-β gene methylation in C33A cell line. After treated with the 5-Aza-cdR, methylated RAR-β gene was partly demethylated, and RAR-β mRNA and protein were re-expressed in the previous three cell lines in which RAR-β gene expression was silent or decreased. The 5-Aza-cdR treatment could supress cell proliferation as well. Conclusion The RAR-β gene expressive defection plays an important role in the carcinogenesis of cervical cancer. The abnormal RAR-β gene methylation in the the promotor region has an important role in gene expressive defection. The cell proliferation can be supressed by demethylated treatment.
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Objective:To study the gene expression and aberrant methylation of FBN2 on lung cancer cell lines.Methods:Three lung cancer cell lines HCC366,H1299 and H2195 were cultured.mRNA was analysed by RT-PCR and methylation status was also investigated by methylation specific PCR.The loss of FBN2 expression cell lines were treated with the demethylating agent,5-aza-2'-deoxycytid-ine(5-aza-cdR).Results:The expression of FBN2 was detected in NHBEC and H2195,whereas it was not detected in HCC366 and H1299 which showed aberrant methylation of FBN2.FBN2 expression was restored after treatment with 5-aza-cdR.Conclusion:Methylation and silencing of FBN2 in tumor cells may play an important role in carcinogenesis of lung cancer.
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Objective: To observe the expression of RAR-β gene in SiHa, HeLa, C33A and CasKi cell lines of cervical carcinoma and to investigate the role of methylated RAR-β in its expressive defection. Methods: Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of RAR-β gene. Immunohistochemistry and Western Blot were used to analyze the protein expression of RAR-β gene in four cervical cancer cell lines as well as the influence of 5-Aza-cdR on gene expressive defection. Methylation specific PCR (MSP) was used to detect whether there was the methylation in RAR-β gene in four cell lines. The change of RAR-β gene methylation state was also observed by MSP. The cell proliferation rate influenced by the 5-Aza-cdR was observed by MTT assay. Results: The expression of RAR-β mRNA and protein in SiHa, HeLa and CasKi cell lines of cervical cancer was silent or decreased, whereas its expression was detected in C33A cell line. By using MSP method, it was found that there was RAR-β gene methylation in those three cell lines, whereas there was no RAR-β gene methylation in C33A cell line. After treated with the 5-Aza-cdR, methylated RAR-β gene was partly demethylated, and RAR-β mRNA and protein were re-expressed in the previous three cell lines in which RAR-β gene expression was silent or decreased. The 5-Aza-cdR treatment could supress cell proliferation as well. Conclusion: The RAR-β gene expressive defection plays an important role in the carcinogenesis of cervical cancer. The abnormal RAR-β gene methylation in the the promoter region has an important role in gene expressive defection. The cell proliferation can be supressed by demethylated treatment.
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Background and purpose:hMLH1 promoter methylation is one of the factors in the development of lung cancer.However,little is known about hMLH1 promoter methylation in association with chemotherapy resistance in lung cancer cells.This study aimed to investigate whether resistance to DDP can be overcame by restoring the expression of hMLH1 in A549/DDP cells after treatment with a demethylating agent,5-Aza-2'-deoxycytidine (5-Aza-CdR).Methods:hMLH1 mRNA was determined by RT-PCR in A549,A549/DDP and A549/DDP treated with 5-Aza-CdR;MSP was carried out to determine the methylation status of hMLH1 in A549 and A549/DDP,A549/DDP treated with 5-Aza-CdR.MTT test,Hoechst 33258 staining and flow cytometry were used to determine the change of cell proliferation,apoptotic morphology and the cell apoptosis index,respectively.Results:hMLH1 mRNA was expressed higher in A549 than in A549/DDP.The unmethylation status of hMLH1 was observed in A549 cells whereas partial methylation status of hMLH1 in A549/DDP cells.After treatment with 5-Aza-CdR,the A549/DDP cells displayed an unmethylation of the hMLH1 gene.After the treatment with cisplatin,the IC50 of A549,A549/DDP and A549/DDP treated by 5-Aza-CdR were (4.7?0.7),(30.1?1.8) and (6.9?0.6)?mol/L,respectively.Apoptotic bodies were less in A549/DDP cells than in A549/DDP cells treated with the 5-Aza-CdR.The rate of apoptosis of A549/DDP treated by 5-Aza-CdR was higher than that of A549/DDP.Conclusion:The methylation of hMLH1 may be relevant to the resistance to platinum-based chemotherapy in the A549/DDP cell line.5-Aza-CdR can inhibit hMLH1 methylation,restore the expression of hMLH1,and enhance cell apoptosis as well as the inhibition of cell proliferation of A549/DDP cells after the treatment of cisplatin.
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Objective To study the association of methylation status of C5 of the cytosine in the CpG di-nucleotide of caspase-8 promoter and expression of caspase-8 mRNA with the resistance to tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) in human hepatocellular carcinoma cell lines,and to evaluate the effect of demethylation agent 5-Aza-2′-deoxycytidine(5-Aza-CdR) on the resistance to TRAIL of human hepatocellular carcinoma cell lines.Methods Methylation status of caspase-8 promoter was measured with methylation-specific PCR method(MSP).Expression of caspase-8 mRNA was detected with RT-PCR.Apoptosis induced by TRAIL was observed by Acridine Orange/Ethidium Bromide(AO/EB) staining.Results Unmethylated status of caspase-8 promoter was found in both HepG2 and SMMC 7721 hepatocellular carcinoma cells.5-Aza-CdR neither up-regulated caspase-8 mRNA expression nor increased the sensitivity of hepatocellular carcinoma cells to TRAIL.Conclusion caspase-8 promoter methylation status and caspase-8 mRNA expression are not related to the resistance to TRAIL.5-Aza-CdR can not increase the sensitivity of hepatocellular carcinoma cells to TRAIL.
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Background and purpose:The MEG3 gene is a imprinted gene, whose loss may be associated with the pathogenesis and progression of several tumor types. This study was done to investigate the transcription of MEG3 mRNA in human mammary cancer cell line MCF7 and cell proliferation, in order to explore the effect of the methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR) on MEG3 gene expression and proliferation in MCF7. Methods:MCF7 was treated with 5 ?mol/L 5-aza-CdR for 2, 4, 6 days, then the alteration of MEG3 gene expression was detected by RT-PCR and Northern blot technology and the proliferation difference in cell growth of MCF7 was observed by MTT. Results:After treated with 5-aza-CdR, the transcription of MEG3 mRNA in MCF7 was increased and the growth of MCF7 was reduced. MCF7 was treated with 5 ?mol/L 5-aza-CdR for 2, 4, 6 days, the inhibitory rates were (23.16?3.93), (49.39?2.38), (64.73?2.24), there were signifi cant differences between them. Conclusion:The growth of MCF7 was possibly inhibited by MEG3 gene, and the downregulation of MEG3 gene might result from the methylation, which was involved in the mammary cancer pathogenesis.
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Background and purpose:Regulation of MMR activity under hypoxia may play an important role in genetic instability of cancer,but the mechanism is still unclear.We investigated the expression of DNA mismatch repair genes MLH1 and MSH2 in human SCLC cell line H446 under hypoxic condition and explore the role of promoter methylation of genes in hypoxia.Methods:RT-PCR and Western blot were applied to detect MLH1 and MSH2 expression in human SCLC cell line H446 at the mRNA and the protein level,respectively,under either hypoxic condition or after 5-Aza-CdR treatment.Meanwhile,methylation-specific PCR(MSP)was used to determine promoter methylation of MLH1 and MSH2.Results:The expression of MLH1 and MSH2 in H446 cells significantly decreased both at the mRNA and the protein level under hypoxic condition.5-Aza-CdR treatment led to the restoration of MLH1 and MSH2 expression,while,both MLH1 and MSH2 were down-regulated again after removing 5-Aza-CdR.Conclusions:The promoter methylation of MLH1 and MSH2 may play an important role in its defective expression in H446 cells under hypoxic condition.And 5-Aza-CdR could restore MLH1 and MSH2 expression.
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Objective To investigate the effect of 5-Aza-CdR on experimental lung metastasis of breast cancer and the possible mechanisms.Methods MDA-MB-231 cells were divided into control group and 5-Aza-CdR-treated group.The mRNA expressions and promotor methylation status of BRMS1 and CXCR4 of the MDA-MB-231 cells were evaluated by SqRT-PCR and MSP respectively.Then,the MDA-MB-231 cells of control group and 5-Aza-CdR-treated group were injected into BALB/c nude mice through lateral tail veins,respectively.five weeks later,the mRNA abundance of the target gene HPRT and internal control gene GAPDH of the lung tissues from the mice were evaluated by FqRT-PCR.Results 5-Aza-CdR upgraded the BRMS1mRNA expression significantly than that in control group(0 versus 0.39?0.001,P0.05) and the status of unmethylated CXCR4 CpG island 1 in promotor were not changed significantly.The Ct values of HPRT and GAPDH in control and 5-Aza-CdR-treated groups were 24.75?1.55,16.19?0.69 versus 27.61?1.67,17.48?0.96 respectively,2-??Ct=0.34.The mRNA abundance of the HPRT was lower and there were fewer metastases in the lungs of 5-Aza-CdR-treated group compared with control group.Conclusions 5-Aza-CdR can activate tumor metastasis suppressor gene BRMS1 by demethylation mechanism,and thus,decreased the ability of breast cancer MDA-MB-231 cells for experimental metastasis to lungs.
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Objective:To evaluate the disruption of FANCF gene by methylation in cervical cancer and analyze the relation between cervical cancer and the methylation of FANCF DNA so as to find a method to inhibit the development of cervical cancer.Methods:The level of FANCF mRNA was detected with reverse transcription polymerase chain reaction(RT-PCR) before and after treatment with 5-aza-2'-deoxycytidine(5-Aza-CdR).Protein was tested by Western Blot.Methylation specific PCR(MSP) was used to check whether it was methylated in exon of FANCF.Normal cervical cancer tissues were used as controls.Results:The expression of FANCF mRNA in cervical cancer cell line SiHa was absent,and so was the expression of protein.The expression of FANCF mRNA and protein was decreased in Hela cell line.After treated with(5-Aza-CdR),SiHa expressed FANCF mRNA and protein,and time for SiHa cells to proliferate double was prolonged.Compared with the controls,the expression of FANCF mRNA in cervical cancer tissues decreased significantly or absolutely defaulted,and expression of FANCF protein also decreased(P