人脑胶质瘤细胞株中RIZ1基因启动子甲基化状态的研究

检测4种人脑成胶质细胞瘤细胞株中成视网膜细胞瘤蛋白结合锌指结构基因1（retinoblastoma protein-interacting zinc finger gene1,RIZ1）基因启动子区的甲基化状态,进一步认识RIZ1在胶质瘤发病机制中的作用。方法：应用甲基化特异性PCR法（methylation specific PCR,MSP）检测4种人脑成胶质细胞瘤细胞株U87、U251、A172和T98中RIZ1基因启动子区的甲基化水平,其中U87细胞发生甲基化,被选为后续实验对象。RT-PCR检测U87细胞经5-Aza-CdR处理前后RIZ1 mRNA表达量的变化,MTT检测5-Aza-CdR对U87细胞生长增殖的影响。结果：4种人脑成胶质细胞瘤细胞株中U87和U251细胞中检测到RIZ1基因启动子区域发生甲基化;U87细胞经5-Aza-CdR处理后其RIZ1 mRNA的表达量上调;MTT法检测显示5-Aza-CdR能抑制U87的生长增殖,且与5-Aza-CdR的浓度和作用时间呈负相关。结论：RIZ1基因启动子高甲基化是人脑成胶质细胞瘤细胞株中RIZ1基因表达下调的重要机制。     

DNA去甲基化对急性B淋巴细胞白血病细胞株中DNA甲基转移酶基因及微RNA作用的研究

目的：探索甲基化转移酶抑制剂5-杂氮-2'-脱氧胞苷（5-Aza-CdR）对急性B淋巴细胞白血病（B-ALL）细胞株NALM-6的作用以及对细胞中微RNA（miRNA）表达水平的影响。方法用不同浓度5-Aza-CdR处理NALM-6细胞,采用四甲基偶氮唑盐（MTT）法检测细胞增殖情况,采用荧光定量反转录聚合酶链反应（RT-PCR）检测5-Aza-CdR处理后细胞DNA甲基转移酶（DNMT）基因mRNA表达水平的变化,采用miScript miRNA PCR Array芯片检测去甲基化后细胞中表达量发生改变的miRNA。结果 NALM-6细胞经不同浓度5-Aza-CdR处理不同时间后,细胞生长受抑,最高抑制率达（74.163±0.381）%。5-Aza-CdR作用浓度与DNMT基因mRNA表达水平呈反比,浓度为1000μmol／L的5-Aza-CdR作用细胞72 h后,DNMT-1的相对表达量降至0.453±0.021,DNMT-3L的相对表达量为0.003±0.001, DNMT-3B的相对表达量为0.395±0.019。miScript miRNA PCR Array筛选出3个miRNA（miR-184、miR-23a-3p、miR-34a-5p）与DNA甲基化相关。结论5-Aza-CdR可下调NALM-6细胞中DNMT基因的表达,并对细胞增殖有抑制作用。miR-184、miR-23a-3p和miR-34a-5p在B-ALL的发生、发展中与DNA甲基化相关。     

5-Aza-2’-deoxycytidine Induces Hepatoma Cell Apoptosis via Enhancing Methionine Adenosyltransferase 1A Expression and Inducing S-Adenosylmethionine Production

In hepatocellular cancer (HCC), lack of response to chemotherapy and radiation treatment can be caused bya loss of epigenetic modifications of cancer cells. Methionine adenosyltransferase 1A is inactivated in HCC andmay be stimulated by an epigenetic change involving promoter hypermethylation. Therefore, drugs releasingepigenetic repression have been proposed to reverse this process. We studied the effect of the demethylating reagent5-aza-2’-deoxycitidine (5-Aza-CdR) on MAT1A gene expression, DNA methylation and S-adenosylmethionine(SAMe) production in the HCC cell line Huh7. We found that MAT1A mRNA and protein expression wereactivated in Huh7 cells with the treatment of 5-Aza-CdR; the status of promoter hypermethylation was reversed.At the same time, MAT2A mRNA and protein expression was significantly reduced in Huh7 cells treated with5-Aza-CdR, while SAMe production was significantly induced. However, 5-Aza-CdR showed no effects onMAT2A methylation. Furthermore, 5-Aza-CdR inhibited the growth of Huh7 cells and induced apoptosis andthrough down-regulation of Bcl-2, up-regulation of Bax and caspase-3. Our observations suggest that 5-Aza-CdR exerts its anti-tumor effects in Huh7 cells through an epigenetic change involving increased expression ofthe methionine adenosyltransferase 1A gene and induction of S-adenosylmethionine production.     

肺腺癌细胞DR4基因启动子甲基化与TRAIL敏感性的相关性研究

目的 探讨DR4基因启动子甲基化水平与肺腺癌细胞株对肿瘤坏死因子相关凋亡诱导配体（TRAIL）敏感性之间的相关性。方法 选取未经5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理和10 μmol/L 5-Aza-CdR处理3 d后的肺腺癌细胞A549、LTEP-α-2,采用CCK-8法检测细胞增殖抑制率,倒置显微镜下观察细胞形态学改变,流式细胞仪检测细胞凋亡率;采用RT-PCR法、免疫印迹法和甲基化特异性PCR（MSP）法分别检测肺腺癌细胞株（A549、LTEP-α-2）DR4基因mRNA、蛋白表达和启动子区甲基化状态。结果 肺腺癌细胞（A549、LTEP-α-2）对低浓度TRAIL高度耐受,提高TRAIL浓度（15.625、31.25、62.5、125、250、500 μg／ml）作用细胞24、48 h后,细胞生长受到不同程度抑制,且呈剂量和时间依赖性;经5-Aza-CdR处理后,TRAIL对肺腺癌细胞的增殖抑制作用均较处理前显著增强（P＜0.05）,倒置显微镜下细胞形态表现出变圆、皱缩甚至脱落等特征。应用5-Aza-CdR处理后,125 μg／ml TRAIL诱导肺腺癌细胞的凋亡率较处理前明显升高（P＜0.05）。A549、LTEP-α-2细胞存在DR4 mRNA及蛋白的低表达,其基因启动子处于甲基化状态;经5-Aza-CdR处理后,肺腺癌细胞中DR4 mRNA及蛋白的表达均显著升高（P＜0.05）,其基因启动子处于非甲基化状态。结论 5-Aza-CdR可以逆转DR4基因启动子甲基化状态,上调基因表达,增强TRAIL诱导肺腺癌细胞凋亡的能力,从而逆转TRAIL耐药。5-Aza-CdR联合TRAIL可能是治疗肺腺癌的一种新策略。     

乳腺癌中NDRG-1基因甲基化及其体外逆转研究

目的:探讨N-myc下游调节基因(N-myc downstream regulated gene-1,NDRG-1)基因在乳腺癌组织中的甲基化状态,研究甲基化酶抑制剂5-氮杂-2'-脱氧胞苷(5-Aza-2'-deoxycytidine, 5-Aza-CdR)对乳腺癌细胞株T47D的生长增殖及NDRG-1 mRNA表达的影响.方法:采用甲基化特异性PCR(methylation specific PCR,MSP)法检测乳腺癌组织、相应癌旁组织和乳腺良性病变组织中NDRG-1基因启动子甲基化状态;5-Aza-CdR处理T47D细胞后,MTT法观察细胞生长活性的变化,RT-PCR法检测处理前后抑癌基因NDRG-1的mRNA表达变化.结果:NDRG-1基因在乳腺癌组织中甲基化率为46.8%,癌旁组织中甲基化率为21.3%,而乳腺良性病变组织均未检测到甲基化.T47D细胞经5-Aza-CdR处理后,与对照组相比,细胞生长受到明显抑制.RT-PCR检测发现,与对照组相比,不同浓度处理组细胞的NDRG-1 mRNA表达增多. 结论:NDRG-1基因甲基化状态与乳腺癌发生有密切关系.5-Aza-CdR逆转T47D细胞NDRG-1基因甲基化,恢复该基因的表达,从而抑制肿瘤细胞生长.     

HPV16E6 siRNA联合5-Aza-CdR对宫颈癌SiHa细胞中E-钙黏蛋白表达和基因甲基化的影响

目的:探讨HPV16E6 siRNA联合5-Aza-CdR(5-aza-2'-deoxycytidine,5-氮-2'-脱氧胞苷,又称地西他滨)对宫颈癌SiHa细胞中E-钙黏蛋白表达和基因甲基化的影响。方法:采用SiHa细胞构建的HPV16E6 沉默细胞株及5-Aza-CdR细胞株,检测细胞中E-cadherin mRNA和蛋白表达,以及E-cadherin甲基化状态。采用细胞黏附、Transwell体外侵袭、迁移实验检测5-Aza-CdR和siRNA E6对SiHa细胞黏附、侵袭迁移的影响。结果:5-Aza-CdR+siRNA E6组较5-Aza-CdR组、siRNA E6组中E-cadherin mRNA 及蛋白表达均上升,细胞的黏附率、侵袭抑制率和迁移抑制率均升高;5-Aza-CdR+siRNA E6组较5-Aza-CdR组、siRNA E6组中E-cadherin基因启动子区甲基化指数明显下降。结论:HPV16E6 siRNA联合5-Aza-CdR可显著引起宫颈癌细胞中E-cadherin 基因低甲基化,并可导致E-cadherin mRNA 及蛋白的表达水平明显上调,肿瘤细胞黏附能力升高,侵袭迁移能力下降,两者在一定程度起到协同作用。     

5-氮杂-2'-脱氧胞苷对肺癌细胞H460生物学行为及抑癌基因RASSF1A表达的影响

 目的　观察5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对肺癌细胞H460的生物学行为及RASSF1A mRNA表达的影响。方法　5-Aza-CdR处理H460细胞,通过MTT方法、平板克隆试验观察细胞生长活性的变化,PCR检测RASSF1A甲基化状态,Western blotting法检测RASSF1A的蛋白表达,流式细胞术进行细胞周期分析。结果　H460细胞经5-Aza-CdR处理后,与未处理组相比,生长速度出现不同程度减慢,克隆形成率明显降低,RASSF1A甲基化程度降低,延缓H460细胞周期G1／S进程,使细胞阻滞于G1期。结论　在肺癌细胞系中,RASSF1A基因甲基化可能导致其表达缺失,而5-Aza-CdR能够恢复RASSF1A基因的表达,为肺癌的去甲基化治疗提供理论依据。     

Statin attenuates cell proliferative ability via TAZ (WWTR1) in hepatocellular carcinoma

Diabetes and obesity are associated with non-alcoholic steatohepatitis and an increased incidence of hepatocellular carcinoma (HCC). TAZ and YAP are equivalently placed downstream effectors of the Hippo pathway with oncogenic roles in human cancers. Statins are commonly used to patients with metabolic problems as hypercholesterolemia. Statins also have anti-cancer properties, and the cross-talk between mevalonate pathway and Hippo pathway was known. The aim of this study is to confirm the statin’s anti-cancer effects on HCC cells and its survival benefits in HCC patients with curative surgery. TAZ expression level in HCC cell lines was analyzed by western blot. Two cell lines (HLF and HuH1) were used in this study. Then the mechanism of statin’s anti-proliferative effect was examined in HLF and HuH1 cells. In clinical setting, overall survival and recurrence-free survival (RFS) rate were examined in comparison between statin intake and statin non-intake group. The proliferation assay using four different statins (atorvastatin, pravastatin, fluvastatin, simvastatin). Simvastatin and fluvastatin showed very strong growth suppressive effects, and induced apoptosis in HLF cells, but not HuH1 cells. TAZ expression was suppressed in HLF cells by fluvastatin and simvastatin treatment. The similar change pattern was confirmed in p-ERK1/2 and ERK. In HuH1 cells, such expression change was not confirmed. In clinical setting, statin intake was significantly associated with longer RFS in the HCC patients with hepatectomy (P = 0.038). The statin had the anti-proliferative effects and induced apoptosis in HCC cells and improved the prognosis of HCC patients.     

启动子区5''CpG岛去甲基化对人肠癌RKO细胞生物学表型的影响

目的:探讨DNA启动子区5'CpG岛甲基化状态与人肠癌RKO细胞生物学特征的关系.方法:应用特异性DNA甲基转移酶抑制剂-5-氮-2'-脱氧胞苷(5-Aza-2'-deoxycytidine,5-Aza-CdR)处理肠癌RKO细胞72小时,甲基化特异性PCR(methylation-specific PCR,MSP)及DNA测序法分析p16/CDKN2抑癌基因5'CpG岛甲基化状态;MTT、FCM、荧光染色及透射电镜检测启动子区去甲基化后对细胞生长、形态和细胞周期凋亡的影响.结果:肠癌RKO细胞p16/CDKN2基因5'CpG岛呈高甲基化状态;DNA甲基转移酶抑制剂(5-Aza-CdR)能较好地逆转启动子区胞嘧啶甲基化状态;CpG岛去甲基化后能明显地抑制肠癌细胞的生长,增加细胞群体倍增时间(P＜0.01),诱导肠癌细胞凋亡,并呈良好的量效依赖关系.结论:通过逆转CpG岛高甲基化能有效地抑制肠癌细胞增殖,为临床治疗大肠癌提供新的作用靶点.     

Synergistic growth inhibition by acyclic retinoid and vitamin K2 in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. However, effective chemopreventive and chemotherapeutic agents for this cancer have not yet been developed. In clinical trials acyclic retinoid (ACR) and vitamin K(2) (VK(2)) decreased the recurrence rate of HCC. In the present study we examined the possible combined effects of ACR or another retinoid 9-cis retinoic acid (9cRA) plus VK(2) in the HuH7 human HCC cell line. We found that the combination of 1.0 microM ACR or 1.0 microM 9cRA plus 10 microM VK(2) synergistically inhibited the growth of HuH7 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VK(2) also acted synergistically to induce apoptosis in HuH7 cells. Treatment with VK(2) alone inhibited phosphorylation of the retinoid X receptor (RXR)alpha protein, which is regarded as a critical factor for liver carcinogenesis, through inhibition of Ras activation and extracellular signal-regulated kinase phosphorylation. Moreover, the inhibition of RXRalpha phosphorylation by VK(2) was enhanced when the cells were cotreated with ACR. The combination of retinoids plus VK(2) markedly increased both the retinoic acid receptor responsive element and retinoid X receptor responsive element promoter activities in HuH7 cells. Our results suggest that retinoids (especially ACR) and VK(2) cooperatively inhibit activation of the Ras/MAPK signaling pathway, subsequently inhibiting the phosphorylation of RXRalpha and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.     

5-氮-2'-脱氧胞嘧啶对U266细胞系P16基因去甲基化作用研究

  目的 探讨5-氮-2'-脱氧胞嘧啶（5-Aza-CdR）诱导骨髓瘤细胞系U266 p16基因 DNA 5′CpG岛去甲基化作用及对U266细胞增生的影响。方法 采用巢式甲基特异性PCR法（n-MSP）、DNA序列分析、RT-PCR、细胞生长曲线、流式细胞仪DNA含量分析法检测5-Aza-CdR对U266细胞 p16基因去甲基化作用及其对U266细胞的生长、增生及细胞周期的影响。结果 （1）5-Aza-CdR能够逆转U266细胞 p16 基因异常甲基化；（2）5-Aza-CdR能激活p16基因沉默的再转录；（3）5-Aza-CdR能下调U266细胞甲基转移酶DNMT1、DNMT3A、DNMT3B 的表达并呈浓度依赖性；（4）5-Aza-CdR作用的U266细胞被阻滞于G0 ~ G1期。结论 5-Aza-CdR可能通过抑制甲基转移酶直接对p16 基因去甲基化，逆转U266 细胞DNA 异常甲基化，并有效地激活因高甲基化所致p16基因沉默的再转录     

Synergistic growth inhibition by 9-cis-retinoic acid plus trastuzumab in human hepatocellular carcinoma cells.

PURPOSE: A malfunction of retinoid X receptor-alpha (RXRalpha) due to phosphorylation by the Ras/mitogen-activated protein kinase signaling pathway is associated with the development of hepatocellular carcinoma (HCC). The humanized anti-HER2 monoclonal antibody trastuzumab inhibits the activation of HER2 and its multiple downstream signaling pathways, including the Ras/mitogen-activated protein kinase pathway. In this study, the effects of phosphorylation of RXRalpha on the ability of RXRalpha ligand 9-cis-retinoic acid (9cRA) and trastuzumab to inhibit growth of HCC cells was examined. EXPERIMENTAL DESIGN: The effects of a combination of 9cRA plus trastuzumab on the inhibition of cell growth in HLF human HCC cells which express constitutive activation of HER2 protein were examined. RESULTS: The combination of 9cRA plus trastuzumab synergistically inhibited the growth of HLF cells without affecting the growth of Hc normal human hepatocytes. Combined treatment with these agents acted synergistically to induce apoptosis in HLF cells. The treatment of HLF cells with trastuzumab alone inhibited the phosphorylation of HER2, RXRalpha, ERK, Akt, and Stat3 proteins and these effects were enhanced when the cells were cotreated with 9cRA. Reporter assays indicated that the combination of 9cRA plus trastuzumab markedly increased both the retinoic acid responsive element and retinoid X responsive element promoter activities in HLF cells. CONCLUSION: 9cRA and trastuzumab cooperatively inhibit the activation of HER2 and its downstream signaling pathways, subsequently inhibiting the phosphorylation of RXRalpha and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.     

Alteration of the CDKN2/p16 gene is not required for HPV-positive uterine cervical cancer cell lines

To determine whether alterations of the CDKN2/p16 might be involved in HPV-positive cervical cancers, we examined for alterations of this gene and function of the protein p16 to interact with CDK4 in 5 cervical cancer cell lines. No alteration of this gene was detected. Proteins for p16 and CDK4 were normally expressed and function of p16 to interact with CDK4 was not abrogated in these cell lines. These cell lines were human papillomavirus (HPV)-positive and carried wild-type p53. These findings suggest that phosphorylation of pRb by CDK4 is not critical in the carcinogenesis or in the establishment of HPV-positive cervical cancer cell lines, since HPV E6 or E7 viral-transforming proteins inactivate p53 and pRb tumor suppressor protein function, resulting in deregulated progression of the cell cycle.     

脱乙酰化酶抑制剂与去甲基化制剂联合诱导U266细胞凋亡和p16基因重新表达

背景与目的：DNA甲基化调节基因表达与组蛋白脱乙酰化的作用密切相关,针对这两大途径用药,观察对肿瘤细胞的影响是有意义的,本文探讨脱乙酰化酶抑制剂苯丁酸钠（sodium phenylbutyrate,SPB)联合去甲基化制剂5-氮杂-2′-脱氧胞苷（5-Aza-CdR)处理人骨髓瘤细胞系U266诱导高甲基化失活的p16基因重新表达的可能性及其对细胞生长的影响。方法：通过透射电镜,DNA梯形条带及流式细胞术分析细胞凋亡及细胞周期的变化;RT-PCR和Western blot检测p16表达水平。结果：单用5-Aza-CdR(1μmol/L）或SPB（1mmol/L)及两药联合诱导的细胞凋亡率分别是15.09%,89.19%和85.18%。单用5-Aza-CdR或SPB对细胞G1期无影响,单用SPB使G2/M期细胞比例增高,联合用药发生明显的G1期阻滞,且出现亚G1期峰达50%。单用PB不能诱导U266细胞p16的表达,单用5-Aza-CdR可诱导p16重新表达,两药联合明显增强p16表达水平。结论：PB与5-Aza-CdR协同可显著诱导U266细胞p16重新表达,细胞阻滞在G1期,并使细胞凋亡发生的时相不同于单独用药组。     

PROMOTER HYPERMETHYLATION OF p16 GENE AND DAPK GENE IN SERA FROM HEPATOCELLULAR CARCINOMA （HCC） PATIENTS

Objective： To analyze the aberrant methylation of p16 gene and DAPK gene in sera from primary liver cancer patients ad to evaluate the clinical significance. Methods： A methylation-specific PCR was performed for the detection of promoter hypermethylation of p16 gene and DAPK gene in blood DNA from 64 cases of HCC patients, and to analyze the relation of the aberrant methylation of p16 gene and KAPK gene and the clinical pathological data. Results： 76.6%（49/64） of the sera from 64 cases of HCC patients showed hypermethylation for p16 promoter and 40.6% （26/64） for KAPK promoter, whereas no methylated p16 gene promoter and DAPK gene promoter were found in sera from benign liver diseases patients and normal control. Methylated p16 gene and KAPK gene promoters in sera did not strongly correlated with HBsAg, stage, metastasis and differentiation in HCC; but strongly correlated with AFP. Conclusion： Detection of the aberrant methylation of p16 gene and KAPK gene in blood DNA from HCC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.