MicroRNA-148b and microRNA-152 reactivate tumor suppressor genes through suppression of DNA methyltransferase-1 gene in pancreatic cancer cell lines

Overexpression of DNA methyltransferase 1 (DNMT-1) is observed mostly in pancreatic cancer and it can cause tumor suppressor genes silencing in this disease. Recent studies suggest that abnormal expressions of microRNAs (miRs) are involved in pathogenesis of different types of human cancers including pancreatic cancer. In this study we aimed to investigate the effect of miR-148b and -152 on reverting the tumorigenic phenotype of pancreatic cancer cell lines. In order to investigate whether miR-148b and -152 are involved in the regulation of DNMT-1, luciferase reporter assay was used and confirmed that the DNMT-1 mRNA could be a target for miR-148b and miR-152. Furthermore, overexpression of miR-148b and -152 in pancreatic cancer cell lines (MIA PaCa-2 and AsPC-1) decreased DNMT-1 expression (53% and 59% respectively), returned DNA methylation to normal patterns and induced re-expression of tumor suppressor genes, like BNIP3 (4.7- and 3.8-fold) and SPARC (5.3- and 2.9-fold) for miR-148b and -152 respectively. Moreover, the introduced miR-148b and -152 could inhibit the proliferation of MIA PaCa-2 (35% and 37% respectively) and AsPC-1 (39% and 40% respectively) cell lines. The apoptosis rates of MIA PaCa-1 after treatment with miR-148b and -152 were 10% and 8% respectively; while these rates in AsPC-1 were 16% and 11% respectively. Conclusively these findings mean that miRs that are targeting DNMT-1 and modifying methylation status of tumor suppressor genes such as BNIP3 and SPARC can be applied in killing the pancreatic cancer cells and decreasing the tumorigenicity of these cells.     

Peperomin E reactivates silenced tumor suppressor genes in lung cancer cells by inhibition of DNA methyltransferase

Advanced lung cancer has poor prognosis owing to its low sensitivity to current chemotherapy agents. Therefore, discovery of new therapeutic agents is urgently needed. In this study, we investigated the antitumor effects of peperomin E, a secolignan isolated from Peperomia dindygulensis, a frequently used Chinese folk medicine for lung cancer treatment. The results indicate that peperomin E has antiproliferative effects, promoting apoptosis and cell cycle arrest in non‐small‐cell lung cancer (NSCLC) cell lines in a dose‐dependent manner, while showing lower toxicity against normal human lung epidermal cells. Peperomin E inhibited tumor growth in A549 xenograft BALB/c nude mice without significant secondary adverse effects, indicating that it may be safely used to treat NSCLC. Furthermore, the mechanisms underlying the anticancer effects of peperomin E have been investigated. Using an in silico target fishing method, we observed that peperomin E directly interacts with the active domain of DNA methyltransferase 1 (DNMT1), potentially affecting its genome methylation activity. Subsequent experiments verified that peperomin E decreased DNMT1 activity and expression, thereby decreasing global methylation and reactivating the epigenetically silenced tumor suppressor genes including RASSF1A, APC, RUNX3, and p16INK4, which in turn activates their mediated pro‐apoptotic and cell cycle regulatory signaling pathways in lung cancer cells. The observations herein report for the first time that peperomin E is a potential chemotherapeutic agent for NSCLC. The anticancer effects of peperomin E may be partly attributable to its ability to demethylate and reactivate methylation‐silenced tumor suppressor genes through direct inhibition of the activity and expression of DNMT1.     

Recent progress in the study of methylated tumor suppressor genes in gastric cancer

Gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide.The pathogenesis mechanisms of gastric cancer are still not fully clear.Inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis.Accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes,particularly caused by hypermethylation of CpG islands in promoters,is critical to carcinogenesis and metastasis.Here,we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer.We also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications.     

抑癌基因甲基化与胃癌研究进展

DNA甲基化是基因表达调控的一种方式，抑癌基因启动子高甲基化可以使其表达受抑，这与肿瘤的发生关系密切。胃癌的发生是由多因素多基因多阶段异常累计的结果，其中抑癌基因甲基化与胃癌的发生具有重要的关系。     

Epigenetic modulation of endogenous tumor suppressor expression in lung cancer xenografts suppresses tumorigenicity

Epigenetic changes involved in cancer development, unlike genetic changes, are reversible. DNA methyltransferase and histone deacetylase inhibitors show antiproliferative effects in vitro, through tumor suppressor reactivation and induction of apoptosis. Such inhibitors have shown activity in the treatment of hematologic disorders but there is little data concerning their effectiveness in treatment of solid tumors. FHIT, WWOX and other tumor suppressor genes are frequently epigenetically inactivated in lung cancers. Lung cancer cell clones carrying conditional FHIT or WWOX transgenes showed significant suppression of xenograft tumor growth after induction of expression of the FHIT or WWOX transgene, suggesting that treatments to restore endogenous Fhit and Wwox expression in lung cancers would result in decreased tumorigenicity. H1299 lung cancer cells, lacking Fhit, Wwox, p16(INK4a) and Rassf1a expression due to epigenetic modifications, were used to assess efficacy of epigenetically targeted protocols in suppressing growth of lung tumors, by injection of 5-aza-2-deoxycytidine (AZA) and trichostatin A (TSA) in nude mice with established H1299 tumors. High doses of intraperitoneal AZA/TSA suppressed growth of small tumors but did not affect large tumors (200 mm(3)); lower AZA doses, administered intraperitoneally or intratumorally, suppressed growth of small tumors without apparent toxicity. Responding tumors showed restoration of Fhit, Wwox, p16(INKa), Rassf1a expression, low mitotic activity, high apoptotic fraction and activation of caspase 3. These preclinical studies show the therapeutic potential of restoration of tumor suppressor expression through epigenetic modulation and the promise of re-expressed tumor suppressors as markers and effectors of the responses.     

KAI1基因转染协同ATRA诱导对肺腺癌细胞株抑制作用的研究

目的 :探讨新抑癌基因KAI1与全反式维甲酸 (all trans retinoicacid ,ATRA)对肺腺癌A5 49细胞株抑制增殖和诱导分化的作用。方法 :用脂质体介导的基因转染方法 ,借助质粒表达载体 (PCMV NEO XhoI) ,将抑癌基因KAI1转入肺腺癌A5 49细胞中 ,经G418筛选 ,获得稳定表达的细胞克隆。用 10 6mol/LATRA作用于转染及未转染KAI1基因的肺腺癌A5 49细胞株 ,MTT法检测细胞体外增殖能力 ,流式细胞仪进行细胞周期和凋亡分析 ,间接免疫荧光染色结合流式细胞仪检测转染前后细胞CD82 蛋白的表达。免疫组化测定myc、基质金属蛋白酶 (MMP 1)的表达 ,放射免疫测定层连蛋白(LN)表达。结果 :ATRA处理KAI1基因转染的肺腺癌细胞CD82 表达降低 ,细胞增殖能力下降 ,凋亡增加 ,更多的细胞被阻止于G1/G0 期 ,myc、MMP 1及LN表达下降 ,比对照组及单纯转染组细胞差异有统计学意义 ,P <0 0 5。结论 :抑癌基因KAI1与ATRA对抑制肺腺癌A5 49细胞株的增殖浸润转移有协同作用。     

PRDM基因及DNA甲基化与肿瘤的关系

PRDI-BF1 and RIZ homology domain containing proteins (PRDM) play a key role in cell differentiation and proliferation. Most members of the PRDM gene family are tumor suppressor genes which involved in tumorigenesis and abnormal expression in a variety of tumors. Aberrant DNA methylation often silences these genes, which may occur in the early stage of tumor. Cancer can be reversed by demethylation, which provides a new way for cancer treatment.     

分泌型卷曲相关蛋白基因甲基化与大肠癌的研究进展

 随着表观遗传学的发展,越来越多的研究证实DNA甲基化与肿瘤的发生关系密切。分泌型卷曲相关蛋白（sFRP）基因甲基化可导致该基因表达的沉默,使得对 Wnt 信号通路的抑制作用减弱,促进肿瘤特别是大肠癌的发生与发展。当重新恢复sFRP 的功能后,Wnt 通路活性受到抑制,进而抑制了肿瘤的形成与进展并促进肿瘤细胞凋亡的发生。因此sFRP基因家族甲基化与大肠癌的发生、发展关系密切。所以通过检测sFRP 基因的甲基化对大肠癌进行早期筛查有望成为诊断大肠癌的一种新途径。     

癌相关基因在结肠癌中的表达及其甲基化调控

目的 探讨癌相关基因APC、p16^INK4A,p21^WAF1和c-myc等在结肠癌中的表达及其受甲基化的调控。方法 培养结肠癌细胞SWlll6、Colo-320、HT29，分别用不同浓度的5-aza-dC干预细胞。提取细胞的RNA，用RT-PCR的方法检测p16INK4A、,021WAFl、APC和c-myc等多种基因的表达情况；同时以流式细胞仪分析SWlll6和Colo-320的细胞周期。结果 (1)三种细胞在干预前有较弱的p16^INK4A和APC的表达，而在用5-aza-dC干预后表达增强，且在不同的结肠癌细胞株5-aza-dC作用发挥最佳的时间与浓度不同。(2)p21WAF1在干预前后均不表达，c-myc在干预前后表达无明显变化。结论 三株人结肠癌细胞中，5-aza-dC均可诱导p16^INK4A和APC的表达，但对p21WAF1和c-myc无明显影响。     

抑癌基因ING1在大肠癌中的表达及预后意义

目的 探讨ING1基因在散发性大肠癌中的表达与预后及多个临床病理变量之间的关系,并分析其在大肠癌预后的危险因素中是否具有显著意义。方法 应用定量RT-PCR方法检测82例大肠癌手术切除标本及相应的癌旁组织中ING1 mRNA的表达水平,分析其表达的差异,研究其表达水平与大肠癌患者临床病理特征及预后的关系。结果 (1)ING1 mRNA在大肠癌及癌旁组织中均可被检出,同一配对组织相比,癌旁组织中ING1表达量明显高于肿瘤原发灶;(2)ING1 mRNA的表达与大肠癌的浸润层次、淋巴结转移、远处转移以及TNM分期密切相关;(3)肿瘤组织与癌旁组织ING1表达量相比,比值越低其DFS也越低(P＜0.0001);(4)经单因素及多因素COX模型分析后显示,ING1作为候选抑癌基因可作为大肠癌预后的独立预测因素(P＜0.0001)。结论 ING1的过度表达是大肠癌发生过程中的分子事件,可能参与大肠癌的发展过程。ING1可作为判断大肠癌预后的重要分子标志。     

DNA甲基转移酶3B4过表达对293A细胞增殖影响及其机制的探讨

背景与目的：DNA甲基转移酶(DNA methyltransferase,DNMT)3B有近40种异构体,它们的表达有组织和疾病的特异性,细胞中这些异构体的功能尚不清楚。本研究旨在探讨外源性DNMT3B4基因过表达对人胚肾细胞株293A增殖的影响及其机制。方法：将携带DNMT3B4基因的质粒pCMV-DNMT3B4及空质粒pCMV-2B转染293A细胞(293A-vector)并形成稳定表达细胞系,培养300 d后用MTT法检测细胞的增殖;流式细胞仪检测细胞的周期分布;real-time PCR蛋白质印迹法(Western blot)检测细胞中p21表达情况;甲基化特异PCR(MS-PCR)检测p21基因启动子区甲基化状态。结果：在接种于96孔板的第4天,2株过表达DNMT3B4的293A细胞(293A-DNMT3B4-1和293A-DNMT3B4-2)吸光度(A)值分别是293A-vector细胞的(58.92±3.47)%和(68.82±5.64)%,过表达DNMT3B4抑制细胞增殖,差异有统计学意义(P<0.05);293A-DNMT3B4-1和293A-DNMT3B4-2的S期细胞比例分别为(35.88±2.00)%和(37.00±1.79)%,293A-vector细胞比例为(40.44±0.91)%,过表达DNMT3B4降低S期细胞比例,差异有统计学意义(P<0.05)。过表达DNMT3B4增加p21的表达,但不改变p21基因启动子区甲基化状态。结论：DNMT3B4基因过表达可抑制293A细胞增殖并增加p21表达。     

胃癌组织中mRNA-375基因高甲基化及其表达的研究

目的:探讨胃癌组织中miRNA-375基因表达与基因甲基化调控的相关性.方法:2011年3月至8月在天津医科大学总医院通过胃镜检查收集90例新鲜组织活检标本,分为2组,胃癌组54例,非癌对照组36例.应用实时荧光定量反转录PCR检测miRNA-375基因表达,甲基化特异性PCR检测miRNA-375基因启动子区CpG岛甲基化.结果:胃癌组miRNA-375基因表达下调,与非癌对照组相比差异有统计学意义(P<0.05);胃癌组和非癌对照组 miRNA-375基因启动子区高甲基化阳性率分别为62.96%(34/54)和22.22%(8/36),差异有统计学意义(χ2=14.405, P<0.05).中高分化胃癌组织中miRNA-375基因表达高于低分化组,差异有统计学意义(t=2.634,P=0.011);miRNA-375基因启动子区甲基化阳性率中高分化组与低分化组分别为44.44%(8/18)和72.22%(26/36),差异有统计学意义(χ2=3.971,P=0.046).结论:癌组织中存在miRNA-375基因异常低表达及启动子区的高甲基化,miRNA-375基因高甲基化可能抑制miRNA-375基因表达,在胃癌发生发展中发挥重要作用.     

结直肠肿瘤中sFRP基因的甲基化及表达调控

目的探讨分泌型卷曲相关蛋白(sFRP)家族基因启动子CpG岛甲基化在结直肠肿瘤发生、发展中的作用。方法应用5-氮杂-2’-脱氧胞苷(DAC)和曲古菌素A(TSA)对结直肠癌细胞系RKO、HCT116和SW480进行去甲基化处理。甲基特异性PCR和逆转录PCR分别检测结直肠肿瘤组织及细胞系中sFRP基因甲基化和mRNA表达。结果正常大肠黏膜不存在sFRP基因甲基化。sFRP1、sFRP2和sFRP5在结直肠腺癌、腺瘤和异常隐窝灶(ACF)中均存在高频率甲基化,sFRP1甲基化率>85%,sFRP2甲基化率>75%,sFRP5甲基化率>50%,腺癌、腺瘤和ACF间sFRP基因甲基化率差异无统计学意义(P>0.05),但均高于正常黏膜及瘤旁正常组织(P<0.05)。3种结直肠癌细胞系RKO、HCT116和SW480均存在sFRP基因甲基化,甲基化的sFRP基因mRNA失表达。联合使用DAC和TSA能有效恢复结直肠癌细胞系的sFRP基因表达。结论在ACF中,sFRP基因家族已出现高频率甲基化,是结直肠肿瘤发生常见的早期事件,sFRP1、sFRP2和sFRP5甲基化可能成为早期发现结直肠肿瘤的生物学标志。去甲基化能有效恢复基因表达,可能成为治疗肿瘤的有效手段。     

Alterations of 3p21.31 tumor suppressor genes in head and neck squamous cell carcinoma: Correlation with progression and prognosis

The aim of our study was to analyze the alterations of some candidate tumor suppressor genes (TSGs) viz. LIMD1, LTF, CDC25A, SCOTIN, RASSF1A and CACNA2D2 located in the chromosomal region 3p21.31 associated with the development of early dysplastic lesions of head and neck. In analysis of 72 dysplastic lesions and 116 squamous cell carcinoma of head and neck, both deletion and promoter methylation have been seen in these genes except for CDC25A and SCOTIN where no methylation has been detected. The alteration of LIMD1 was highest (50%) in the mild dysplastic lesions and did not change significantly during progression of tumor indicating its association with this stage of the disease. It was evident that alterations of LTF, CDC25A and CACNA2D2 were associated with development of moderate dysplastic lesions, while alterations in RASSF1A and CACNA2D2 were needed for progression. Novel somatic mutations were seen in exon 1 of LIMD1 (7%), intron 3/exon4 splice junction of LTF (2%) and exon 7 of cdc25A (10%). Quantitative RT-PCR analysis revealed mean reduced expression of the genes in the following order: LTF (67.6 +/- 16.8) > LIMD1 (53.2 +/- 20.1) > CACNA2D2 (23.7 +/- 7.1) > RASSF1A (15.1 +/- 5.6) > CDC25A (5.3 +/- 2.3) > SCOTIN (0.58 +/- 0.54). Immunohistochemical analysis of CDC25A showed its localization both in cytoplasm and nucleus in primary lesions and oral cancer cell lines. In absence of HPV infection, LTF and RASSF1A alterations jointly have adverse impact on survival of tobacco addicted patients. Thus, our data suggested that multiple candidate TSGs in the chromosomal 3p21.31 region were differentially associated with the early dysplastic lesions of head and neck.     

DNA甲基转移酶1在子宫颈病变组织和子宫颈癌细胞中的异常表达及其意义

 目的　探讨DNA甲基转移酶1（DNMT1）在子宫颈病变组织和子宫颈癌细胞中的异常表达及其意义。方法　选择经病理学确诊的子宫颈鳞状细胞癌（SCC）患者80例、子宫颈上皮内瘤样变（CIN）患者105例（CINⅠ 52例,CINⅡ／Ⅲ 53例）和子宫颈正常（NC）者53名,收集其手术或活组织检查子宫颈组织标本。选择子宫颈癌Caski细胞（HPV16阳性）和C33A细胞（HPV阴性）进行体外实验。分别采用Western blot和实时荧光聚合酶链反应（qRT-PCR）法检测子宫颈组织和细胞中DNMT1蛋白和mRNA的表达。结果　DNMT1蛋白和mRNA的表达水平在CIN及SCC组织中均较NC组织高,差异均有统计学意义（F＝110.57,P＜0.001;F＝2.68,P＝0.048）;随着子宫颈病变程度的加重,DNMT1蛋白表达水平呈逐渐上升趋势（χ2趋势＝50.80,P＜0.001）,但DNMT1 mRNA的表达在调整子宫颈癌相关因素后,未显示相同趋势（χ2趋势＝3.63,P＞0.05）。Caski 细胞DNMT1蛋白和mRNA的表达水平均较C33A细胞高,特别是mRNA的表达水平差异有统计学意义（t＝7.134,P＝0.002）。结论　DNMT1蛋白和mRNA高表达均是导致子宫颈癌和癌前病变发生的危险因素,HPV16感染与DNMT1转录功能异常对子宫颈癌的发生可能有协同效应。     

SEMA3B和SEMA3F基因在宫颈癌中的表达及其临床意义

目的:探讨新型候选抑癌基因SEMA3B和SEMA3F在宫颈癌组织中的表达及其与宫颈癌发生、发展的关系。方法:采用逆转录-聚合酶链反应(RT-PCR)方法检测40例宫颈癌组织、10例宫颈上皮内瘤样病变(CIN)组织及10例正常宫颈组织中SEMA3B和SEMA3F基因产物的表达水平。结果:宫颈癌组织、CIN及正常宫颈组织中SEMA3B的表达缺失率分别为65·0%、20·0%和0,P<0·05;SE-MA3F的表达缺失率分别为37·5%、20·0%和0,P>0·05。晚期(Ⅲ~Ⅳ期)宫颈癌组织中SEMA3B的表达缺失率(85·0%)明显高于其在早期(Ⅰ~Ⅱ期)宫颈癌组织中的表达缺失率(45·0%),χ2=7·03,P=0·008;SEMA3F与宫颈癌的临床分期无明显关系,χ2=0·10,P=0·744;SEMA3F在宫颈鳞癌中的表达缺失率(52·0%)明显高于其在宫颈腺癌中的表达缺失率(13·3%),χ2=5·98,P=0·014;而SEMA3B的表达异常与宫颈癌的组织类型无关,χ2=3·55,P=0·06。两者与组织学分级均无明显关系,P>0·05。结论:SEMA3B表达异常可能在宫颈癌发生、发展及预后中起重要作用。     

抑癌基因ING3在大肠癌中表达及意义

目的：探讨ING3在大肠癌中的表达及意义。方法收集82例大肠癌临床手术标本,通过荧光定量PCR检测癌旁正常黏膜中与癌组织中ING3的表达水平,分析ING3的表达水平与临床病理特征及预后的关系。结果51％的大肠癌患者中,ING3 mRNA在癌组织中的表达水平显著低于癌旁组织, ING3的表达与临床T分期、M 分期及TNM分期密切相关（P＜0．05）。单因素分析显示出T、N、M分期、TNM分期及ING3的表达均与患者术后总无病生存率相关（ P＜0．05）。 ING3低表达组患者长期无病生存率较中高表达组患者显著降低（ P＜0．05）。结论 ING3的表达与大肠癌发生发展相关,有望成为大肠癌早期诊断早期治疗的分子靶点。