14-3-3sigma expression is an independent prognostic parameter for poor survival in colorectal carcinoma patients.

PURPOSE: 14-3-3sigma is an intracellular, dimeric, phosphoserine binding protein that is expressed in epithelial cells and involved in cancer development. In this study, we examined the expression of 14-3-3sigma and evaluated its clinical significance in colorectal carcinoma. EXPERIMENTAL DESIGN: Expression of 14-3-3sigma was analyzed by Western blot in nine colorectal carcinoma cell lines, eight paired colorectal carcinoma tissues, and normal mucosas. Immunohistochemistry was used to evaluate expression of 14-3-3sigma in tissues of 121 colorectal carcinoma patients and to correlate it with clinical parameters. RESULTS: Western blot analysis of colorectal carcinoma cell lines and tissues revealed strong 14-3-3sigma expression in four of eight cell lines and 14-3-3sigma overexpression in carcinomas compared with normal mucosa in six of eight colorectal carcinoma tissue pairs. Immunohistochemical analysis revealed 14-3-3sigma overexpression in 38.8% of colorectal carcinoma samples. Furthermore, highly positive immunoreactivity was significantly correlated with tumor differentiation (P < 0.001) and pT stage (P < 0.003). In Kaplan-Meier analysis, 14-3-3sigma overexpression was associated with a significantly decreased survival time compared with negatively stained or low stained cases (P < 0.0096). In multivariate regression analysis, 14-3-3sigma expression emerged as a significant independent parameter (P < 0.037). CONCLUSIONS: These results provide evidence that 14-3-3sigma expression increases during carcinoma progression in a subset of colorectal carcinoma. The overexpression of this antigen identifies patients at high risk. It is tempting to suggest that 14-3-3sigma overexpression either promotes tumor proliferation and/or prevents apoptotic signal transduction in colorectal carcinoma. Thus, targeting 14-3-3sigma might be a new therapeutic strategy in colorectal carcinoma.     

上皮性卵巢组织G3BP和OPN表达及相关性研究

目的 卵巢癌是妇科肿瘤中死亡率居首的常见恶性肿瘤,寻找新的生物学标志物以提高卵巢癌诊治的敏感性与特异性至关重要.本研究探讨GTP酶激活蛋白SH3功能区结合蛋白(Ras-GTPase-activating protein SH3 domainbinding protein,G3BP)和骨桥蛋白(osteopontin,OPN)在上皮性卵巢组织中的表达、意义及其表达的相关性,为卵巢肿瘤的临床诊断及治疗提供依据.方法 采用免疫组织化学方法检测G3BP和OPN在来源于中国医科大学盛京医院2009-10-01-2015-05-31的11例正常卵巢组织、20例卵巢上皮性良性肿瘤组织、31例卵巢上皮性交界性肿瘤组织及60例卵巢上皮性恶性肿瘤组织中的表达情况;并对二者表达的相关性进行统计分析.结果 G3BP在正常卵巢上皮组织、卵巢上皮性良性肿瘤组织、卵巢上皮性交界性肿瘤组织及卵巢上皮性恶性肿瘤组织中的表达呈递增趋势,其中上皮性良性肿瘤组织与正常卵巢上皮组织比较,x2=66.38,P＜0.01;上皮性交界性肿瘤组织与上皮性良性肿瘤组织比较,x2 =41.183,P＜0.01;上皮性恶性肿瘤组织与上皮性交界性肿瘤组织比较,x2=73.342,P＜0.01.OPN在正常卵巢上皮组织、卵巢上皮性良性肿瘤组织、卵巢上皮性交界性肿瘤组织及卵巢上皮性恶性肿瘤组织中的表达呈递增趋势,其中上皮性良性肿瘤组织与正常卵巢上皮组织比较,x2=60.005,P＜0.01;上皮性交界性肿瘤组织与上皮性良性肿瘤组织比较,x2 =43.844,P＜0.01;上皮性恶性肿瘤组织与上皮性交界性肿瘤组织比较,x2=73.429,P＜0.01.G3BP与OPN在卵巢上皮性恶性肿瘤组织中的表达具有正相关性,r=0.351,P＜0.01.结论G3BP和OPN表达升高与上皮性卵巢癌的发生发展有密切关系,且二者可能存在协同作用.     

Inactivation of the 14-3-3 sigma gene is associated with 5' CpG island hypermethylation in human cancers

The cell cycle checkpoint plays an important role in maintaining the integrity of cells. Recently, one of the 14-3-3 protein family members, 14-3-3sigma, was shown to be regulated by p53 and to play a role in the G2-M-phase checkpoint. To determine whether 14-3-3sigma is inactivated in human cancers, the methylation status of the 5' region of 14-3-3sigma was investigated in a series of gastric, colorectal, and hepatocellular cancer cell lines. Of 22 cell lines examined, 6 showed aberrant methylation. The methylation status of 14-3-3sigma was found to be correlated with loss of expression, which was restored by 5-aza-2'-deoxycytidine treatment. Furthermore, normal G2 arrest after DNA damage was not demonstrated in the cell lines with methylation. In primary gastric cancers, 14-3-3sigma hypermethylation was observed frequently in 26 of 60 (43%) cases and observed more frequently in poorly differentiated adenocarcinomas (P = 0.0017). Our findings suggest that 14-3-3sigma is inactivated by aberrant methylation of the 5' region in various human cancers and that it might play an important role in the development of undifferentiated gastric cancers.     

上皮性卵巢肿瘤RASSF1A基因甲基化与蛋白表达的关系及意义

目的:检测上皮性卵巢肿瘤中RASSF1A基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与蛋白表达的关系及其意义。方法:运用甲基化特异性PCR(MSP)方法对62例上皮性卵巢癌、21例交界性囊腺瘤及30例良性囊腺瘤的RASSF1A基因启动子甲基化状态进行检测,免疫组化S-P法检测上述标本中RASSF1A蛋白表达,并结合肿瘤生物学行为进行分析。结果:上皮性卵巢癌组织、卵巢交界性囊腺瘤组织中RASSF1A基因启动子区甲基化率(58.06%、42.85%)显著高于卵巢良性囊腺瘤组织(13.33%),差异有统计学意义(P<0.05)。RASSF1A基因启动子区甲基化与上皮性卵巢癌的细胞分化程度和临床分期密切相关(P<0.05)而与组织类型和患者年龄及绝经与否无相关性(P>0.05);RASSF1A基因甲基化与其蛋白表达下降一致。结论:卵巢癌组织中存在RASSF1A基因启动子CpG岛的异常甲基化,可能和该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一。     

Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma

The 14-3-3 sigma gene has been implicated in G2/M cell cycle arrest by p53. Frequent inactivation of the 14-3-3 sigma gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of this study was to examine the methylation status of CpG islands of the 14-3-3 sigma gene in hepatocellular carcinoma (HCC). The methylation status of the 14-3-3 sigma gene was evaluated in four normal liver tissues and 19 paired specimens of carcinoma and adjacent non-tumorous liver tissues using bisulfite-single strand conformation polymorphism (bisulfite-SSCP), a combination of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. The 14-3-3 sigma protein expression was examined by immunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 sigma gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samples analysed by immunohistochemistry showed loss of 14-3-3 sigma expression, while both of the methylation-negative HCC samples retained the expression, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumorous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis tissues), but the 14-3-3 sigma expression was retained in all of these tissues. In a methylation-positive HCC cell line, HLE, 5-aza-2'-deoxycytidine (5-aza-dC)-induced demethylation of CpG islands led to reactivation of gene expression, indicating that hypermethylation plays a causal role in inactivation of the 14-3-3 sigma gene in HCC. Hypermethylation and the resulting loss of expression of the 14-3-3 sigma gene corresponds to one of the most common abnormalities reported to date in HCC, suggesting their crucial role in the development and/or progression of HCC.     

上皮性卵巢肿瘤RASSF1A基因甲基化与蛋白表达的关系及意义

目的：检测上皮性卵巢肿瘤中RASSF1A基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与蛋白表达的关系及其意义。方法：运用甲基化特异性PCR（MSP）方法对62例上皮性卵巢癌、21例交界性囊腺瘤及30例良性囊腺瘤的RASSF1A基因启动子甲基化状态进行检测,免疫组化S-P法检测上述标本中RASSF1A蛋白表达,并结合肿瘤生物学行为进行分析。结果：上皮性卵巢癌组织、卵巢交界性囊腺瘤组织中RASSF1A基因启动子区甲基化率（58.06%、42.85%）显著高于卵巢良性囊腺瘤组织（13.33%）,差异有统计学意义（P〈0.05）。RASSF1A基因启动子区甲基化与上皮性卵巢癌的细胞分化程度和临床分期密切相关（P〈0.05）而与组织类型和患者年龄及绝经与否无相关性（P〉0.05）;RASSF1A基因甲基化与其蛋白表达下降一致。结论：卵巢癌组织中存在RASSF1A基因启动子CpG岛的异常甲基化,可能和该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一。     

微管相关蛋白LC3和自噬基因Beclin1在上皮性卵巢癌中的表达及其意义

背景与目的:有研究表明自噬活性的改变与肿瘤的发生、发展有关。诱导自噬性细胞死亡已经成为杀死肿瘤细胞的新策略。本研究检测微管相关蛋白LC3和自噬基因Beclin1在不同卵巢肿瘤组织中的表达情况,探讨其与上皮性卵巢癌发生、发展的相关性及意义。方法:用免疫组化法检测25例良性卵巢肿瘤、25例交界性卵巢肿瘤及75例上皮性卵巢癌组织的LC3和Beclin1表达,并结合临床病理因素进行分析。结果:良性肿瘤组LC3和Beclin1的阳性率(100%、100%)和交界性卵巢肿瘤组LC3和Beclin1的阳性率(96%、84%)都明显高于上皮性卵巢癌组(57%、57%),且差异具有统计学意义(P均<0.001)。LC3在上皮性卵巢癌中的表达与FIGO分期和肿瘤分化程度相关(P=0.017;0.001)。Beclin1表达与上皮性卵巢癌患者FIGO分期相关(P=0.04)。LC3和Beclin1在卵巢上皮癌中的表达无相关性(P=0.875)。结论:LC3和Beclin1在上皮性卵巢癌中的表达下调,自噬活性的改变可能与上皮性卵巢癌的发生、发展相关。     

Regulation of 14-3-3sigma expression in human thyroid carcinoma is epigenetically regulated by aberrant cytosine methylation

Increased 14-3-3sigma expression has been observed by immunohistochemistry in papillary and anaplastic tumors, but not follicular thyroid cancers. 14-3-3sigma mRNA expression and methylation status was examined in tumor cell lines and primary thyroid tissues using real-time RT-PCR, bisulfite sequencing and methylation-specific PCR. Most of the 27 CpG's in the gene's CpG island were methylated in normal thyroid, TPC-1, NPA, FTC-238 and 2-7, which did not express 14-3-3sigma. In contrast, they were unmethylated in KAK-1 and anaplastic lines KAT4 and DRO-90. 14-3-3sigma expression was not increased in thyroid carcinomas, the majority of which had a methylated CpG island. In addition, 5-aza-dC treatment increased 14-3-3sigma expression in the FTC-238 and NPA cell lines, which had low baseline expression. We conclude 14-3-3sigma expression in thyroid carcinomas is regulated by CpG island hypermethylation.     

Regulation of 14-3-3σ expression in human thyroid carcinoma is epigenetically regulated by aberrant cytosine methylation

Increased 14-3-3sigma expression has been observed by immunohistochemistry in papillary and anaplastic tumors, but not follicular thyroid cancers. 14-3-3sigma mRNA expression and methylation status was examined in tumor cell lines and primary thyroid tissues using real-time RT-PCR, bisulfite sequencing and methylation-specific PCR. Most of the 27 CpG's in the gene's CpG island were methylated in normal thyroid, TPC-1, NPA, FTC-238 and 2-7, which did not express 14-3-3sigma. In contrast, they were unmethylated in KAK-1 and anaplastic lines KAT4 and DRO-90. 14-3-3sigma expression was not increased in thyroid carcinomas, the majority of which had a methylated CpG island. In addition, 5-aza-dC treatment increased 14-3-3sigma expression in the FTC-238 and NPA cell lines, which had low baseline expression. We conclude 14-3-3sigma expression in thyroid carcinomas is regulated by CpG island hypermethylation.     

卵巢癌组织SEMA3F表达及甲基化初步研究

目的研究神经轴突导向分子SEMA3F在不同卵巢上皮肿瘤组织的表达及其与上皮性卵巢癌各临床病理参数及预后的关系,并初步探讨SEMA3F启动子区甲基化与上皮性卵巢癌的关系。方法选取2004-12-01-2008-12-30南通大学附属医院收集的95例卵巢癌、30例交界性卵巢上皮肿瘤、30例良性卵巢上皮肿瘤和10例正常卵巢组织标本,采用免疫组织化学方法检测SEMA3F的表达。应用半定量RT-PCR法检测2013-05-01-2013-12-31南通大学附属医院收集的12例新鲜上皮性卵巢癌、10例新鲜良性卵巢上皮肿瘤、10例新鲜正常卵巢组织中SEMA3F的表达水平,并通过亚硫酸氢盐测序法（BSP）检测上皮性卵巢癌中SEMA3F启动子区的甲基化情况,用SPSS 17.0统计软件分析数据。结果免疫组化显示,SEMA3F在不同卵巢上皮肿瘤组织中的表达水平不同,在上皮性卵巢癌组织中表达下降或缺失明显,结合临床资料分析显示,SEMA3F表达与上皮性卵巢癌的临床分期（χ2=27.100,P〈0.001）、组织学分级（χ2=10.673,P=0.005）及有无盆腔外腹膜转移（z=-4.447,P〈0.001）有关,但与患者的年龄（z=-0.670,P=0.503）、组织分类（χ2=3.729,P=0.444）和淋巴结转移（z=-0.584,P=0.559）无关。Kaplan-Meier分析显示,SEMA3F阴性或低表达组患者5年生存率为23.5%（16/68）,明显低于SEMA3F高表达组的52.6%（10/19）,差异有统计学意义,χ2=7.460,P=0.006。RT-PCR结果显示,SEMA3FmRNA相对表达量在正常卵巢组织（1±0.080）、良性卵巢上皮肿瘤（0.927±0.116）和上皮性卵巢癌中（0.436±0.122）依次下降,前两者表达差异无统计学意义,t=0.533,P〉0.05,但正常卵巢与上皮性卵巢癌组织中SEMA3F的表达差异有统计学意义,t=3.820,P〈0.05,且良性卵巢上皮肿瘤与上皮性卵巢癌组织中SEMA3F表达差异亦有统计学意义,t=2.979,P〈0.05。上皮性卵巢癌和正常卵巢组织中SEMA3F启动子区的平均甲基化率分别为23.17%和20.00%,两?     

鼻咽癌细胞株14-3-3σ启动子去甲基化与基因表达研究

目的 研究鼻咽癌细胞株14-3-3σ基因启动子甲基化状况及去甲基化处理对其表达的影响.方法 用甲基化特异性聚合酶链反应(PCR)和逆转录PCR方法检测鼻咽癌细胞株CNEI、CNE2、5-8F、6-10B和非肿瘤人鼻咽上皮细胞NP69细胞14-3-3σ基因启动子甲基化状况和mRNA表达水平.用不同浓度5-杂氮-2'-脱氧胞苷(5-aza-2dC)处理鼻咽癌细胞株72 h,检测处理组和对照组14-3-3σ基因启动子甲基化状况和mRNA表达水平,并用Western-blot检测14-3-3σ蛋白表达情况.结果 NP69细胞14-3-3σ启动子未检测到甲基化,CNE1、CNE2、5-8F、6-10B细胞14-3-3σ基因启动子都存在甲基化,4株肿瘤细胞的14-3-3σ mRNA表达水平显著低于NP69细胞.5-aza-2dC处理能剂量依赖性地逆转鼻咽癌细胞14-3-3σ启动子的甲基化状态并上调其mRNA和蛋白质的表达水平,高分化细胞株(CNE1)对药物的敏感性高于低分化细胞株(CNE2、5-8F和6-10B).结论 启动子甲基化是导致鼻咽癌细胞株14-3-3σmRNA和蛋白质表达降低的直接原因,14-3-3σ启动子去甲基化可能成为鼻咽癌治疗的新靶点.     

卵巢上皮性癌组织中DAPK基因启动子甲基化及其蛋白表达的研究

  目的  研究卵巢上皮性癌组织中DAPK基因启动子甲基化及其蛋白表达在卵巢癌发生发展过程中的作用及意义。  方法  应用甲基化特异性PCR(MSP)检测55例卵巢上皮性癌(恶性组)、25例卵巢交界性上皮性肿瘤(交界组)、30例卵巢良性上皮性肿瘤(良性组)、25例正常卵巢上皮组织(正常组)的石蜡包埋组织中DAPK基因启动子的甲基化情况。应用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连接(S-P)法检测上述蜡块组织中DAPK蛋白的表达情况。  结果  DAPK启动子在正常组、良性组、交界组、恶性组的甲基化率分别为0(0/25)、6.7%(2/30)、16.0%(4/25)、47.3%(26/55), 差异有统计学意义(P < 0.001), 恶性组的甲基化率高于其他三组; DAPK蛋白在正常组、良性组、交界组、恶性组的阳性表达率分别为96.0%(24/25)、90.0%(27/30)、48.0%(12/25)、30.9%(17/55), 差异有统计学意义(P < 0.001), 恶性组、交界组的阳性表达率均低于正常组和良性组; DAPK基因启动子甲基化和DAPK蛋白表达呈负相关。  结论  DAPK基因启动子甲基化导致基因沉默、失活, 引起蛋白表达下调或缺失, 并参与了卵巢上皮性癌的发生发展。      

Proteomic differential display analysis shows up-regulation of 14-3-3 sigma protein in human scirrhous-type gastric carcinoma cells

This study performed proteomic differential display analysis of human scirrhous-type gastric carcinoma (SGC) cell lines and normal gastric mucosa (NGM) tissues by using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The human SGC cell lines were OCUM-1, OCUM-2M, OCUM-2MLN, OCUM-2D, OCUM-D3, OCUM-9 and OCUM-12. Among the SGC cell lines and the NGM tissues, 28 protein spots were found whose expression levels were different from the results of 2-DE: 19 protein spots appeared higher, and 9 other protein spots appeared lower in SGCs than in NGM tissues. These spots were analysed by LC-MS/MS analysis and identified by a peptide sequence tag. Identified increased spots included elongation factor 1-beta, 14-3-3 sigma, tropomyosin alpha-4 chain, protein DJ-1, nucleoside diphosphate kinase A, elongation factor Tu and peroxiredoxin-1. Western blot analysis showed increased protein level of 14-3-3 sigma in SGCs. Although OCUM-1 and AGS (gastric cancer) showed up-regulation of 14-3-3 sigma, MiaPaca-2 (pancreatic cancer), Huh-7 (HCC) and NCI-H2052 (malignant pleural mesothelioma) showed very weak expression of 14-3-3 sigma. The up-regulation of 14-3-3 sigma may play an important role in SGC carcinogenesis and progression and may be used as a diagnostic biomarker of SGC.