肿瘤中TOB1功能相关的调控及其潜在的临床应用价值

TOB1基因编码蛋白是TOB/BTG抗增殖蛋白家族成员之一，目前已在多种肿瘤中发现TOB1表达下调、磷酸化积累和亚细胞分布异常。诸多研究表明TOB1基因的表达既受多种上游转录因子的调控，又能通过多种途径调控其下游靶基因的表达来影响肿瘤细胞的增殖、侵袭、转移和凋亡等，与肿瘤的发生发展密切相关。因此，深入探讨肿瘤中TOB1基因表达调控及相关功能改变在临床中的价值具有重要意义。本文旨在阐述TOB1基因及其相关功能的调控机制在临床肿瘤诊疗和预后评估中的潜在应用。     

TOB1基因功能及其与肿瘤关系的研究进展

摘 要：人类ErbB2转录因子1（TOB1）基因编码抗增殖蛋白Tob1,是一种多功能蛋白质。TOB1基因不仅涉及抑制细胞增殖和维持T细胞静止,还在诱导细胞凋亡、骨形成、学习记忆、精子发生和胚胎发育等过程中发挥重要作用。TOB1基因的表达失调或磷酸化累积可使其抗增殖功能明显下降或缺失,促进肿瘤形成和发展。全文对TOB1基因功能及其与肿瘤关联的研究进展进行综述。     

JUNB基因和TOB1基因在肝细胞癌中的表达及意义

目的：探讨JUNB基因和TOB1基因的表达在原发性肝细胞癌（HCC）发生发展中的作用.方法：应用半定量逆转录聚合酶链（RT-PCR）技术检测55例肝癌患者、55例其它肿瘤患者和33例对照组患者外周血JUNB基因和TOB1基因的表达.结果：各组间外周血JUNB基因和TOB1基因阳性表达率无差异,JUNB基因和TOB1基因在肝癌患者外周血中的表达水平明显高于其他组（分别为P=0.003和P=0.000）,其他组之间无差异.结论：JUNB基因和TOB1基因在外周血中的mRNA表达水平可能与肝癌发生密切相关,提示其表达水平可能与预测HCC发生、发展有明显相关性.     

miR-25在人胃癌组织中的表达分析及其靶基因TOB1的鉴定

  目的   分析miR-25在胃癌组织中的表达水平并鉴定其新的靶基因。   方法   选取10例胃癌患者的胃癌及癌旁正常组织样本, 用qRT-PCR法检测miR-25的表达水平。利用miRNA靶基因预测数据库预测miR-25的靶基因。构建荧光素酶载体(pMIR-TOB1)及绿色荧光蛋白报告载体(pMIR-GFP-TOB1)(含有miR-25结合位点的TOB1的3'UTR片段), 利用荧光素酶活性实验和GFP荧光强度报告实验鉴定miR-25的预测靶基因(TOB1)。qRT-PCR法和Western blot法分别检测TOB1的mRNA和蛋白质的表达水平。   结果   miR-25在80%(8/10)的胃癌组织样本中表达上调。miR-25模拟物显著抑制了荧光素酶的活性(P < 0.01)和GFP荧光强度。miR-25模拟物显著下调了AGS细胞中TOB1的mRNA(P < 0.05)和蛋白质的表达。   结论   miR-25在人胃癌组织中表达上调, miR-25能结合TOB1的3'UTR并下调TOB1的mRNA和蛋白质的表达。      

TOB1高表达抑制肺癌A549细胞体外迁移和侵袭

目的:探讨TOB1 (transducer of ErbB 2,1)高表达对人肺癌A549细胞体外迁移和侵袭的影响.方法:应用RT-PCR法检测人支气管上皮细胞HBE和10种肺癌细胞中TOB1、PTEN (phosphatase and tensin homolog deleted on chromosome ten)、乳腺癌转移抑制基因1(breast cancer metastasis suppressor 1,BRMS1)、RHOC(ras homolog gene family,member C)和NME1 (non-metastatic cells 1)mRNA的表达水平;将重组表达载体质粒pcDNA3.0-TOB1和“空载体”质粒pcDNA3.0转染到A549细胞中,建立TOB1高表达的A549/TOB1细胞和对照组A549/PC3细胞,应用体外划痕和Transwell实验检测细胞的体外迁移和侵袭能力,RT-PCR法检测细胞中PTEN、BRMS1、RHOC和NME1 mRNA的表达,蛋白质印迹法检测细胞中α-N-catenin、β-catenin、γ-catenin、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)和MMP9蛋白的表达.结果:10种肺癌细胞中TOB1 mRNA的表达水平明显低于HBE细胞(P＜0.05),不同细胞株中TOB1 mRNA与PTEN、BRMS1和NME1 mRNA的表达水平间呈正相关(P＜0.05).A549/TOB1细胞的体外迁移和侵袭能力明显低于A549细胞(P＜0.05).A549/TOB1细胞中PTEN、BRMS1和NME1 mRNA的表达水平明显高于A549细胞(P＜0.05),而RHOC mRNA的表达水平明显低于A549细胞(P＜0.05); A549/TOB1细胞中α-N-catenin、β-catenin、γ-catenin、MMP2和MMP9蛋白的表达水平明显低于A549细胞(P＜0.05).结论:外源性TOB1高表达可以明显抑制肺癌A549细胞的体外转移和侵袭,其机制可能与TOB1调控PTEN、BRMS1、RHOC和NME1的表达,进一步下调α-N-catenin、β-catenin、γ-catenin、MMP2和MMP9蛋白的表达水平有关.     

乳腺癌中PTEN蛋白表达及其与p27kip1和cyclin D1表达的相关性

目的 探讨抑癌基因PTEN蛋白在人乳腺癌中的表达情况,及其与抑癌基因p27^kiP1、癌基因cyclin D1表达的相关性。方法 应用免疫组织化学染色S-P法,检测6l例乳腺癌组织石蜡切片中PTEN蛋白、p27^kiP1和cyclin D1蛋白的表达情况,比较PTEN蛋白表达与临床病理指标的关系,及其与p27^kiP1、cyclin D1蛋白表达的相关性。结果 PTEN蛋白表达定位于细胞质。PTEN蛋白阴性占6．6％(4／61),表达减低者占41．0％(25／61),阳性表达者占52．5％(32／61)。PTEN与淋巴结状态、ER状态及局部复发、远处转移相关。单因素生存分析显示,PTEN蛋白高表达组的5年生存率(87．5％)高于低表达组(58．6％,P=0．0101)。PTEN蛋白表达与p27^kiP1、cyclin D1表达未显示有明显相关性。结论 乳腺癌患者PTEN蛋白低表达与预后不良有关,PTEN对乳腺癌肓一定的预后价值。在人乳腺癌中,p27^kiP1和cvclin D1可能不是PTEN的主要下游基因。     

WAF1基因蛋白在贲门癌中的表达及其预后价值

目的：检测贲门癌组织中WAF1基因蛋白的表达，分析其表达与临床病理参数之间的关系，并探讨其预后价值。方法：应用免疫组化二步法检测104例贲门癌组织中WAF1基因蛋白表达的状况，结合临床病理资料进行分析。结果：贲门癌组织中WAF1基因蛋白的表达主要定位于细胞核，其癌组织的阳性率为26．0％(27／104)。WAF1基因蛋白的表达与肿瘤的大小、分化程度、淋巴结转移及外侵密切相关，而且WAF1基因蛋白的表达率与不同的PTNM分期有关。结合随访资料分析表明WAF1基因蛋白的表达与预后有关。WAF1基因蛋白的表达与年龄、性别、大体类型、组织类型、浆膜是否受累及食管是否受累均无关。结论：贲门癌组织中WAF1基因蛋白的表达缺失与病变的进展相关，WAF1基因蛋白表达预后好，表达缺失预后差。通过免疫组化法检测WAF1基因蛋白在贲门癌中的表达可能有助于预测贲门癌患的预后。     

lncRNA TOB1-AS1在上皮性卵巢癌组织中的表达及其临床意义

目的：探讨 ERBB2.1 转导蛋白反义 RNA1（transducer of ERBB2.1 antisense RNA 1,TOB1-AS1）在上皮性卵巢癌（epithelial ovarian cancer,EOC）组织中的表达情况及其临床意义,初步探讨TOB1-AS1对EOC细胞体外增殖、迁移和侵袭的影响。方法：使用TCGA数据库对EOC组织中TOB1-AS1表达情况进行分析;收集2017年7月至2018年1月在河北医科大学第四医院妇科行肿瘤切除并经病理检查证实为EOC的67例患者的肿瘤组织,收集同期因其他妇科疾病接受手术的30例患者的非肿瘤卵巢组织作为对照。采用qPCR法检测EOC组织和非肿瘤卵巢组织中TOB1-AS1的表达水平,χ2检验分析TOB1-AS1的表达与不同临床病理特征之间的相关性,Kaplan-Meier和Cox比例风险回归模型分析患者生存及预后的潜在影响因素。CCK-8实验、划痕实验和Transwell实验分别检测敲低TOB1-AS1表达对EOC细胞SKOV3和A2780增殖、迁移和侵袭的影响。结果：TCGA数据库中资料和qPCR检测结果均显示,在EOC组织中TOB1-AS1的表达水平显著高于非肿瘤卵巢组织（均P<0. 01）。TOB1-AS1的高表达与EOC患者较晚的FIGO分期、较差的组织分级、淋巴结转移及腹膜转移有关（均P<0.05）。Kaplan-Meier生存分析结果显示,TOB1-AS1 高表达组患者术后 DFS 和 OS 均短于 TOB1-AS1 低表达组（均 P<0.05）。Cox 比例风险回归模型分析结果显示,FIGO分期、淋巴结转移、腹膜转移及TOB1-AS1表达是EOC患者预后的独立影响因素（均P<0.05）。TOB1-AS1在EOC细胞系SKOV3、A2780中的表达水平也显著高于正常卵巢上皮细胞系IOSE80（均P<0.01）。细胞功能实验结果显示,敲低TOB1-AS1可抑制SKOV3和A2780细胞的增殖、迁移和侵袭（均P<0.05）。结论：TOB1-AS1在EOC组织中高表达,与患者的不良预后显著相关。TOB1-AS1可能通过促进EOC细胞SKOV3、A2780的增殖、迁移和侵袭来影响EOC的恶性进展。     

钟基因hClock、hBmal1在结直肠肿瘤中的 表达

 目的 探讨人类生物钟基因hClock及hBmal1在不同Dukes分期结直肠肿瘤中的表达,研究它们的表达与结直肠肿瘤侵袭及转移的关系。方法 采用原位杂交法检测结直肠肿瘤与相应瘤旁组织中hClock及hBmal1基因mRNA的表达,并采用免疫组织化学检测相应标本中hClock基因蛋白产物（CLOCK蛋白）的表达。结果 32例结直肠肿瘤中hClock mRNA阳性表达率93.75%（30／32）,中或强阳性表达率78.13%（25／32）;hBmal1 mRNA阳性表达率71.88%（23／32）,中或强阳性表达率40.63%（13／32）。两者的中或强阳性率与Dukes分期相关（P<0.05）。阳性表达的肿瘤组织对应肿瘤旁组织hClock及hBmal1基因mRNA的弱阳性表达。CLOCK蛋白中或强阳性表达率87.50%（28／32）,瘤旁组织中CLOCK蛋白弱阳性率93.75%（30／32）（P<0.01）。结论 hClock、hBmal1基因与结直肠肿瘤的发生、发展及侵袭、转移有一定的相关性。     

转染外源CC10对肺腺癌细胞系A549的CyclinD1蛋白及mRNA的影响(英文)

目的研究转染外源性CC10基因对肺腺癌A549细胞株的细胞周期及周期蛋白CyclinD1蛋白和mRNA表达影响。方法用脂质体法分别将外源性抑癌基因CC10转染到人肺腺癌细胞株A549中,8h、16h、24h、36h、48h后,分别用Western blot法检测转染CC10基因的A549细胞CC10蛋白表达,用流式细胞仪观察细胞周期的变化,用免疫细胞化学及RT-PCR检测转染CC10基因对周期蛋白CyclinD1蛋白、mRNA的影响。结果转染外源CC10基因对A549细胞生长具有抑制作用,细胞生长阻止于G0／G1,细胞周期S期+G2／M期比例下降。转染外源 CC10基因的A549细胞CC10蛋白表达增高,而CyclinD1的蛋白及mRNA表达明显降低。结论转染外源CC10基因对肺癌细胞G0／G1周期的抑制作用可能与CyclinD1基因表达下调有关。     

Alteration of expression or phosphorylation status of tob, a novel tumor suppressor gene product, is an early event in lung cancer

Tob is a member of the Tob/BTG family, a novel class of anti-proliferative proteins. To investigate the involvement of tob as a tumor suppressor gene in human lung cancer, we analyzed the expression of tob mRNA and protein in lung cancer tissue and adjacent normal lung tissue. Immunohistochemical analysis using anti-Tob antibody showed decreased expression of Tob in 72% (31/43) of lung cancer tissues. Furthermore, 95% (19/20) of squamous cell carcinoma patients showed an apparent decrease in Tob in cancer tissues, associated with smoking status. The phosphorylated form of Tob, an inactive form of Tob, was detected in 76% (16/21) of cancer tissues of adenocarcinoma patients, but not in normal alveolar epithelial cells. Either a decrease in Tob expression or an accumulation of phosphorylated Tob was observed from early clinical stages, even in bronchial dysplasia, a premalignant lesion of squamous cell carcinoma. The above findings suggest that the disruption of anti-proliferative Tob plays a distinct part in the early stage of lung carcinogenesis.     

High expression of Tob1 indicates poor survival outcome and promotes tumour progression via a Wnt positive feedback loop in colon cancer

Tob1, a Tob/BTG anti-proliferative protein family member, functions as a tumour suppressor in many cancers. Here, we reveal a unique oncogenic role of Tob1 in colon cancer. Tob1 expression was upregulated during colon cancer progression, was significantly correlated with tumour size and tumour differentiation, and was a prognostic indicator of colon cancer. Unlike in other cancers, where nuclear Tob1 performs anticancer activity, Tob1 is predominantly localized in the cytosol of colon cancer cells, where this protein binds and stabilizes β-catenin to activate Wnt/β-catenin signalling, which in turn enhances Tob1 expression, thus forming a positive feedback loop to promote cell proliferation. Moreover, Tob1 deficiency led to reduced tumourigenesis in AOM/DSS-treated and Apc^Min/+ mice. Our findings provide important insights into a previously unrecognized oncogenic role of Tob1 in colon cancer and suggest that Tob1 is an adverse prognostic factor and therapeutic target for colon cancer.     

TOB suppresses breast cancer tumorigenesis

Transducer of ErbB‐2 (TOB) is a member of the TOB/Btg gene family. A role for TOB in the suppression of human tumorigenesis has been proposed, based on the observations that TOB‐knockout mice spontaneously form tumors and TOB expression is lost in human lung and thyroid cancers. However, the role of TOB in human breast cancer remains unknown. To evaluate the this role, we screened a panel of breast cancer cell lines for TOB expression levels and found that they are inversely correlated with the tumorigenicity and metastatic potential of the cell lines. In addition, we demonstrated for the first time that TOB expression is inversely correlated with breast cancer progression in clinical specimens. These results strongly indicate that the loss of TOB expression plays a role in breast cancer progression. We have also provided the first evidence that TOB functions as a tumor suppressor in breast cancer MCF‐7 cells, using gain‐of‐function and loss‐of‐function approaches to manipulate TOB expression. Cell‐cycle analysis further revealed that TOB can prolong the G1‐S phase transition by inducing arrest at G1‐S phase. Moreover, upregulation of the cyclin‐dependent kinase inhibitor p27 and downregulation of the antiapoptotic proteins Bcl‐2 and Bcl‐XL were observed in MCF7/TOB transfectants. Conversely, opposite results were observed in shRNA‐TOB transfectants. Furthermore, decreased activity of Erk2, AKT, CrkL, PDK1, and Smads were observed in TOB‐overexpressing cells. Taken together, these data provide evidence that TOB can function as a tumor suppressor in breast cancer through modulation and regulation of multiple signaling pathways. © 2009 UICC     

Degradation of Tob1 mediated by SCFSkp2-dependent ubiquitination

Tob1, a member of the Tob/BTG family, is involved in the control of G(1)-S progression by suppressing cyclin D1 expression and acts as a tumor suppressor gene. Tob1 was reported to have a quick turnover through the ubiquitin-proteasome pathway, but proteins involved in this process are still unknown. We showed that Skp2, a substrate-targeting subunit of the SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complex, was involved in ubiquitin-dependent degradation of Tob1. Skp2 interacted with Tob1 and facilitated ubiquitination of Tob1 in intact cells as well as in vitro. Skp2 mutants without the F-box or leucine rich repeat were not able to bind to Tob1 and did not enhance ubiquitination of Tob1. Tob1 was stabilized in both Skp2(-/-) mouse fibroblasts and Skp2 knockdown HeLa cells. Moreover, cyclin D1 expression was suppressed in Skp2 knockdown HeLa cells. These data suggest that Tob1 is a novel target for degradation by the SCF-Skp2 ubiquitin ligase.     

PGE2通过EP1受体促进肝癌细胞生长和侵袭的研究进展

前列腺素E2（PGE2）是一种参与肝癌细胞生长与侵袭的重要细胞因子。PGE2通过与细胞膜表面的EP受体结合来发挥其调控肿瘤发生、发展的作用。在肝癌中,PGE2通过前列腺素E受体1（EP1）受体调节EGFR／PI3K／Akt、Src／EGFR／p44／42 MAPK／mTOR等信号通路影响Survivin、YB-1等关键物质,促进肝癌细胞生长和侵袭。现就近年来PGE2通过EP1受体促进肝癌细胞生长和侵袭作用机制的研究进行综述。