Decreased expression levels of DAL-1 and TOB1 are associated with clinicopathological features and poor prognosis in gastric cancer

PurposeWe previously demonstrated that the functional inactivation of DAL-1 and TOB1 promotes an aggressive phenotype in gastric cancer cells, but the links between both genes and the survival of patients with gastric cancer are unknown. Here, we investigated the correlations of the expression levels of DAL-1 and TOB1 with the progression of gastric cancer.MethodsA total of 270 patients who underwent resectable gastrectomy were included. The expression of DAL-1 and TOB1 was detected by immunohistochemistry.ResultsLow expression of DAL-1 in cancer tissue was significantly associated with tumor site (p < 0.05), histological grade (p < 0.01), depth of invasion (p < 0.05), lymph node metastasis status (p < 0.05), Lauren classification (p < 0.001), and clinical stage (p < 0.01). A lower level of TOB1 was observed in gastric cancer patients with diffuse type disease compared to patients with either intestinal or mixed type disease (p < 0.001). Additionally, Spearman’s correlation analysis revealed that decreased expression of DAL-1 was positively correlated with low TOB1 expression (r=0.304, p < 0.001). The survival analysis showed that low levels of DAL-1 and TOB1 were significantly associated with poor survival of gastric cancer patients (p <0.001 and p < 0.05, respectively).ConclusionThe downregulation of DAL-1 and TOB1 expression is associated with shorter survival of gastric cancer patients. Hence, DAL-1 and TOB1 may be considered potential novel markers for predicting the outcomes of patients with gastric cancer.     

抗增殖蛋白TOB1影响乳腺癌细胞MDA-MB-231体外转移的作用研究

目的研究抗增殖蛋白家族成员TOB1对乳腺癌细胞MDA-MB-231体外侵袭与转移的生物学作用与机制。方法采用脂质体介导的质粒转染和G418筛选培养法获得稳定高表达TOB1的高转移性乳腺癌细胞系MDA-MB-231。通过人工基底膜侵袭实验和划痕实验检测TOB1过表达对MDA-MB-231细胞体外侵袭能力和转移能力的影响。采用super array基因芯片法检测TOB1引起的肿瘤转移相关基因表达的变化。结果与对照组相比,外源性TOB1高水平表达使MDA-MB-231的体外侵袭能力显著降低（P〈0.05）,同时抑制MDA-MB-231的体外迁移能力;TOB1表达水平的增加引起MDA-MB-231细胞中肿瘤转移抑制基因BRMS1表达的明显增加,同时显著抑制肿瘤转移相关基因HSP90、FXYD5、RHOC、S100A4等的表达（均P〈0.05）。结论 TOB1过表达抑制乳腺癌细胞MDA-MB-231的体外侵袭、转移,涉及的机制可能与TOB1对多种重要的肿瘤转移相关基因的表达调控有关。     

川芎嗪对妥布霉素肾毒性保护作用及机理的实验研究

目的：为减轻妥布霉素的肾毒性．应用川芎嗪进行预防其肾毒性的实验研究。方法：实验大鼠分三组：Ⅰ组正常对照组、Ⅱ组中毒模型组、Ⅲ组川芎嗪预防组。给大鼠腹腔注射妥布霉紊制成中毒模型。注射川芎嗪预防肾毒性。用药前、后检测大鼠的肾功能、尿酶等指标,实验结束后进行肾组织学检查。结果：发现模型组尿NAG酶（N-乙酰-β—D氨基葡萄糖苷酶）比正常组增高．血清中超氧化物歧化酶（SOD）活性低于正常组．血中的尿素氮（BUN）及肌酐（Cr）含量均比正常组增高。形态学检查可见模型组肾近曲小管上皮细胞广泛性坏死。川芎嗪预防组各项功能指标有所改善．形态学检查变性情况减轻。结论：川芎嗪对妥布霉素的肾毒性有预防保护作用。     

miR-32-5p靶向TOB1基因调控结直肠癌细胞放射增敏性及迁移侵袭能力机制研究

目的 探讨miR-32-5p对结直肠癌细胞放射敏感性及迁移侵袭能力的影响及其潜在作用机制。方法 培养人结直肠癌SW480细胞和正常结肠上皮NCM460细胞,将结直肠癌细胞分为未转染组和转染组(分别转染anti-miR-NC、anti-miR-32-5p、pcDNA、pcDNA-TOB1、anti-miR-32-5p+si-NC、nti-miR-32-5p+si-TOB1)。RT-qPCR和Western blot分别检测细胞中miR-32-5p、TOB1 mRNA和蛋白表达,克隆形成实验检测转染各组细胞放射敏感性,Transwell实验检测转染各组细胞迁移、侵袭能力,双荧光素酶报告基因实验和Western blot验证miR-32-5p是否靶向TOB1。结果 与人正常结肠上皮细胞相比,结肠癌细胞中miR-32-5p表达明显上调(P<0.05),TOB1 mRNA和蛋白表达明显下调(P<0.05)。与anti-miR-NC比较,anti-miR-32-5p组细胞放射增敏比1.801,细胞迁移和侵袭数目均明显减少(P<0.05);与pcDNA组比较pcDNA-TOB1组细胞放射增敏比1.764,细胞迁移数目和侵袭数目均明显减少(P<0.05)。双荧光素酶报告基因实验和Western blot结果证实miR-32-5p靶向负调控TOB1蛋白表达。与anti-miR-32-5p+si-NC组比较,anti-miR-32-5p+si-TOB1组细胞放射增敏比为0.591,细胞迁移数目和侵袭数目均明显增多(P<0.05)。结论 抑制miR-32-5p表达能够明显增强结直肠癌细胞放射敏感性,抑制细胞迁移和侵袭,其作用机制可能与靶向促进TOB1表达有关。     

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。     

Suppression of human lung cancer cell proliferation and metastasis in vitro by the transducer of ErbB-2.1 (TOB1)

Aim:

To investigate the effects of the transducer of ErbB-2.1 (TOB1) on the proliferation, migration and invasion of human lung cancer cells in vitro.

Methods:

Human lung cancer cell lines (95-D, A549, NCI-H1299, NCI-H1975, NCI-H661, NCI-H446, NCI-H1395, and Calu-3) and the normal human bronchial epithelial (HBE) cell line were tested. The expression levels of TOB1 in the cells were determined with Western blot and RT-PCR analyses. TOB1-overexpressing cell line 95-D/TOB1 was constructed using lipofectamine-induced TOB1 recombinant plasmid transfection and selective G418 cell culture. The A549 cells were transcend-transfected with TOB1-siRNA. MTT assay, flow cytometry and Western blot analysis were used to examine the effects of TOB1 on cancer cell proliferation and wound healing. Transwell invasive assay was performed to evaluate the effects of TOB1 on cancer cell migration and invasion. The activity of MMP2 and MMP9 was measured using gelatin zymography assay.

Results:

The expression levels of TOB1 in the 8 human lung cancer cell lines were significantly lower than that in HBE cells. TOB1 overexpression inhibited the proliferation of 95-D cells, whereas TOB1 knockdown with TOB1-siRNA promoted the growth of A549 cells. Decreased cell migration and invasion were detected in 95-D/TOB1 cells, and the suppression of TOB1 enhanced the metastasis in A549 cells. TOB1 overexpression not only increased the expression of the phosphatase and tensin homolog (PTEN), an important tumor suppressor, but also regulated the downstream effectors in the PI3K/PTEN signaling pathway, including Akt, ERK1/2, etc. In contrast, decreased expression of TOB1 oppositely regulated the expression of these factors. TOB1 also regulates the gelatinase activity of MMP2 and MMP9 in lung cancer cells.

Conclusion:

The results demonstrate that the PI3K/PTEN pathway, which is essential for carcinogenesis, angiogenesis, and metastasis, may be one of the possible signaling pathways for regulation of proliferation and metastasis of human lung cancer cells by TOB1 in vitro.     

miR-32-5p靶向TOB1基因调控结直肠癌细胞放射增敏性及迁移侵袭能力机制研究

目的 探讨miR-32-5p对结直肠癌细胞放射敏感性及迁移侵袭能力的影响及其潜在作用机制。方法 培养人结直肠癌SW480细胞和正常结肠上皮NCM460细胞,将结直肠癌细胞分为未转染组和转染组(分别转染anti-miR-NC、anti-miR-32-5p、pcDNA、pcDNA-TOB1、anti-miR-32-5p+si-NC、nti-miR-32-5p+si-TOB1)。RT-qPCR和Western blot分别检测细胞中miR-32-5p、TOB1 mRNA和蛋白表达,克隆形成实验检测转染各组细胞放射敏感性,Transwell实验检测转染各组细胞迁移、侵袭能力,双荧光素酶报告基因实验和Western blot验证miR-32-5p是否靶向TOB1。结果 与人正常结肠上皮细胞相比,结肠癌细胞中miR-32-5p表达明显上调(P<0.05),TOB1 mRNA和蛋白表达明显下调(P<0.05)。与anti-miR-NC比较,anti-miR-32-5p组细胞放射增敏比1.801,细胞迁移和侵袭数目均明显减少(P<0.05);与pcDNA组比较pcDNA-TOB1组细胞放射增敏比1.764,细胞迁移数目和侵袭数目均明显减少(P<0.05)。双荧光素酶报告基因实验和Western blot结果证实miR-32-5p靶向负调控TOB1蛋白表达。与anti-miR-32-5p+si-NC组比较,anti-miR-32-5p+si-TOB1组细胞放射增敏比为0.591,细胞迁移数目和侵袭数目均明显增多(P<0.05)。结论 抑制miR-32-5p表达能够明显增强结直肠癌细胞放射敏感性,抑制细胞迁移和侵袭,其作用机制可能与靶向促进TOB1表达有关。     

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     

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。