中国科技论文统计源期刊 中文核心期刊  
美国《化学文摘》《国际药学文摘》
《乌利希期刊指南》
WHO《西太平洋地区医学索引》来源期刊  
日本科学技术振兴机构数据库(JST)
第七届湖北十大名刊提名奖  
医药导报, 2017, 36(10): 1119-1123
doi: 10.3870/j.issn.1004-0781.2017.10.008
Y-box结合蛋白1与人肝癌耐药细胞Bel-7402/ADM P糖蛋白的相关性*
Correlation Between Y-box Binding Protein-1 Expression and P-Glycoprotein in Drug-resistant Hepatocellular Carcinoma Bel-7402/ADM Cells
王姗姗1,, 汪选斌2,

摘要:

目的 研究Y-box结合蛋白1(YB-1)与人肝癌耐药细胞Bel-7402/ADM中P糖蛋白 (P-gp)的关系,推测其诱导癌细胞产生耐药性的相关机制。方法 以浓度梯度递增联合大剂量间断冲击诱导法建立人肝癌耐药细胞株Bel-7402/ADM;运用实时荧光定量聚合酶链反应(RT-PCR)检测YB-1、P-gp在人肝癌耐药细胞株Bel-7402/ADM 和相应敏感株 mRNA含量;采用Western blotting检测YB-1、P-gp在两种细胞株的表达量;将YB-1 siRNA转染耐药细胞,检测转染前后YB-1和MDR1在 mRNA水平和蛋白水平的变化。结果 多柔比星对Bel-7402和Bel-7402/ADM的半数抑制浓度(IC50)值分别为(2.23±0.07)和(7.02±0.03) μmol·L-1。耐药组MDR1 mRNA的表达量高于亲本细胞(P<0.01),YB-1 mRNA的表达量也显著高于亲本细胞(P<0.01)。耐药组P-gp表达量高于亲本细胞(P<0.05),YB-1表达量也显著高于亲本细胞(P<0.01)。耐药细胞转染siRNA后YB-1 mRNA、MDR1 mRNA的表达显著降低(P<0.01),YB-1、MDR1蛋白水平较转染前显著降低(均P<0.05)。结论 YB-1表达与P-gp表达密切相关,YB-1与人肝癌耐药细胞株Bel-7402/ADM多药耐药相关。

关键词: 多药耐药 ; P糖蛋白 ; Y-box结合蛋白1

Abstract:

Objective To explore the correlation between Y-box binding protein-1 (YB-1) and P-glycoprotein (P-gp) in drug-resistant hepatocellular carcinoma (HCC) Bel-7402/ADM cells, and speculate the related mechanism of drug resistance. Methods Bel-7402/ADM cells were developed by concentration gradient escalation and intermittent administration of large dose. The levels of YB-1 mRNA and MDR1 mRNA were detected by means of RT-PCR.Western blotting was used to detect the protein expression of YB-1 and P-gp in the Bel-7402 cells and doxorubicin resistant Bel-7402 (Bel-7402/ADM) cells. Bel-7402/ADM cells were transfected with small interfering RNA (siRNA) targeting human YB-1. Expression levels of YB-1 and MDR1 mRNA and protein were detected by means of RT-PCR and Western blotting. Results IC50 values of ADM on hepatoma carcinoma cells Bel-7402 and Bel-7402/ADM were (2.23±0.07) and (7.02±0.03) μmol·L-1. The mRNA expression levels of MDR1 and YB-1 were all significantly higher in Bel-7402/ADM cells than in Bel-7402 (P<0.01). The mRNA expression levels of MDR1 and YB-1 in Bel-7402/ADM cells transfected with YB-1 siRNA were reduced significantly (P<0.01). The protein levels of YB-1 and MDR1 in Bel-7402/ADM cells transfected with YB-1 siRNA were reduced significantly (P<0.05). Conclusion These results suggest that the high expression level of YB-1 is probably correlated with multidrug resistance in HCC Bel-7402/ADM cells.

Key words: Multidrug resistance ; P-glycoprotein ; Y-box binding protein-1

肿瘤多药耐药(multidrug resistance,MDR)是指一种药物作用于肿瘤使之产生耐药性后,该肿瘤对未接触过、结构无关、机制各异的多种抗肿瘤药也具有交叉耐药的现象,是肿瘤耐药的主要原因。其机制与拓扑异构酶(TOPOⅡ)、P-糖蛋白(P-glycoprotein,P-gp)、谷胱甘肽S转移酶(GST-α)等的表达、凋亡相关通路改变等因素有关[1]。ABC(ATP-binding cassette)超家族转运体蛋白能够将药物分子排移至胞外,减弱其对细胞的毒性,导致肿瘤细胞耐药性的产生。此类蛋白包括P-gp、MRP、BCRP/ABCG2等,其中P-gp是最重要的耐药相关蛋白,由MDR1(即ATP-binding cassette sub-family B member 1,ABCB1)基因编码。Y-box 结合蛋白是一类特异性结合目的基因启动子和增强子内部Y-box 序列(CTGATTGGCCAA)的转录因子,广泛存在于从细菌到人类的许多物种中[2-3],发挥多种重要生物学功能。Y-box结合蛋白1(Y-box binding protein-1,YB-1)是Y-box 结合蛋白家族的成员之一,现已有大量证据表明,YB-1 与肿瘤的发生和维持存在广泛密切的联系[4-5]。YB-1 在肿瘤的发生中发挥多种重要作用,不仅促进癌细胞过度增殖和抗凋亡,还协助肿瘤扩散和转移,并直接参与染色体异常和癌组织耐药性的产生。诸多学者研究提示YB-1与人乳腺癌、肺癌、结直肠癌等的MDR密切相关[6-8],但YB-1的表达与人肝癌耐药细胞株Bel-7402/ADM(多柔比星)多药耐药的关系尚未检索到有关报道。笔者在本研究比较YB-1siRNA转染耐药细胞前后YB-1和MDR1在 mRNA水平和蛋白水平的变化,探讨YB-1与Bel-7402/ADM多药耐药的关系,为改善耐药状况的新型策略提供基础研究支持和参考。

1 材料与方法
1.1 材料

人肝癌亲本细胞Bel-7402,人肝癌耐药细胞株Bel-7402/ADM;RPMI1640培养液(GIBCO公司);胎牛血清(FBS,杭州四季青生物工程材料有限公司);P-gp单抗(Cell Signaling Technology 公司)、YB-1抗体(Cell Signaling Technology);SYBR RT-PCR kits(Toyobo Japan),TRIzol(Invitrogen);注射用多柔比星(ADM,浙江海正药业股份有限公司,规格:每支10 mg,批号:131105 );Lipofectamine 2000 Reagent(Invitrogen);细胞裂解液(碧云天生物技术研究所);辣根过氧化物酶标记羊抗兔 IgG、辣根过氧化物酶标记羊抗小鼠 IgG (美国Jackson 免疫研究实验室)。

1.2 仪器

二氧化碳(CO2)培养箱(Thermo Scientific Forma Series ⅡWater-Jacketed CO2 Incubators,HEPA filter,USA);BCM-100生物洁净工作台(苏州安泰空气技术有限公司);离心机(BR4i multifunction centrifuge,Berkman,USA);冰箱(海尔公司);MLS-3780高压灭菌锅(日本三洋公司);Sunrise Remote F039300型酶联免疫仪器(澳大利亚TECAN公司);DYY-7型转移电泳仪(北京市六一仪器厂);Mx3000P实时定量荧光PCR仪(Stratagene 公司);FD201型稳压电泳仪(上海医用分析仪器厂);凝胶成像分析系统(美国Bio-Rad公司)。

1.3 细胞的培养

人肝癌亲本细胞株Bel-7402培养于10% FBS的RPMI1640培养基中,置37 ℃、5%CO2培养箱中。耐药细胞株Bel-7402/ADM 经复苏后,培养于含有ADM的10%FBS RPMI1640培养基中,ADM的浓度从50 nmol·L-1起培养,逐步增加ADM浓度至终浓度2 000 nmol·L-1

1.4 噻唑蓝(MTT)法检测ADM对两种细胞株的抑制率

胰酶消化处理对数生长期细胞,用含10% FBS的RPMI 1640培养液配成细胞悬液,调整细胞浓度为4×103个,每孔接种200 μL。空白对照组没有细胞,只加培养液,阴性对照组不加任何药物,ADM浓度梯度组1,2,4,8,10 μmol·L-1。每组4个复孔,在培养箱中培养48 h后,弃掉培养液,每孔加入5 mg·mL-1MTT溶液 20 μL,含 10% FBS的RPMI 1640新鲜培养液180 μL置于培养箱中孵育4 h。弃培养液,每孔加入DMSO150 μL,振荡混匀,待紫色结晶完全溶解后,在酶联免疫仪检测波长为570 nm的吸光度值(A)。按下列公式计算细胞生长抑制率、半数抑制浓度(IC50)值、耐药倍数。生长抑制率(% )=(1-给药组平均A值/对照组平均A值) ×100%。 lgIC50=Xm-I[P-(3-Pm-Pn) /4 ][9],其中Xm:lg[最大剂量];I:lg[最大剂量/相邻剂量];P:阳性反应率之和;Pm:最大阳反应率;Pn:最小阳性反应率。耐药倍数=耐药细胞组IC50/亲本细胞组IC50

1.5 实时荧光定量聚合酶链反应(real-time PCR)

按照说明书用TRIzol提取细胞的总RNA,用逆转录试剂盒将RNA逆转录为cDNA,94 ℃ 5 min预变性,94 ℃ 10 s、58 ℃ 20 s和72 ℃ 15 s,40个循环,72 ℃5 min。所用引物序列见表1。本 实 验 采 用 目 标 基 因 定 量 分 析 比 较 ΔCt 值,以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)为内参,ΔΔCt=ΔCt(target)-ΔCt(GAPDH),实验结果用2-ΔΔCt值表示。

表1 PCR扩增所用引物及其序列
Tab.1 Primer sequence of PCR
引物 序列
GAPDH 5'-TCATTGACCTCAACTACATGGTTT-3'
5'-GAAGATGGTGATGGGATTTC-3'
MDR1 5'-AGCTCATCGTTTGTCTACAGTTCG-3'
5'-TCCACGGACACTCCTACGAGT-3'
YB-1 5'-CAGACTGCCATAAAGAAGAATAACC-3'
5'-TACTGCCTTGAACTGGAACACC-3'
siYB-1 5'-GGAGGCAGCAAAUGUUACATT-3'
5'-UGUAACAUUUGCUGCCUCCTT-3'

表1 PCR扩增所用引物及其序列

Tab.1 Primer sequence of PCR

1.6 Western blotting检测蛋白表达

提取两种细胞株的蛋白,BCA法绘制蛋白浓度标准曲线,测定提取的蛋白浓度。按照每泳道30 μg蛋白上样,蛋白样品置于10%十二烷基硫酸钠聚丙烯酰氨凝胶中电泳,再转到聚偏氟乙烯膜上。转膜成功后,用5% BSA室温孵育2 h。在4 ℃环境中一抗孵育过夜,TBST漂洗4次,每次5 min。然后用结合有辣根过氧化物的二抗37 ℃孵育膜1 h。二抗孵育结束参照说明书用化学发光试剂盒对膜显色后用Bio-Rad成像系统成像。

1.7 siRNA转染

转染前1天,将Bel-7402/ADM以2×105细胞数量接种在6孔板中,以转染时细胞汇合度在30%~50%为宜。在Opti-DMEM培养基250 μL中加入siRNA100 pmol,轻柔混匀;用Opti-DMEM培养基250 μL稀释lipofectamin试剂5 μL,轻轻混匀,室温放置5 min。将稀释好的siRNA和lipofectamin混合,室温放置20 min,以便形成siRNA/lipofectamin复合物。将转染复合物500 μL加到含有培养基的6孔板中,来回轻柔摇晃细胞培养板。细胞在CO2培养箱中37 ℃温育,6 h后,更换新鲜10%FBS1640培养基。细胞继续培养48 h后收集细胞检测 mRNA 和蛋白表达。

1.8 统计学方法

釆用SPSS 17.0版统计软件进行分析。计量资料以均数±标准差( x ¯ ±s)表示,组间均数比较采用方差分析,以 P<0.05为差异有统计学意义。

2 结果
2.1 MTT法检测ADM对两种细胞株的抑制率

ADM对两种细胞株的抑制率结果见图1,Bel-7402和Bel-7402/ADM的IC50值分别为(2.23±0.07)和(7.02±0.03) μmol·L-1。耐药倍数为3.15,两者比较差异有统计学意义(P<0.01)。

图1 ADM对 Bel-7402和Bel-7402/ADM细胞的抑制率(x¯±s,n=3)
与Bel-7402/ADM比较,*1P<0.01

Fig.1 Inhibition ratio of ADM on Bel-7402 cells and Bel-7402/ADM cells (x¯±s, n=3)
Compared with Bel-7402/ADM,*1P<0.01

2.2 YB-1、MDR1在两种细胞株 mRNA水平的检测

结果见图2。耐药组MDR1 mRNA的表达量高于亲本细胞(P<0.01),YB-1 mRNA的表达量也显著高于亲本细胞,均差异有统计学意义(P<0.01)。

2.3 YB-1、MDR1在两种细胞株蛋白水平的检测

结果见图3。耐药组P-gp的表达量高于亲本细胞(P<0.05),YB-1的表达量也显著高于亲本细胞,均差异有统计学意义(P<0.01)。

图2 MDR1 mRNA 和YB-1 mRNA在两种细胞株的表达(x¯±s,n=3)
与Bel-7402/ADM比较,*1P<0.01

Fig.2 Expression of MDR1 mRNA and YB-1 mRNA in two cell lines(x¯±s,n=3)
Compared with Bel-7402/ADM,*1P<0.01

图3 YB-1、MDR1在两种细胞株蛋白水平的表达(x¯±s,n=3)
与Bel-7402/ADM比较,*1P<0.05,*2P<0.01

Fig.3 Protein expression of YB-1 and MDR1 in two cell lines(x¯±s,n=3)
Compared with Bel-7402/ADM,*1P<0.05,*2P<0.01

2.4 YB-1、MDR1在Bel-7402/ADM转染siRNA后 mRNA水平的检测

耐药细胞转染siRNA后,通过RT-PCR验证YB-1的表达显著降低,差异有统计学意义(P<0.01),MDR1的表达也较转染之前显著降低,差异有统计学意义(P<0.01)。见图4。

图4 MDR1 mRNA 和YB-1 mRNA分别在Bel-7402/ADM 转染siRNA前后的水平(x¯±s,n=3)
与Bel-7402/ADM比较,*1P<0.01

Fig.4 Expression of MDR1 mRNA and YB-1 mRNA in Bel-7402/ADM cells before or after siRNA transfection(x¯±s,n=3)
Compared with Bel-7402/ADM , *1P<0.01

2.5 YB-1、MDR1在Bel-7402/ADM 转染siRNA蛋白水平的检测

耐药细胞转染siRNA后,YB-1、MDR1蛋白水平较转染前显著降低,均差异有统计学意义(均P<0.05)。见图5。

图5 MDR1和YB-1在Bel-7402/ADM 转染siRNA前后蛋白水平(x¯±s,n=3)
与Bel-7402/ADM比较,*1P<0.05

Fig.5 Protein expression of YB-1 and MDR1 in Bel-7402/ADM cells before or after siRNA transfection (x¯±s, n=3)
Compared with Bel-7402/ADM,*1P<0.05

3 讨论

肝细胞癌是我国常见消化系统恶性肿瘤,该肿瘤生长速度快,恶性程度高,侵袭转移程度高,预后凶险。化疗是治疗肝癌的重要手段。然而,MDR的产生是降低化疗效果重要原因,且MDR的发生机制十分复杂,其具体机制也没有完全明确。因此,进一步研究其发生机制并寻找逆转对策是目前肿瘤研究领域极其关注的热点。

本实验运用肝细胞癌亲本细胞Bel-7402和耐药细胞Bel-7402/ADM作为体外实验的研究对象,结果显示YB-1、P-gp在人肝癌耐药细胞株Bel-7402/ADM 和相应敏感株中,无论是RNA水平,还是蛋白水平的表达量,差异有统计学意义,P-gp的高表达总是伴随着YB-1的高表达。

结果显示,MDR1的表达随着YB-1的表达下调显著受到抑制。提示YB-1的表达与P-gp的表达是密切相关的,YB-1与人肝癌耐药细胞株Bel-7402/ADM多药耐药相关。YB-1 在肿瘤的发生中发挥多种重要作用,不仅促进癌细胞过度增殖和抗凋亡,还协助肿瘤扩散和转移,并直接参与染色体异常和癌组织耐药性的产生[10-15]。诸多学者研究提示YB-1与人乳腺癌、肺癌、结直肠癌等的MDR密切相关[6-8]。本研究结果显示YB-1介导肝癌细胞对ADM的耐药,其更深入的机制有待于进一步研究,为今后从基因水平逆转肝癌耐药、开发肝癌耐药逆转剂提供新靶点。

The authors have declared that no competing interests exist.

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Abstract Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.
DOI:10.5713/ajas.2013.13356      PMID:9095180      URL    
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[5] PISAREV A V,SKABKIN M A,THOMAS A A,et al .Positive and negative effects of the major mammalian messenger ribonucleoprotein p50 on binding of 40s ribosomal subunits to the initiation codon of beta-globin mRNA[J].J Biol Chem,2002,277(18):15445-15451.
Abstract p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these phenomena using the in vitro assembly of 48 S preinitiation complexes from fully purified translational components in the presence or absence of p50 as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3, and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for comparison) strongly stimulates formation of the 48 S preinitiation complexes with beta-globin mRNA. This stimulation is observed when just a few molecules of p50 are bound per molecule of the mRNA. When the amount of p50 in solution is increased over some threshold p50/mRNA ratio, a remarkable repression is observed that can still be relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50, the inhibitory effect becomes irreversible. The threshold ratio depends upon the extent of secondary structure of the 5'-untranslated region linked to the beta-globin coding region. Chemical probing has confirmed that the binding of p50 to mRNA involves only the sugar-phosphate backbone of the mRNA leaving nucleotide bases free for interaction with other messenger ribonucleoprotein (mRNP) components. These data are best compatible with the functional role of p50 as a "manager" of mRNA-protein interactions in mammalian mRNPs.
DOI:10.1074/jbc.M111954200      PMID:11854282      URL    
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[6] DE SOUZA-PINTO N C,MASON P A,HASHIGUCHI K,et al.Novel DNA mismatch-repair activity involving YB-1 in human mitochondria[J].DNA Repair (Amst),2009,8(6):704-719.
Maintenance of the mitochondrial genome (mtDNA) is essential for proper cellular function. The accumulation of damage and mutations in the mtDNA leads to diseases, cancer, and aging. Mammalian mitochondria have proficient base excision repair, but the existence of other DNA repair pathways is still unclear. Deficiencies in DNA mismatch repair (MMR), which corrects base mismatches and small loops, are associated with DNA microsatellite instability, accumulation of mutations, and cancer. MMR proteins have been identified in yeast and coral mitochondria; however, MMR proteins and function have not yet been detected in human mitochondria. Here we show that human mitochondria have a robust mismatch-repair activity, which is distinct from nuclear MMR. Key nuclear MMR factors were not detected in mitochondria, and similar mismatch-binding activity was observed in mitochondrial extracts from cells lacking MSH2, suggesting distinctive pathways for nuclear and mitochondrial MMR. We identified the repair factor YB-1 as a key candidate for a mitochondrial mismatch-binding protein. This protein localizes to mitochondria in human cells, and contributes significantly to the mismatch-binding and mismatch-repair activity detected in HeLa mitochondrial extracts, which are significantly decreased when the intracellular levels of YB-1 are diminished. Moreover, YB-1 depletion in cells increases mitochondrial DNA mutagenesis. Our results show that human mitochondria contain a functional MMR repair pathway in which YB-1 participates, likely in the mismatch-binding and recognition steps.
DOI:10.1016/j.dnarep.2009.01.021      PMID:19272840      URL    
[本文引用:2]
[7] FUJII T,SEKI N,NAMOTO-MATSUBAYASHI R,et al.YB-1 prevents apoptosis via the mTOR/STAT3 pathway in HER-2-overexpressing breast cancer cells[J].Future Oncol,2009,5(2):153-156.
Evaluation of: Lee C, Dhillon J, Wang MY et al.: Targeting YB-1 in HER-2 overexpressing breast cancer cells induces apoptosis via the mTOR/STAT3 pathway and suppresses tumor growth in mice. Cancer Res. 68 (21), 8661-8666 (2008). The transcription factor Y-box binding protein (YB)-1 is highly expressed in breast cancer cells and is strongly linked with breast cancer patient prognosis. In this paper, siRNA knockdown of YB-1 was used to investigate breast cancer cell proliferation. Six breast cancer cell lines that either overexpress HER-2 or were triple negative demonstrated growth inhibition following YB-1 knockdown. In particular, YB-1 knockdown induced apoptosis in BT-474-m1 and Au565 cells. Knockdown of YB-1 also decreased phosphorylation of STAT3S727, ERK1/2T202/Y204, mTORS2448 and total mTOR expression. When STAT3 was knocked down by siSTAT3, apoptosis was induced and constitutively active phosphorylated STAT3 was found to rescue YB-1-induced apoptosis. Furthermore, YB-1 knockdown remarkably suppressed colony formation in a soft agar assay, while delayed tumor formation was observed in mice. YB-1 knockdown inhibited cell growth and it is thought to involve induction of apoptosis via the mTOR/STAT3 intracellular signaling pathway. YB-1 is a promising molecular target for HER-2-overexpressing or triple-negative breast cancer cells.
DOI:10.2217/14796694.5.2.153      PMID:19284373      URL    
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[8] OHGA T,KOIKE K,ONO M,et al.Role of the human Y box-binding protein YB-1 in cellular sensitivity to the DNA-damaging agents cisplatin,mitomycin C,and ultraviolet light[J].Cancer Res,1996,56(18):4224-4228.
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[10] EL-NAGGAR A M,VEINOTTE C J,CHENG H,et al.Translational activation of HIF1 alpha by YB-1 promotes sarcoma metastasis[J].Cancer Cell,2015,27(5):682-697.
YB-1 binds DNA and RNA and has been shown to promote epithelial-to-mesenchymal transition and metastasis of carcinomas. El-Naggar et02al. show that YB-1 also contributes to metastasis of high-risk sarcomas by binding to HIF1A mRNA and enhancing its translation.
DOI:10.1016/j.ccell.2015.04.003      PMID:25965573      URL    
[本文引用:1]
[11] HA B,LEE E B,CUI J,et al.YB-1 overexpression promo-tes a TGF-beta1-induced epithelial-mesenchymal transition via Akt activation[J].Biochem Biophys Res Commun,2015,458(2):347-351.
The Y-box binding protein-1 (YB-1) is a transcription/translation regulatory protein, and the expression thereof is associated with cancer aggressiveness. In the present study, we explored the regulatory effects of YB-1 during the transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma cells. Downregulation of YB-1 increased E-cadherin promoter activity, and upregulation of YB-1 decreased promoter activity, suggesting that the YB-1 level may be correlated with the EMT. TGF-β1 induced YB-1 expression, and TGF-β1 translocated cytosolic YB-1 into the nucleus. YB-1 overexpression promoted TGF-β1-induced downregulation of epithelial markers, upregulation of mesenchymal markers, and cell migration. Moreover, YB-1 overexpression enhanced the expression of E-cadherin transcriptional repressors via TGF-β1-induced Akt activation. Our findings afford new insights into the role played by YB-1 in the TGF-β1 signaling pathway.
DOI:10.1016/j.bbrc.2015.01.114      PMID:25645014      URL    
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[12] JUNG K,WU F,WANG P,et al.YB-1 regulates Sox2 to coordinately sustain stemness and tumorigenic properties in a phenotypically distinct subset of breast cancer cells[J].Bmc Cancer,2014,14(1):1-13.
Background Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase ?? subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated. Methods Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis. Results Using gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models. Conclusion These results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression.
DOI:10.1186/1471-2407-14-1      PMID:3880410      URL    
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[13] WANG X,GUO X B,SHEN X C,et al.Prognostic role of YB-1 expression in breast cancer:a meta-analysis[J].Int J Clin Exp Med,2015,8(2):1780-1791.
In published studies, Y-box binding protein-1 (YB-1) correlated with the prognosis of patients with breast cancer (BC), but the specific role of YB-1 is still unclear. Our study aimed to evaluate the prognostic value of YB-1 in BC patients using meta-analysis based on the published studies.We searched the relevant literatures deadline for June 2014 in databases, including PubMed, Embase, Medline and Cochrane library, and finally 8 studies were included in our study. Our study contained 1094 BC patients with 398 YB-1 positive and 696 YB-1 negative.Our results showed that YB-1 abnormal expression did not correlated with the lymph node status [OR = 1.258, 95% CI = 0.895-1.769, P = 0.186], high histological grade [OR = 2.709, 95% CI = 0.861-8.530, P = 0.089], histological type [OR = 0.837, 95% CI = 0.526-1.331, P = 0.452], P53 status [OR = 2.006, 95% CI 0.686-5.865, P = 0.203] and PR [OR = 0.607, 95% CI = 0.347-1.061, P = 0.080] in BC patients. But YB-1 over-expression was associated with other unfavorable factors: ER negativity [OR = 0.604, 95% CI = 0.388-0.941, P = 0.026], HER2 positivity [OR = 3.841, 95% CI = 2.637-5.594, P = 0.000], and high tumorous T stage [OR = 2.169, 95% CI = 1.295-3.632, P = 0.003]. In addition, our data suggested that high YB-1 expression had an adverse impact on 5-year OS [RR = 2.767, 95% CI = 2.054-3.727, P = 0.000] in BC patients.Our findings implied that YB-1 might a novel biomarker to predict the prognosis of BC, and could be a potential direction for developing diagnostic and therapeutic approaches in BC.
DOI:10.1186/1471-2407-12-573      PMID:4402753      URL    
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[14] NISHIO S,USHIJIMA K,YAMAGUCHI T,et al.Nuclear Y-box-binding protein-1 is a poor prognostic marker and related to epidermal growth factor receptor in uterine cervical cancer[J].Gynecol Oncol,2014,132(3):703-708.
Y-box binding protein-1 (YB-1) is a member of the cold shock protein family and functions in transcription and translation. Many studies indicate that YB-1 is strongly expressed in tumor cells and is considered a marker of tumor aggressiveness and clinical prognosis. Overexpression of epidermal growth factor receptor (EGFR) has been associated with poor outcomes in cervical cancer. Clinical trials of EGFR family-base therapy are currently being initiated in cervical cancer. Nuclear YB-1 expression correlates with EGFR expression in various types of cancer. However, the clinical significance of nuclear YB-1 expression in different settings, the correlation with EGFR, and the prognostic implications of YB-1 expression in cervical cancer remain elusive.Nuclear YB-1 expression was immunohistochemically analyzed in tissue specimens obtained from 204 patients with cervical cancer who underwent surgery. Associations of nuclear YB-1 expression with clinicopathological factors such as survival, EGFR expression, and human epidermal growth factor receptor 2 (HER2) expression were investigated.Nuclear YB-1 expression was found in 41 (20.2%) of 204 cases of cervical cancer and correlated with disease stage, tumor diameter, stromal invasion, and lymph-node metastasis. Nuclear YB-1 expression also correlated with EGFR expression (P=0.0114) as well as HER2 expression (P=0.0053). Kaplan-Meier survival analysis showed that nuclear YB-1 expression was significantly associated with poor progression-free survival (P=0.0033) and overall survival (P=0.0003), respectively.Nuclear YB-1 expression is a prognostic marker and correlates with EGFR expression in cervical cancer.
DOI:10.1016/j.ygyno.2014.01.045      PMID:24486603      URL    
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[15] GUO T T,YU Y,YIP W C,et al.Y-box binding protein 1 is correlated with lymph node metastasis in intestinal type of gastric cancer[J].Histopathology,2015,66(4):491-499.
Y-box binding protein-1 (YB-1) is known to modulate gene transcription and protein translation, as well as cellular response to drug treatment. The aim of this study is to correlate YB-1 protein expression levels with clinicopathological parameters in intestinal-type gastric cancer tissue samples (as categorized by the Lauren classification) and substantiate the findings with in vitro experimentation.Paraffin-embedded samples from 167 patients with intestinal-type gastric cancer were used for the construction of tissue microarrays (TMAs). TMA slides were immunostained and YB-1 immunoreactivity score was based on the weighted average intensity score. Univariate analysis revealed that YB-1 immunohistochemical expression was correlated significantly with lymph node status (P = 0.054, borderline significance) and perforation (P = 0.043). YB-1 expression was also found to be an independent predictor of lymph node spread by multivariate analysis. Furthermore, siRNA-mediated YB-1 gene knockdown in MKN7 gastric cancer cells (which is known to originate from an intestinal-type gastric cancer tissue) inhibited cell migration (P = 0.0002) and invasion in vitro (P = 0.0129) significantly.YB-1 expression is associated with lymph node spread in intestinal-type gastric cancer and is a potential prognostic biomarker in this subtype of gastric cancer.
DOI:10.1111/his.12570      PMID:25270600      URL    
[本文引用:1]
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关键词(key words)
多药耐药
P糖蛋白
Y-box结合蛋白1

Multidrug resistance
P-glycoprotein
Y-box binding protein-1

作者
王姗姗
汪选斌

WANG Shanshan
WANG Xuanbin