结直肠癌组织中HNF4α的细胞内定位及临床意义

引用本文

张梁, 元辉雄, 黄永秩. 结直肠癌组织中HNF4α的细胞内定位及临床意义. 2013, 28(3): 215-217
ZHANG Liang, YUAN Huixiong, HUANG Yongzhi. The intracellular localization and clinical significance of HNF4α in tissues of colorectal cancer. Labratory Medicine, 2013, 28(3): 215-217 复制到剪切板

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《检验医学》编辑部

结直肠癌组织中HNF4α的细胞内定位及临床意义

张梁¹, 元辉雄¹, 黄永秩²

1.广西右江民族医学院附属医院检验科,广西百色 533000

2.广西右江民族医学院附属医院病理科,广西百色 533000

通讯作者:元辉雄,联系电话:0776-2828361。

作者简介:张梁,女,1963年生,学士,副主任技师,主要从事临床生物化学检验与教学工作。

基金:百色市科学技术局资助项目(百科计字[2010]5号);

摘要

目的

检测肝细胞核因子4α(HNF4α)在结直肠癌组织中的细胞内定位及其表达情况,探讨HNF4α定位及其表达与结直肠癌临床各病理因素的关系。

方法

应用免疫组化方法检测132例结直肠癌组织标本及其癌旁正常组织中HNF4α的细胞内定位及其表达情况。

结果

结直肠癌组织中HNF4α蛋白分子的表达定位于细胞浆或细胞核;结直肠癌组织细胞浆HNF4α的阳性率(76.5%)显著高于癌旁正常组织(3.8%,P<0.05);结直肠癌组织细胞核HNF4α的阳性率(12.9%)显著低于癌旁正常组织(64.4%,P<0.05);HNF4α在结直肠癌组织细胞浆中的表达与组织分化程度及Dukes分期有关(P=0.02、P=0.03),与性别、年龄、肿瘤位置、有无淋巴结转移等临床病理特征无明显相关性(P均>0.05)。

结论

结直肠癌组织中HNF4α在细胞浆中高表达或在细胞核中低表达,HNF4α的细胞内定位异常可为结直肠癌的诊断及预后判断提供客观依据。

关键词: 肝细胞核因子4α; 细胞内定位; 免疫组化; 结直肠癌

中图分类号:R446.63 文献标志码:A 文章编号:1673-8640(2013)03-215-03

The intracellular localization and clinical significance of HNF4α in tissues of colorectal cancer

ZHANG Liang¹, YUAN Huixiong¹, HUANG Yongzhi²

1.Department of Clinical Laboratory, the Affiliated Hospital of Youjiang Medical University for Nationalities , Guangxi Baise 533000, China

2. Department of Pathology, the Affiliated Hospital of Youjiang Medical University for Nationalities , Guangxi Baise 533000, China

Abstract

Objective

To detect the intracellular localization and expression of hepatocyte nuclear factor-4 alpha (HNF4α) in tissues of colorectal cancer, and to investigate its relationship with clinical pathological parameters in patients with colorectal cancer.

Methods

Immunohistochemistry was used to detect the intracellular localization and expression of HNF4α in 132 specimens of colorectal cancer tissues and adjacent normal intestinal mucosa.

Results

The intracellular localization of HNF4α in colorectal cancer tissues was located in the cell plasma or in the cell nuclear. The positive rate of HNF4α expression in the cell plasma was significantly higher in colorectal cancer tissues than that in adjacent normal intestinal mucosa (76.5% and 3.8%,P<0.05). The positive rate of HNF4α expression in the cell nuclear was significantly lower in colorectal cancer tissues than that in adjacent normal intestinal mucosa (12.9% and 64.4%,P<0.05). The expression of HNF4α in the cell plasma of colorectal cancer tissues was related to tissue differentiation degree and Dukes staging(P=0.02,P=0.03), but not to sex, age, tumor location, or lymph node metastasis(P>0.05).

Conclusions

The high expression of HNF4α in the cell plasma or low expression in the cell nuclear of colorectal cancer tissues is related with the development of colorectal cancer. The abnormal localization of HNF4α in colorectal cancer can serve as an objective index for the diagnosis and prognosis of colorectal cancer.

Keyword: Hepatocyte nuclear factor-4 alpha; Intracellular localization; Immunohistochemistry; Colorectal cancer

Show Figures

引言

肝细胞核因子4α(hepatocyte nuclear factor-4alpha,HNF4α)属于核受体超家族成员之一,是一种多功能的转录因子,在生长发育、代谢和疾病中控制着许多基因的表达^{[ 1]}。HNF4α在人体肝、肾、心、脾、肠等器官中均有表达^{[ 2]},至今已有许多关于其参与恶性肿瘤发生、发展过程的报道,但HNF4α在结直肠癌中的表达及其作用的研究报道较少^{[ 3, 4, 5, 6]},其研究结果也存在较大差异。为此,本研究采用免疫组化方法,对HNF4α在结直肠癌及癌旁正常组织中的细胞内定位及表达进行检测,探讨其与结直肠癌发生、发展的关系。

材料和方法

一、病例和材料

收集右江民族医学院附属医院 2008年3月至2011年3月行手术治疗的结直肠癌患者132例,其中男83例,女 49例,年龄 4371岁。高分化11例,中分化31例,低分化90例;临床Dukes分期,A期19例,B期27例,C期56例,D期30例;有淋巴结转移53例,无淋巴结转移79例。所有病例术前未采用任何放化疗或生物治疗,标本均经病理学诊断证实。取癌组织和配对癌旁正常组织(经病理学诊断证实^{[ 7]}的正常组织,距肿瘤边缘约5 cm)各132份。

二、试剂与检测方法

HNF4α石蜡切片的免疫组化染色采用Santa Cruz公司Immuno Cruz^TM staining system SP法免疫组化试剂盒产品,HNF4α抗体为羊来源的多克隆抗体,工作浓度为1∶75,用磷酸盐缓冲液(PBS)代替一抗进行空白对照,HNF4α阴性对照加试剂盒附带的正常羊血清。

三、免疫组化结果判定

以出现棕黄色颗粒染色为阳性,计分标准:Ⅰ类根据染色细胞总数,<5%为0分,5%25%为1分,25% 50%为2分,>50%为3分 ;Ⅱ类根据着色强度,细胞染色呈阴性为0分,呈淡黄色为1分,呈棕黄色为2分,呈棕褐色为3分。Ⅰ类与Ⅱ类分数相加的总分作为判断结果,总分为0视为阴性(-) ,1 2为弱阳性(+) ,3 4为中等阳性(+ +) ,5 6为强阳性(+ + +)。每张切片随机抽取5个高倍视野,每个视野计数100个细胞,计算平均得分作为最终结果^{[ 3]}。

四、统计学方法

所有数据采用SPSS 17.0软件进行统计学分析,计数资料采用 χ²检验,检验水准 α=0.05。

结果

一、HNF4α蛋白的表达及细胞内定位

免疫组化结果显示,HNF4α蛋白分子的表达定位于细胞浆或细胞核,呈棕黄色。结直肠癌组织细胞浆中HNF4α表达阳性率为76.5%,而癌旁正常组织细胞浆中HNF4α表达阳性率为3.8%;结直肠癌组织细胞核中HNF4α表达阳性率为12.9%,而癌旁正常组织细胞核中HNF4α表达阳性率为64.4%。与癌旁正常组织相比,结直肠癌组织细胞浆和细胞核中HNF4α的表达阳性率差异均有统计学意义( χ²=145.3, χ²=73.9, P<0.05)。见表1。

表1 HNF4α在结直肠癌组织及相应的癌旁正常组织中的细胞内定位[例(%)]

二、HNF4α蛋白表达与结直肠癌临床病理特征间的关系

细胞浆中HNF4α蛋白表达阳性率在不同肿瘤分化程度、Dukes分期间差异有统计学意义( P=0.02、 P=0.03);与患者性别、年龄(≥55岁或<55岁)、肿瘤位置、有无淋巴结转移等临床病理特征无明显相关性( P均>0.05)。见表2。

表2 细胞浆HNF4α表达与结直肠癌临床病理特征的关系

讨论

结直肠癌是消化系统常见的恶性肿瘤,近年来发病率有逐年上升的趋势^{[ 3]},但其发生、发展是多步骤、多环节、多因素、多分子参与的复杂过程。HNF4α是一种核转录因子,对物质代谢及细胞分化的许多基因都起着重要的调节作用,在恶性肿瘤中扮演重要角色,已有研究证明HNF4α具有抑制肝细胞癌的作用^{[ 4]}。近年来HNF4α与结直肠肿瘤的关系日益受到重视,但HNF4α在结直肠肿瘤的表达及其意义一直存在争议。

Schwartz等^{[ 8]}通过配体拮抗剂抑制HNF4α的转录与蛋白表达,发现结肠癌细胞的生长被抑制,HNF4α具有促进肿瘤形成的作用。Darsigny等^{[ 4]}通过ApcMin小鼠的对比实验发现,缺乏HNF4α时肠肿瘤被抑制,也说明HNF4α能促进肠肿瘤形成。而宁北芳^{[ 9]}对大鼠的研究则表明,HNF4α具有抑制肿瘤细胞增殖和肿瘤生长的作用。Chellappa等^{[ 5]}通过研究HNF4α的启动子亚型发现,近80%结肠癌样本的核HNF4α P1启动子亚型是缺失的,核HNF4α表达的缺失,可能是结肠癌的早期迹象,核HNF4α具有预防结肠癌的作用。本研究结果显示,结直肠癌组织细胞浆中HNF4α的表达显著高于癌旁正常组织,而细胞核中的表达显著低于癌旁正常组织,结合Chellappa等^{[ 5]}的研究结果分析,可能是由于结直肠癌组织中的Src酪氨酸激酶使细胞核内的HNF4α 磷酸化,在配体结合结构域(ligand binding domain,LBD)的磷酸化模拟突变(phosphomimetic mutants)又使磷酸化的HNF4α蛋白的稳定性、核表达和转活功能下降;而HNF4α的表达与Fas配体的表达直接相关^{[ 10]},HNF4α表达减少,Fas配体的表达也相应减少,使Fas配体的促癌细胞凋亡及阻止癌细胞迁移的作用减弱,结直肠组织细胞的恶变因此而未能受到抑制,导致结直肠癌的发生。而结直肠组织HNF4α的细胞内定位异常的机制目前尚不明了,有待更深入的研究。

进一步分析结直肠癌组织细胞浆中HNF4α的表达与临床病理特征的关系,结果发现结直肠癌组织细胞浆中HNF4α表达与患者的年龄、性别、肿瘤部位无明显相关性,而与分化程度和Dukes分期密切相关,因而可为结直肠癌的诊断及预后提供客观依据。可能由于样本量偏少,本研究尚不足以证实HNF4α与结直肠癌淋巴结转移存在联系,因此HNF4α与结直肠癌发生、发展的关系尚需更深入的研究。

The authors have declared that no competing interests exist.

参考文献

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[1]	Weltmeier F, Borlak J. A high resolution genome-wide scan of HNF4α recognition sites infers a regulatory gene network in colon cancer[J]. PLoS One, 2011, 6(7): e21667. [本文引用:1] [JCR: 3.73]
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[4]	Darsigny M, Babeu JP, Seidman EG, et al. Hepatocyte nuclear factor-4alpha promotes gut neoplasia in mice and protects against the production of reactive oxygen species[J]. Cancer Res, 2010, 70(22): 9423-9433. [本文引用:3] [JCR: 8.65]
[5]	Chellappa K, Jankova L, Schnabl JM, et al. Src tyrosine kinase phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer[J]. Proc Natl Acad Sci USA, 2012, 109(7): 2302-2307. [本文引用:3] [JCR: 9.737]
[6]	Garrison WD, Battle MA, Yang C, et al. Hepatocyte nuclear factor 4alpha is essential for embryonic development of the mouse colon[J]. Gastroenterology, 2006, 130(4): 1207-1220. [本文引用:1] [JCR: 12.821]
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[9]	宁北芳. 肝细胞核因子4α预防大鼠肝癌及其分子机制研究[D]. 上海: 第二军医大学, 2009. [本文引用:1]
[10]	Osanai M, Chiba H, Kojima T, Hepatocyte nuclear factor(HNF)-4alpha induces expression of endothelial Fas ligand (FasL) to prevent cancer cell transmigration: a novel defense mechanism of endothelium against cancer metastasis[J]. Jpn J Cancer Res, 2002, 93(5): 532-541. [本文引用:1] [JCR: 2.225]

2011

3.73

0.0

PLoS ONE. 2011, 6(7):e21667-null

A High Resolution Genome-Wide Scan of HNF4α Recognition Sites Infers a Regulatory Gene Network in Colon Cancer

Fridtjof Weltmeier 1 ,Juergen Borlak 1,2

1 Department of Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany 2 Centre for Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany

The hepatic nuclear factor HNF4α is a versatile transcription factor and controls expression of many genes in development, metabolism and disease. To delineate its regulatory gene network in colon cancer and to define novel gene targets a comprehensive genome-wide scan was carried out at a resolution of 35 bp with chromatin IP DNA obtained from the human colon carcinoma cell line Caco-2 that is a particularly rich source of HNF4α. More than 90% of HNF4α binding sites were mapped as promoter distal sequences while enhancer elements could be defined to foster chromatin loops for interaction with other promoter-bound transcription factors. Sequence motif analysis by various genetic algorithms evidenced a unique enhanceosome that consisted of the nuclear proteins ERα, AP1, GATA and HNF1α as cooperating transcription factors. Overall >17,500 DNA binding sites were identified with a gene/binding site ratio that differed >6-fold between chromosomes and clustered in distinct chromosomal regions amongst >6600 genes targeted by HNF4α. Evidence is presented for nuclear receptor cross-talk of HNF4α and estrogen receptor α that is recapitulated at the sequence level. Remarkably, the Y-chromosome is devoid of HNF4α binding sites. The functional importance of enrichment sites was confirmed in genome-wide gene expression studies at varying HNF4α protein levels. Taken collectively, a genome-wide scan of HNF4α binding sites is reported to better understand basic mechanisms of transcriptional control of HNF4α targeted genes. Novel promoter distal binding sites are identified which form an enhanceosome thereby facilitating RNA processing events.

... )属于核受体超家族成员之一,是一种多功能的转录因子,在生长发育、代谢和疾病中控制着许多基因的表达^[1] ...

2006

0.0

... 在人体肝、肾、心、脾、肠等器官中均有表达^[2],至今已有许多关于其参与恶性肿瘤发生、发展过程的报道,但HNF4#cod#x003b1 ...

2011

0.0

... 在结直肠癌中的表达及其作用的研究报道较少^[3,4,5,6],其研究结果也存在较大差异 ...

... 每张切片随机抽取5个高倍视野,每个视野计数100个细胞,计算平均得分作为最终结果^[3] ...

... 讨论结直肠癌是消化系统常见的恶性肿瘤,近年来发病率有逐年上升的趋势^[3],但其发生、发展是多步骤、多环节、多因素、多分子参与的复杂过程 ...

2010

8.65

0.0

... 在结直肠癌中的表达及其作用的研究报道较少^[3,4,5,6],其研究结果也存在较大差异 ...

... 具有抑制肝细胞癌的作用^[4] ...

... Darsigny等^[4]通过ApcMin小鼠的对比实验发现,缺乏HNF4#cod#x003b1 ...

2012

9.737

0.0

PNAS. 2012, 109(7):2302-2307

Src tyrosine kinase phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer

Karthikeyani Chellappa a , Lucy Jankova b , Jake M. Schnabl a , Songqin Pan c , Yann Brelivet d , Caroline L-S. Fung e , Charles Chan e , Owen F. Dent f , Stephen J. Clarke g , Graham R. Robertson b , and Frances M. Sladek a , 1

a Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521; b Cancer Pharmacology Unit, ANZAC Research Institute, University of Sydney, Sydney, New South Wales, Australia; c W. M. Keck Proteomics Laboratory, Institute for Integrative Genome Biology, University of California, Riverside, CA 92521; d Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch,France; e Department of Anatomical Pathology, f Department of Colorectal Surgery, Concord Hospital; and g Northern Clinical School, University of Sydney, Sydney, New South Wales, Australia

Src tyrosine kinase has long been implicated in colon cancer but much remains to be learned about its substrates. The nuclearreceptor hepatocyte nuclear factor 4α (HNF4α) has just recently been implicated in colon cancer but its role is poorly defined.Here we show that c-Src phosphorylates human HNF4α on three tyrosines in an interdependent and isoform-specific fashion. Theinitial phosphorylation site is a Tyr residue (Y14) present in the N-terminal A/B domain of P1- but not P2-driven HNF4α. Phospho-Y14interacts with the Src SH2 domain, leading to the phosphorylation of two additional tyrosines in the ligand binding domain(LBD) in P1-HNF4α. Phosphomimetic mutants in the LBD decrease P1-HNF4α protein stability, nuclear localization and transactivationfunction. Immunohistochemical analysis of approximately 450 human colon cancer specimens (Stage III) reveals that P1-HNF4αis either lost or localized in the cytoplasm in approximately 80% of tumors, and that staining for active Src correlates withthose events in a subset of samples. Finally, three SNPs in the human HNF4α protein, two of which are in the HNF4α F domainthat interacts with the Src SH3 domain, increase phosphorylation by Src and decrease HNF4α protein stability and function,suggesting that individuals with those variants may be more susceptible to Src-mediated effects. This newly identified interactionbetween Src kinase and HNF4α has important implications for colon and other cancers.

... 在结直肠癌中的表达及其作用的研究报道较少^[3,4,5,6],其研究结果也存在较大差异 ...

... Chellappa等^[5]通过研究HNF4#cod#x003b1 ...

... 的表达显著高于癌旁正常组织,而细胞核中的表达显著低于癌旁正常组织,结合Chellappa等^[5]的研究结果分析,可能是由于结直肠癌组织中的Src酪氨酸激酶使细胞核内的HNF4#cod#x003b1 ...

2006

12.821

0.0

... 在结直肠癌中的表达及其作用的研究报道较少^[3,4,5,6],其研究结果也存在较大差异 ...

2012

0.0

... 取癌组织和配对癌旁正常组织(经病理学诊断证实^[7]的正常组织,距肿瘤边缘约5 cm)各132份 ...

2009

0.0

. 2009, 124(5):1081-1089

Inhibition of colorectal cancer by targeting hepatocyte nuclear factor-4α

Betty Schwartz 1,†,* ,Anna Algamas-Dimantov 1 ,Rachel Hertz 2 ,Jennifer Nataf 1 ,Ayelet Kerman 1 ,Irena Peri 1 andJacob Bar-Tana 2

1 The Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 2 Department of Human Nutrition and Metabolism; Hebrew University Medical School, The Hebrew University of Jerusalem, Jerusalem, Israel * Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel

> Hepatocyte nuclear factor-4α (HNF-4α) serves as target for fatty acid nutrients and xenobiotic amphipathic carboxylates and may account for the differential effects of dietary fatty acids on colorectal cancer (CRC). The putative role played by HNF-4α in CRC has been verified here by evaluating the effect of HNF-4α antagonists and HNF-4α siRNA on CRC growth and proliferation in cultured CRC cells and xenotransplanted nude mice in vivo . HNF-4α ligand antagonists of the MEDICA series, namely, β,β'-tetramethylhexadecanedioic acid (M16ββ) and γ,γ'-tetramethyloctadocanedioic acid (M18γγ) as well as HNF-4α siRNA are shown here to inhibit growth and proliferation of HT29 and Caco2 CRC cells, accompanied by increased subG1 cell population, downregulated PCNA, activation of caspase-3, upregulation of Bak and cytoplasmic cytochrome-c, and downregulation of Bcl-2 resulting in apoptotic death. Inhibition of CRC growth with concomitant apoptosis was further confirmed in nude mice xenotransplanted with HT29 CRC cells. CRC suppression by HNF-4α ligand antagonists and by HNF-4α siRNA was accounted for by suppression of HNF-4α transcription and protein expression. α,α'-tetrachlorotetradecanedioic acid (Cl-DICA), a MEDICA analogue that fails to suppress HNF-4α, was ineffective in suppressing growth of cultured or xenotransplanted HT29 CRC cells. Hence, increased transcriptional activity of HNF-4α converging onto genes coding for antiapoptotic oncogenes and cytokines may promote CRC development. Suppression of HNF-4α activity by natural or xenobiotic HNF-4α ligand antagonists or by HNF-4α siRNA may offer a treatment mode for CRC. © 2008 Wiley-Liss, Inc.

... Schwartz等^[8]通过配体拮抗剂抑制HNF4#cod#x003b1 ...

2009

0.0

... 而宁北芳^[9]对大鼠的研究则表明,HNF4#cod#x003b1 ...

2002

2.225

0.0

. 2002, 93(5):532-541

Hepatocyte Nuclear Factor (HNF)-4α Induces Expression of Endothelial Fas Ligand (FasL) to Prevent Cancer Cell Transmigration: A Novel Defense Mechanism of Endothelium against Cancer Metastasis

Makoto Osanai 1,3,* ,Hideki Chiba 1 ,Takashi Kojima 1 ,Masato Fujibe 1 ,Kazuhide Kuwahara 1 ,Hiromichi Kimura 2 ,Masaaki Satoh 1 andNorimasa Sawada 1

1 Department of Pathology, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060-8556 2 Department of Surgery, Sapporo Tsukisamu Hospital, Tsukisamu, East-1-13, Toyo-hira-ku, Sapporo 062-0051 3 Department of Biochemistry and Pathology, Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada K7L 3N6 * E-mail: osanaim@post.queensu.ca

> Endothelial Fas ligand (FasL) contributes to the “immune privilege” of tissues such as testis and eye, in which apoptosis is induced in infiltrating Fas-positive activated T cells and results in the inhibition of leukocyte extravasation. In this study, we examined the role of endothelial FasL in controlling cancer cell transmigration using rat lung endothelial (RLE) cell line bearing a doxycycline-inducible hepatocyte nuclear factor (HNF)-4α expression system. We showed that a detectable level of FasL was expressed in RLE cells and that this expression was markedly up-regulated and well correlated to the degree of HNF–4α expression in a time-dependent manner. When various cancer cells were overlaid on an RLE monolayer sheet, we examined the ability of endothelial FasL to induce massive apoptosis in Fas-expressing cancer cells and found a causal link to inhibition of the transmigration. Finally, we showed that FasL was expressed in capillaries of the rat brain by immunohistochemical staining, suggesting that FasL serves its functions not only in vitro , but also in vivo. These results raise the possibility that HNF–4α is involved in regulating cancer cell transmigration by modulating the Fas-FasL system.

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