DEC1、HIF1α、STAT3在胃癌中的表达及其意义
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摘要
胃癌是一种多基因多因素的异质性疾病,是全球常见的恶性肿瘤形式,其发生发展的分子机制一直是生命科学研究的热点问题。转录因子的异常改变与肿瘤的发生发展密切相关。从胃癌发生发展的分子网络入手,研究其调控机制,寻找其分子标记及其可能的干预位点,这是目前国内外肿瘤发生发展机制研究的热点和难点。
分化型胚胎软骨发育基因1(differentiated embryo-chondrocyte expressedgene1,DEC1)是bHLH蛋白家族的一个新成员,含有碱性螺旋-环-螺旋(bHLH)结构域,它属于一个转录因子。近来发现它是一个与肿瘤的发生、发展密切相关的基因,其功能涉及肿瘤细胞的增殖、分化、凋亡及早老,但是相关研究尚处于起步阶段。DEC1广泛表达于大多数正常组织,包括软骨、肺、脾和肠,但较少表达于心、脑、肝、胃。已有研究发现DEC1在部分肿瘤组织中的表达升高,如乳腺癌、结肠癌、少突胶质瘤等;在多种肿瘤细胞系中也存在高表达,如白血病、结肠癌、肺腺癌、神经胶质瘤、肾癌等。但是在胃癌细胞株及胃癌组织中DEC1的表达情况尚未有研究,其在胃癌的发生发展中的作用尚未明确。
多项研究认为DEC1与HIF-1α关系密切,例如肺腺癌A549、膀胱癌EJ-28、乳腺上皮细胞HBL-100、肾细胞癌RCC4、胰腺癌Capan-2、神经胶质瘤、ATDC5等细胞系中,缺氧可以诱导DEC1 mRNA的表达。在肿瘤细胞系293细胞(肾母细胞癌)及Hela细胞(宫颈癌)中,DEC1与HIF-1α有较高的亲和力。在肺癌及食管癌组织中也发现DEC1与HIF-1α的表达显著相关。而在胃癌细胞系及组织中DEC1与HIF-1α的关系尚未有研究,二者在胃癌发生中的作用及机制尚不清楚。
lvanova等的一项研究在分子水平揭示了STRA13(DEC1在小鼠中的同源基因)与另一在肿瘤的发生发展中作用重要的转录因子STAT3密切相关,STRA13是STAT信号途径的重要组成部分,可与磷酸化的STAT3结合,促进下游基因的表达,推测DEC1、STAT3两个转录调节因子可能相互调节,共同在胃癌的发生过程中发挥重要作用。
目的
本实验以胃癌细胞系BGC823、SGC7901为研究对象,探讨DEC1、HIF1α、STAT3在胃癌细胞株中的表达情况,并进一步检测三种基因在胃癌组织中的表达情况,以及DEC1与临床病理变量、DEC1与HIF1α、DEC1与STAT3之间的关系,初步探讨三者在胃癌发生发展中的作用。
方法
(1) RT-PCR检测胃癌细胞系BGC823、SGC7901中DEC1、HIF1α、STAT3mRNA水平的表达;
(2)应用流式、细胞免疫组化的方法初步检测胃癌细胞系BGC823、SGC7901中DEC1、HIF1α、STAT3蛋白的表达情况;
(3)以正常永生化人胃黏膜上皮细胞系GES-1作为对照,采用WesternBlot的方法比较DEC1蛋白在正常胃黏膜上皮细胞与胃癌细胞株中的表达情况;
(4)应用免疫组织化学方法检测DEC1、HIF1α、STAT3三种基因在59例胃癌组织中的表达情况,以19例癌旁正常组织作为对照;并且分析DEC1与临床病理变量之间的关系以及DEC1与HIF1α、DEC1与STAT3之间的相关性。
结果
(1) DEC1mRNA及蛋白在胃癌细胞株BGC823、SGC7901中均有表达;
(2) STAT3、HIF1αmRNA及蛋白在BGC823、SGC7901细胞株中也存在着表达;
(3) Western Blot发现BGC823、SGC7901胃癌细胞株中DEC1的表达显著高于正常永生化人胃黏膜上皮细胞GES-1;
(4)免疫组化发现DEC1在胃癌组织中的表达率显著高于手术切线正常组织,并且与胃癌组织的分化程度密切相关,但与患者性别年龄、肿瘤大小、TNM分期、浸润深度、有无淋巴结转移及远处转移无显著相关;
(5) STAT3在胃癌组织中存在着高表达,显著高于手术切线正常对照组织;
(6) HIF1α在胃癌组织中的表达也显著高于切线正常对照组织;
(7) Spearman相关性分析发现DEC1在胃癌组织中的表达与STAT3蛋白的表达呈显著正相关,DEC1与HIF1α蛋白的表达具有显著相关性。
结论
(1)本研究首次证实胃癌组织及胃癌细胞株中DEC1表达水平显著高于对照组织及正常胃黏膜上皮细胞,且在胃癌组织中DEC1的表达随恶性程度的增高而增强,提示DEC1在胃癌的发生发展中具有重要作用。
(2)利用免疫组化方法,本研究首次在胃癌组织中证实DEC1与HIF1α密切相关,其可能的分子机制为HIF1α与DEC1启动子区域的功能性低氧应答元件(HRE)结合,从而引起DEC1的转录活化,继而诱导下游基因的表达而导致肿瘤的发生及发展,为DEC1在肿瘤作用中机制的研究提供思路。
(3)首次在胃癌组织中发现DEC1和与肿瘤密切相关的STAT3分子的表达显著相关,二者均为在肿瘤中高表达(活化)且作用广泛的转录调节因子,本研究结果提示二者的不适当激活有可能是胃癌发生发展的重要因素之一。
Objective
To investigate the expression of differentiated embryo-chondrocyte expressed gene1(DEC1),the signal tranducer and activator of transcription3 (STAT3) and hypoxia-inducible factor1α(HIF1α) in gastric cancer cell lines, such as BGC823 and SGC7901.Then to explore the expression of this three gene in gastric cancer tissues and to analyze the relationship between DEC1 and clinicopathological variables,that between DEC1 and STAT3,as well as HIF1 a.So we can detect the significance of DEC1 in gastric cancer and the role among the three gene in signal pathway taking part in carcinogenisis and development of gastric cancer.
Methods
(1) RT-PCR was used to detect mRNA expression of DEC1,STAT3 and HIF1αin gastric cancer cell lines,BGC823 and SGC7901;
(2) Flowcytometry and immunocytochemistry were used to examine the protein expression of this three gene;
(3) Western Blot was used for semiquantitive analysis of DEC1,GES-1 as controlled;
(4) Immunohistochemistry was used to detect the expression of DEC1、STAT3 and HIF1αin 59 gastric cancer tissues,and 19 normal tissues depart from the tumor as controlled;then the relationship between DEC1 and clinicopathological variables was analyzed.Spearman correlation analysis was used to analyze the relationship between DEC1 and STAT3, and that between DEC1 and HIF1α.
Result
We found the mRNA expression of DEC1,STAT3 and HIF1αthrough RT-PCR,and the protein level expression by flowcytometry and immunocytochemistry in gastric cancer cell lines.Western Blot semiquantitive analysis found that DEC1 expression in gastric cancer cell lines was higher than that in GES-1.And the positive expression rate of DEC1 was significantly higher in gastric cancer tissues than that in benign lesion tissues.DEC1 expression was correlated with tumor grade(p＜0.05),but not with patients'age, sex,tumor size,TNM staging,invasion depth,lymph node involvement and distant metastases(p＞0.05).In normal tissues,DEC1 expression mostly lies in cytoplasm,but in gastric cancer tissues nuclear staining was stronger with tumor grade from well to poorly differentiated,especially in gastric signet-ring cancer. STAT3 was expressed in gastric cancer tissues,especially in poorly differeiated gastric cancer tissues and gastric signet-ring cancer tissues in which nuclear staining was evident.HIF1αwas also expressed in gastric cancer tissues, higher than that in benigh lesion tissues.Spearman correlation analysis showed that the expression of DEC1 in gastric cancer was related to STAT3 protein level,as well as HIF1αexpression.
Conclusion
(1) Our research confirmed that the expression of DEC1 in gastric cancer tissues and gastric cancer cell lines was significantly higher than that in normal tissued and gastric epithelial cells firstly.And the expression of DEC1 was stronger with tumor grade from well to poorly differentiated in gastric cancer tissues.It implied that DEC1 played an important role in the carcinogenesis and development of gastric cancer.
(2) The result of Immunohistochemistry firstly showed the close relationship between DEC1 and HIF1αin gastric cancer tissues.Molecular mechanism may be that HIF1αinduced DEC1 expression by binding to HRE of DEC1 promoter,then target genes of down stream upregulated which maybe result in gastric carcinogenesis,providing a new mind for carcinogenesis.
(3) The significant correlation between DEC1 and STAT3 which participated in carcinogenesis,was found in gastric cancer tissues firstly.The two genes belong to transcription factor,expressed higher in tumor.Our study implicated that the activation of the two genes inadequately may be one of the most important factors in gastric carcinogenesis and development.
分化型胚胎软骨发育基因1(differentiated embryo-chondrocyte expressedgene1,DEC1)是bHLH蛋白家族的一个新成员,含有碱性螺旋-环-螺旋(bHLH)结构域,它属于一个转录因子。近来发现它是一个与肿瘤的发生、发展密切相关的基因,其功能涉及肿瘤细胞的增殖、分化、凋亡及早老,但是相关研究尚处于起步阶段。DEC1广泛表达于大多数正常组织,包括软骨、肺、脾和肠,但较少表达于心、脑、肝、胃。已有研究发现DEC1在部分肿瘤组织中的表达升高,如乳腺癌、结肠癌、少突胶质瘤等;在多种肿瘤细胞系中也存在高表达,如白血病、结肠癌、肺腺癌、神经胶质瘤、肾癌等。但是在胃癌细胞株及胃癌组织中DEC1的表达情况尚未有研究,其在胃癌的发生发展中的作用尚未明确。
多项研究认为DEC1与HIF-1α关系密切,例如肺腺癌A549、膀胱癌EJ-28、乳腺上皮细胞HBL-100、肾细胞癌RCC4、胰腺癌Capan-2、神经胶质瘤、ATDC5等细胞系中,缺氧可以诱导DEC1 mRNA的表达。在肿瘤细胞系293细胞(肾母细胞癌)及Hela细胞(宫颈癌)中,DEC1与HIF-1α有较高的亲和力。在肺癌及食管癌组织中也发现DEC1与HIF-1α的表达显著相关。而在胃癌细胞系及组织中DEC1与HIF-1α的关系尚未有研究,二者在胃癌发生中的作用及机制尚不清楚。
lvanova等的一项研究在分子水平揭示了STRA13(DEC1在小鼠中的同源基因)与另一在肿瘤的发生发展中作用重要的转录因子STAT3密切相关,STRA13是STAT信号途径的重要组成部分,可与磷酸化的STAT3结合,促进下游基因的表达,推测DEC1、STAT3两个转录调节因子可能相互调节,共同在胃癌的发生过程中发挥重要作用。
目的
本实验以胃癌细胞系BGC823、SGC7901为研究对象,探讨DEC1、HIF1α、STAT3在胃癌细胞株中的表达情况,并进一步检测三种基因在胃癌组织中的表达情况,以及DEC1与临床病理变量、DEC1与HIF1α、DEC1与STAT3之间的关系,初步探讨三者在胃癌发生发展中的作用。
方法
(1) RT-PCR检测胃癌细胞系BGC823、SGC7901中DEC1、HIF1α、STAT3mRNA水平的表达;
(2)应用流式、细胞免疫组化的方法初步检测胃癌细胞系BGC823、SGC7901中DEC1、HIF1α、STAT3蛋白的表达情况;
(3)以正常永生化人胃黏膜上皮细胞系GES-1作为对照,采用WesternBlot的方法比较DEC1蛋白在正常胃黏膜上皮细胞与胃癌细胞株中的表达情况;
(4)应用免疫组织化学方法检测DEC1、HIF1α、STAT3三种基因在59例胃癌组织中的表达情况,以19例癌旁正常组织作为对照;并且分析DEC1与临床病理变量之间的关系以及DEC1与HIF1α、DEC1与STAT3之间的相关性。
结果
(1) DEC1mRNA及蛋白在胃癌细胞株BGC823、SGC7901中均有表达;
(2) STAT3、HIF1αmRNA及蛋白在BGC823、SGC7901细胞株中也存在着表达;
(3) Western Blot发现BGC823、SGC7901胃癌细胞株中DEC1的表达显著高于正常永生化人胃黏膜上皮细胞GES-1;
(4)免疫组化发现DEC1在胃癌组织中的表达率显著高于手术切线正常组织,并且与胃癌组织的分化程度密切相关,但与患者性别年龄、肿瘤大小、TNM分期、浸润深度、有无淋巴结转移及远处转移无显著相关;
(5) STAT3在胃癌组织中存在着高表达,显著高于手术切线正常对照组织;
(6) HIF1α在胃癌组织中的表达也显著高于切线正常对照组织;
(7) Spearman相关性分析发现DEC1在胃癌组织中的表达与STAT3蛋白的表达呈显著正相关,DEC1与HIF1α蛋白的表达具有显著相关性。
结论
(1)本研究首次证实胃癌组织及胃癌细胞株中DEC1表达水平显著高于对照组织及正常胃黏膜上皮细胞,且在胃癌组织中DEC1的表达随恶性程度的增高而增强,提示DEC1在胃癌的发生发展中具有重要作用。
(2)利用免疫组化方法,本研究首次在胃癌组织中证实DEC1与HIF1α密切相关,其可能的分子机制为HIF1α与DEC1启动子区域的功能性低氧应答元件(HRE)结合,从而引起DEC1的转录活化,继而诱导下游基因的表达而导致肿瘤的发生及发展,为DEC1在肿瘤作用中机制的研究提供思路。
(3)首次在胃癌组织中发现DEC1和与肿瘤密切相关的STAT3分子的表达显著相关,二者均为在肿瘤中高表达(活化)且作用广泛的转录调节因子,本研究结果提示二者的不适当激活有可能是胃癌发生发展的重要因素之一。
Objective
To investigate the expression of differentiated embryo-chondrocyte expressed gene1(DEC1),the signal tranducer and activator of transcription3 (STAT3) and hypoxia-inducible factor1α(HIF1α) in gastric cancer cell lines, such as BGC823 and SGC7901.Then to explore the expression of this three gene in gastric cancer tissues and to analyze the relationship between DEC1 and clinicopathological variables,that between DEC1 and STAT3,as well as HIF1 a.So we can detect the significance of DEC1 in gastric cancer and the role among the three gene in signal pathway taking part in carcinogenisis and development of gastric cancer.
Methods
(1) RT-PCR was used to detect mRNA expression of DEC1,STAT3 and HIF1αin gastric cancer cell lines,BGC823 and SGC7901;
(2) Flowcytometry and immunocytochemistry were used to examine the protein expression of this three gene;
(3) Western Blot was used for semiquantitive analysis of DEC1,GES-1 as controlled;
(4) Immunohistochemistry was used to detect the expression of DEC1、STAT3 and HIF1αin 59 gastric cancer tissues,and 19 normal tissues depart from the tumor as controlled;then the relationship between DEC1 and clinicopathological variables was analyzed.Spearman correlation analysis was used to analyze the relationship between DEC1 and STAT3, and that between DEC1 and HIF1α.
Result
We found the mRNA expression of DEC1,STAT3 and HIF1αthrough RT-PCR,and the protein level expression by flowcytometry and immunocytochemistry in gastric cancer cell lines.Western Blot semiquantitive analysis found that DEC1 expression in gastric cancer cell lines was higher than that in GES-1.And the positive expression rate of DEC1 was significantly higher in gastric cancer tissues than that in benign lesion tissues.DEC1 expression was correlated with tumor grade(p＜0.05),but not with patients'age, sex,tumor size,TNM staging,invasion depth,lymph node involvement and distant metastases(p＞0.05).In normal tissues,DEC1 expression mostly lies in cytoplasm,but in gastric cancer tissues nuclear staining was stronger with tumor grade from well to poorly differentiated,especially in gastric signet-ring cancer. STAT3 was expressed in gastric cancer tissues,especially in poorly differeiated gastric cancer tissues and gastric signet-ring cancer tissues in which nuclear staining was evident.HIF1αwas also expressed in gastric cancer tissues, higher than that in benigh lesion tissues.Spearman correlation analysis showed that the expression of DEC1 in gastric cancer was related to STAT3 protein level,as well as HIF1αexpression.
Conclusion
(1) Our research confirmed that the expression of DEC1 in gastric cancer tissues and gastric cancer cell lines was significantly higher than that in normal tissued and gastric epithelial cells firstly.And the expression of DEC1 was stronger with tumor grade from well to poorly differentiated in gastric cancer tissues.It implied that DEC1 played an important role in the carcinogenesis and development of gastric cancer.
(2) The result of Immunohistochemistry firstly showed the close relationship between DEC1 and HIF1αin gastric cancer tissues.Molecular mechanism may be that HIF1αinduced DEC1 expression by binding to HRE of DEC1 promoter,then target genes of down stream upregulated which maybe result in gastric carcinogenesis,providing a new mind for carcinogenesis.
(3) The significant correlation between DEC1 and STAT3 which participated in carcinogenesis,was found in gastric cancer tissues firstly.The two genes belong to transcription factor,expressed higher in tumor.Our study implicated that the activation of the two genes inadequately may be one of the most important factors in gastric carcinogenesis and development.
引文
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[47]. Rolfs, A., Kvietikova, I., Gassmann, M., et al.Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1. (1997) J. Biol. Chem. 272,20055 - 20062.
[48]. Iyer, N. V., Kotch, L. E., Agani, F., et al. Cellular and developmental control of 02 homeostasis by hypoxia-inducible factor 1 alpha.(1998) Genes Dev.12,149- 162.
[49].Ryan,H.E.,Lo,J.,and Johnson,R.S.HIF-1 alpha is required for solid tumor formation and embryonic vascularization.(1998)EMBO J.17,3005- 3015.
[50].Carmeliet,P.,Dor,Y.,Herbert,J.M.,et al.Role of HIF-lalpha in hypoxia-mediated apoptosis,cell proliferation and tumour angiogenesis.(1998) Nature 394,485-490.
[51]程春生,贺克俭,吴盛州等.HIF-1α在胃癌组织中的表达及临床意义.胃肠病学和肝病学杂志,2006,15(5):448-450.
[52]韩冰,徐瑞华,史艳侠等.HIF-1α在胃癌组织中的表达及其临床意义.癌症,2006,25(11):1439-1442.
[53]Darnell JE Jr.Transcription factors as targets for cancer therapy.Nat Rev Cancer.2002 0ct;2(10):740-9.
[54]Schindler C,Levy DE,Decker T,et al.JAK-STAT signaling:from interferons to cytokines.J Biol Chem.2007 Jul 13;282(28):20059-63.
[55]Ivanova A.V.,Ivanov S.V.,Zhang X,et al.STRA13 Interacts with STAT3 and Modulates Transcription of STAT3-dependent Targets[J].J.Mol.Biol.,2004,340,641 - 653.
[56]Groner B,Lucks P,Borghouts c.The function of Stat3 in tumor cells and their microenvironment.Semin Cell Dev Biol.2008Aug;19(4):341-50.
[57]Dauer DJ,Ferraro B,Song L et al.Star3 regulates genes common to both wound healing and cancer.Oncogene 2005;24:3397 -408.
[58]Gritsko T,Williams A,Turkson J et al.Persistent activation of stat3 signaling induces survivin gene expression and confers resistance to apoptosis in human breast cancer cells.Clin.Cancer Res.2006;12:11-19.
[59]Kiuchi N,Nakajima K,Ichiba M et al.STAT3 is required for the gp130-mediated full activation of the c-myc gene.J.Exp.Med.1999;189:63-73.
[60]Dechow TN,Pedranzini L,Leitch A et al.Requirement of matrix metalloproteinase-9 for the transformation of human mammary epithelial cells by Stat3-C.Proc.Natl Acad.Sci.USA 2004;101:10602 - 7.
[61]Ivanov VN,Bhoumik A,Krasilnikov M et al.Cooperation between STAT3 and c-jun suppresses Fas transcription.Mol.Cell 2001;7:517 - 28.
[62]Naoki Kanda,Hiroshi Seno,Yoshitaka Konda,et al.STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells.Oncogene(2004) 23,4921 -4929.
[63]俞丽芬,朱延波,乔敏敏等.Stat3在人胃癌细胞株和组织中的组成性激活及其临床意义.中华医学杂志,2004,84(24):2064-2069.
[64]Naoki Kanda,Hiroshi Seno,Yoshitaka Konda,et al.STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells.Oncogene(2004) 23,4921 - 4929
[65]Gong W D,Wang L W,Yao J C,et al.Expression of Activated Signal Transducer Transcription 3 Predicts Expression Endothelial Growth Factor in and Phenotype of Human Oastric Cancer[J].Clin Cancer Res.2005 Feb 15;11(4):1386-93.
[66]Kanda N,Seno H,Konda Y,et al.STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells[J].Oncogene(2004)23,4921 - 4929.
[67]Zhong Z,Wen Z,Damell J.Stat3:a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and Interleukin-6[J]. Science, 1994,264(5155):95-98.
[68] Raz R, Durbin J, Levy D. Acute phase response factor and additional members of interferon-stimulated gene factor 3 family integrate diverse signals from cytokines, interferons and growth factors[J]. J Biol Chem. 1994, 269(39): 24391-24395.