14-3-3zeta在T_1期非小细胞肺癌中的表达及与肺癌侵袭、转移和凋亡关系的研究
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摘要
肺癌是严重危害人类健康的恶性肿瘤,目前居我国城市居民恶性肿瘤死亡率的首位。因此,探讨肺癌侵袭、转移相关的分子机制,对肺癌的预防、诊断和治疗具有重要意义。近年来,14-3-3蛋白与肺癌关系的研究逐渐成为热点。
14-3-3蛋白是一种高度保守的可溶性酸性蛋白家族。研究表明,14-3-3家族可以和许多信号蛋白,包括激酶、磷酸酶和跨膜受体等结合,在信号传导、细胞周期以及细胞凋亡的调控等方面发挥重要作用。哺乳动物中,14-3-3蛋白有7种亚型(β,γ,ε，η,σ,τ和ζ)。研究显示,在正常肺组织中只能检测到两种亚型：即ε和ζ,而在肺癌中β,γ,σ,τ,ε,ζ均有表达。14-3-3蛋白的亚型与肿瘤的发生密切相关,并被作为潜在的肿瘤抑制基因或致癌基因。因此,深入研究14-3-3蛋白的不同亚型可以为抗癌治疗提供新的思路。
14-3-3ζ是14-3-3家族中一个亚型。目前,14-3-3ζ的确切功能还不明了,对14-3-3ζ的研究大多集中在对凋亡的调控上。细胞凋亡程序失控与恶性肿瘤的发生、发展及预后密不可分。Bcl-2家族是调控凋亡过程最重要的蛋白家族之一其中的促凋亡因子Bad在肿瘤中的表达具有重要意义。Ichinose等[6]发现,Bad蛋白磷酸化后失活,失活的Bad加剧胶质母细胞瘤和前列腺癌的恶性转化。研究表明,14-3-3ζ可以通过不同机制对凋亡产生抑制效应,其中之一就是对Bcl-2家族的调控。14-3-3ζ可与磷酸化的Bad (p-Bad)结合,将其隔离在胞浆,从而阻止Bad与Bcl-2和Bcl-XL的结合,抑制凋亡。Akt是一个丝-苏氨酸激酶,位于磷脂酰肌醇-3-激酶(PI3-K)的下游,可被许多促细胞生存信号蛋白所激活。Taso等的研究提示Akt的活化参与癌变的早期阶段,是NSCLC(Non- small Cell LungCancer,非小细胞肺癌)发生中的重要事件。Powell等在体外及实体细胞中均发现Akt可以使14-3-3ζ的Ser-58磷酸化。14-3-3ζ对细胞凋亡的调控也可能在一定程度上对肿瘤的发生、发展、侵袭和转移进行调控,因此研究非小细胞肺癌中凋亡相关因子的表达及其与14-3-3ζ的相互关系具有重要意义。
恶性肿瘤往往伴有对邻近组织的浸润和远处转移。肿瘤的侵袭和转移是一个多步骤、多因素参与的极其复杂的过程,而细胞间粘附功能丧失则是其先决条件。研究表明,上皮型钙粘素(E-cad)与细胞间粘附功能异常、上皮肿瘤的增殖、浸润和转移关系最为密切[11]。E-cad的细胞粘附作用与其细胞内的连环蛋白有关,其中β-连环蛋白(β-catenin)对E-cad的功能调节尤为重要。近年来有关14-3-3ζ与粘附关系的研究开始出现。由于粘附在肿瘤侵袭、转移过程中的作用重要而且复杂,因而深入了解14-3-3ζ与粘附之间的关系,可以进一步加深我们对肺癌侵袭、转移机制的认识。
本课题应用免疫组织化学技术、免疫双标、激光共聚焦显微镜技术、Western Blotting技术及RT-PCR技术,并结合完整的临床资料,探讨14-3-3ζ在T1期非小细胞肺癌中的表达及与肺癌侵袭、转移、预后和凋亡的关系。了解14-3-3ζ蛋白与肺癌的关系并揭示其作用机制,将有可能为人类战胜这种疾病开辟一条新的途径。
实验方法
110例T1期非小细胞肺癌及癌旁组织均来自辽宁医学院2000～2006年手术切除存档蜡块。所有病例均有完整的随访记录,随访截尾日期为2008年4月。生存期的计算从手术日期到随访截尾日期,或由于复发、转移而死亡的日期为止。50例新鲜T1期非小细胞肺癌及癌旁正常肺组织来自中于国医科大学附属第一临床医院胸外科2007年11月～2008年11月手术切除标本。上述所有患者术前均未接受过放、化疗。
应用免疫组织化学技术及免疫印迹技术检测14-3-3ζ、β-catenin、E-cad以及p-Bad和p-Akt在T1期NSCLC及正常肺组织中的表达；应用RT-PCR技术检测14-3-3ζmRNA在T1期NSCLC及正常肺组织中含量的变化；应用免疫双标、共聚焦激光扫描显微镜技术观察T1期NSCLC中14-3-3ζ与β-catenin及14-3-3ζ与E-cad的表达和共存。
实验结果
一、14-3-3ζ在T1期NSCLC中的表达
我们通过免疫组化、Western Blotting及RT-PCR技术检测14-3-3ζ蛋白及14-3-3ζmRNA在T1期NSCLC及正常肺组织中的表达。结果显示,T1期NSCLC中14-3-3ζ表达较正常肺组织明显增强。14-3-3ζ的表达与患者的年龄、性别及病理学分型无关。但随着肺癌分化程度的降低,14-3-3ζ的表达逐渐增强。在淋巴结转移阳性的癌组织中,14-3-3ζ表达高于淋巴结转移阴性的癌组织。生存曲线分析显示14-3-3ζ表达阴性患者的5年生存率及平均生存时间均高于表达阳性者。
二、P-catenin在T1期NSCLC中的表达及与14-3-3ζ的关系
我们通过免疫组化、Western Blotting、免疫荧光双标及激光扫描共聚焦显微镜技术检测β-catenin蛋白在T1期NSCLC及正常肺组织中的表达。结果显示,T1期NSCLC中β-catenin的异常表达率明显增高。β-catenin的异常表达与患者的年龄、性别及病理学分型无关,而与癌组织分化程度相关。在高分化的癌组织中,β-catenin异常表达率较低；在低分化癌组织中,β-catenin异常表达率较高。在淋巴结转移阳性的癌组织中,β-catenin异常表达率明显高于淋巴结转移阴性的癌组织。统计学分析显示β-catenin异常表达与14-3-3ζ阳性表达呈显著负相关。
三、E-cad在T1期NSCLC中的表达及与14-3-3ζ的关系
我们通过免疫组化、Western Blotting、免疫荧光双标及激光扫描共聚焦显微镜技术检测E-cad蛋白在T1期NSCLC及正常肺组织中的表达。结果显示,在NSCLC中E-cad多表现为膜表达减弱或缺失。E-cad的异常表达与患者的年龄、性别及病理学分型无关,而与癌组织分化程度相关。在高分化癌组织中,E-cad异常表达率较低；在低分化癌组织中,E-cad异常表达率较高。在淋巴结转移阳性的癌组织中E-cad异常表达率明显高于淋巴结转移阴性的癌组织。统计学分析显不E-cad的异常表达与14-3-3ζ阳性表达呈显著负相关。
四、p-Bad在T1期NSCLC中的表达及与14-3-3ζ的关系
我们通过免疫组化及Western Blotting技术检测p-Bad蛋白在T1期NSCLC及正常肺组织中的表达。结果显示,在正常肺组织中p-Bad轻微表达,而在NSCLC中表达显著增强。p-Bad的表达与患者的年龄、性别及病理学分型无关,但与癌组织分化程度、淋巴结转移相关。随着肺癌分化程度的降低,p-Bad的表达逐渐增强；在淋巴结转移阳性的癌组织中,p-Bad表达高于淋巴结转移阴性的癌组织。统计学分析显示p-Bad蛋白与14-3-3ζ在T1期NSCLC中的表达明显正相关。
五、p-Akt在T1期NSCLC中的表达及与14-3-3ζ的关系
我们通过免疫组化及Western Blotting技术检测p-Akt蛋白在T1期NSCLC及正常肺组织中的表达。结果显示,在正常肺组织中p-Akt无表达,而在NSCLC中表达显著增强。p-Akt的表达与患者的年龄、性别及病理学分型无关,但与癌组织分化程度、淋巴结转移相关。随着肺癌分化程度的降低,p-Akt的表达逐渐增强；而在淋巴结转移阳性的癌组织中,p-Akt表达高于淋巴结转移阴性的癌组织。统计学分析显示p-Akt蛋白与14-3-3ζ在T1期NSCLC中的表达明显正相关。
结论
1.14-3-3ζ在T1期NSCLC中高表达,且其表达与患者的年龄、性别及病理学分型无关,而与癌组织分化程度、淋巴结转移及患者预后密切相关,提示14-3-3∈有可能作为判断T1期NSCLC发展与预后的生物学指标之一。
2.β-catenin、E-cad在T1期NSCLC表达明显低于正常肺组织。β-catenin、E-cad异常表达与患者的年龄、性别及病理学分型无关,而与肺癌分化程度及淋巴结转移密切相关,提示β-catenin、E-cad可能参与了NSCLC的发展和转移。
3.β-catenin、E-cad在T1期NSCLC中的异常表达与14-3-3ζ阳性表达呈明显负相关,提示14-3-3ζ的高表达可能导致了E-cad、β-catenin异常表达,从而对非小细胞肺癌的侵袭和转移产生影响。
4. p-Bad、p-Akt在T1期NSCLC中高表达,且与癌组织分化程度及淋巴结转移密切相关,提示p-Bad、p-Akt可能参与了NSCLC的发展和转移。
5.14-3-3ζ与p-Bad、p-Akt在T1期NSCLC中表达呈正相关,提示14-3-3ζ可能与p-Bad和p-Akt相互作用,共同参与NSCLC凋亡的调控。
Lung cancer is a malignant tumor and does harm to human health seriously.lt has been one of the leading causes of cancer death for urban residents in China.So it is important to detect the molecular mechanism of lung cancer invation and metastasis. Better understanding of the biological mechanisms in non-small cell lung cancer (NSCLC) is still needed to clarify the patients’progression,prognosis and achieve the most effective treatments. Research on relationship between 14-3-3 protein and lung cancer has become the focus now.
14-3-3 proteins are highly conserved small acidic proteins.Study shows 14-3-3 proteins can bind many signal proteins,such as kinase,phosphatase and transmembrane receptor et al. The 14-3-3 proteins play crucial roles in many biological processes including regulation of signaling,cell cycle and apoptosis.There are seven isoforms in mammalian genes:ζ,β,γ,ε，σ，ηandτ. Isoform s andζcan be found in normal lung tissue butβ,γ,σ,ε,ζandτare expressed in lung cancer tissue all.14-3-3 protein isoforms are relevant to tumorgenesis and regarded as potential tumor suppressor gene or oncogene. Thus intensive understanding the function of different 14-3-3 isoforms may offer us a new idea in aiticancer therapies.
14-3-3ζis one of the isoform of 14-3-3 proteins and its exact function is unclear now. Research of 14-3-3ζfocus on its regulation function in apoptosis. Incontrollable of apoptosis program is associated with the occurrence, development and prognosis in malignant tumor. Bcl-2 family is one of the most important protein family controlling apoptosis. Bad is one of the promoting apoptosis factor and its expression is of important significance in tumor. Ichinose reported that phosphorylated Bad lost its activity and inactive Bad(p-Bad) could enhance vicious transformation of glioblastoma and prostatic cancer.Research shows that 14-3-3ζ, can inhibit apoptosis through different mechanisms,such as by regulating the Bcl-2 family.14-3-3ζbinds the phosphorylated BAD and insulates it in the cytoplasm, prevents its binding with Bcl-2 and Bcl-XL and so as to inhibiting cell apoptosis. Akt is a serine-threonine kinase located in the downstream of phosphatidylinositol-3 - kinase (PI3-K) and can be activated by many cell survival promoting signal protein.Taso found that activation of Akt involved in early stage of canceration and may be the important event in NSCLC origination. Powell found that Akt phosphorylated Ser-58 on 14-3-3zeta both in vitro and in intact cells.Apoptosis regulated by 14-3-3ζmight control tumor occurrence, progression, invation and matastasis to some extent. So it is important for us to study the expression of apoptosis facters and its association with 14-3-3ζin NSCLC.
Malignancy often followed by invation and matastasia. Tumor invation and matastasia is a comolicated process involves many multiple steps and facters and its precondition is the loss of cell adherency. Study showed that E-cadherin had the most close relationship with the abnormal cell-cell adhesion and the proliferation,invation and matastasis of epithelial tumors.Function of cell-cell adhesion mediated by E-cadherin is associated with catenins andβ-catenin plays a key role in the regulation of cell adherency.Research on the relationship between 14-3-3ζand adhension occurs recently and thorough understanding of this relationship may intensify our knowledge and research on the invation and matastasis in NSCLC.
To test 14-3-3ζexpression and its relationship with invation, matastasis, prognosis and apoptosi in stage T1NSCLC tissues, western blotting, double labeling immunofluorescence and confocal laser scanning microscopy, immunohistochemical and RT-PCR analysis were carried out. Understanding the relationship between 14-3-3ζ protein and lung cancer and revealing the molecular mechanism will offer us a new idea to beat the disease.
MATERIALS AND METHODS
The study population included 110 patients who underwent resection of tumor for pathological stage T1 NSCLC between 2000 and 2006 at Department of Thoracic Surgery, First Affiliated Hospital of Liaoning Medical colledge. All cases were full of follow-up and cut-off date for April 2008, calculation of surial from surgery to cut-off date or to date dead of recurrence and metastasis.50 patients who underwent resection of tumor for pathological stage T1 NSCLC between November,2007 and November, 2008 at Department of Thoracic Surgery, First Affiliated Hospital of China Medical University. None of the patients received chemotherapy or radiotherapy before the operation.
To test 14-3-3ζ,β-catenin, E-cadherin, p-Bad and p-Akt expression in stage T1 NSCLC and normal lung tissues, western blotting, immunohistochemical analysis were carried out. To test 14-3-3ζmRNA content in NSCLC and normal lung tissues,RT-PCR analysis were carried out. Double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to test the coexistence of 14-3-3ζwithβ-catenin and E-cadherin.
RESULTS
一、Expression of 14-3-3ζin stage Ti NSCLC
Immunohistochemical, western blotting and RT-PCR analysis were carried out to test 14-3-3ζprotein and 14-3-3ζmRNA expression in stage T1 NSCLC and normal lung tissues. Result shows that more strong expression of 14-3-3ζwas observed in stage T1 NSCLC tissues than in normal lung cancer tissue. Overexpression of 14-3-3ζprotein did not show any correlation with age, gender and histological types,but was significantly correlated with histological grades and lymph node metastasis. Companied with lower lung cancer differentiation,14-3-3ζexpression increased. The 14-3-3ζ expression was higher in the lymph node metastasis positive cancer tissues than in the negatie cases. The survival curves analysis showed that 14-3-3ζexpression is closely related to the prognosis of patients.
二、Expression ofβ-catenin in stage T1 NSCLC and Relationship between 14-3-3ζand P-catenin
Immunohistochemical, western blotting, double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to testβ-catenin protein expression in stage T1 NSCLC and normal lung tissues. Result shows most NSCLC samples showed reduced membranous staining or increased nuclear or cytoplasmic staining. Abnormal expression ofβ-catenin protein did not show any correlation with age, gender and histological types but showed significantly correlation with poor differentiation and lymph node metastasis. Abnormalβ-catenin expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negatie cases. Statistical analysis showed significantly negative correlation between P-catenin protein and 14-3-3ζexpression.
三、Expression of E-cad in stage T1 NSCLC and Relationship between 14-3-3ζand E-cad
Immunohistochemical,western blotting, double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to test E-cad protein expression in stage T1 NSCLC and normal lung tissues. Result indicated most NSCLC samples showed reduced or deletion membranous staining. Abnormal expression of E-cad protein did not show any correlation with age, gender and histological types,but showed significantly correlation with poor differentiation and lymph node metastasis. Abnormal E-cad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negatie cases. Statistical analysis showed significantly negative correlation betweenβ-catenin protein and 14-3-3ζexpression in stage T, NSCLC.
四、Expression of p-Bad in stage T1 NSCLC and Relationship between 14-3-3ζand p-Bad
Immunohistochemical,western blotting analysis were carried out to test p-Bad protein expression in stage T1 NSCLC and normal lung tissues. Result showed increased expression of p-Bad in stage T1 NSCLC tissues. Expression of p-Bad protein did not show any correlation with age, gender and histological types but showed significantly correlation with poor differentiation and lymph node metastasis. Increased p-Bad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negative cases. Statistical analysis showed significantly positive correlation between p-Bad protein and 14-3-3ζexpression in stage T1 NSCLC.
五、Expression of p-Akt in stage T1 NSCLC and Relationship between 14-3-3ζand p-Akt
Immunohistochemical,western blotting analysis were carried out to test p-Akt protein expression in stage T1 NSCLC and normal lung tissues. Result showed no expression of p-Akt was observed in normal lung tissue but increased expression of p-Akt was observed in stage T1 NSCLC tissues. Expression of p-Akt protein did not show any correlation with age, gender and histological types,but showed significantly correlated with poor differentiation and lymph node metastasis. Increased p-Bad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negative cases. Statistical analysis showed significantly positive correlation between p-Akt protein and 14-3-3ζexpression in stage T1 NSCLC.
CONCLUSION
1. The upregulated expression of 14-3-3∫in stage T1 NSCLC indicate its possibility of promoting NSCLC dovelopment. Overexpression of 14-3-3ζcorrelated with histological grades, lymph node metastasis and poor clinical outcome but not with age, gender and histological types in stage T1NSCLC.lt seems that 14-3-3ζmight be used as develpomental and prognostic biomarkers for NSCLC.
2. Expression ofβ-catenin and E-cadherin is increased obviously in stage T1 NSCLC than in normel lung tissue.Abnormal expression ofβ-catenin and E-cadherin correlated with histological grades, lymph node metastasis but not with age, gender and histological types. It seems thatβ-catenin and E-cadherin might be involoved in development and metastasis of NSCLC.
3. Abnormalβ-catenin and E-cadherin expression was associated signigicantly with 14-3-3ζexpression in stage T1 NSCLC. It seems that overexpression of 14-3-3ζmight induce abnormal expression ofβ-catenin and E-cadherin so as to affect invation and metastasis of NSCLC.
4. Expression of p-Bad and p-Akt is increased obviously in stage T1 NSCLC and is correlated with histological grades, lymph node metastasis signigicantly. It seems that p-Bad and p-Akt might be involoved in development and metastasis of NSCLC.
5. Upregulated p-Bad and p-Akt expression was associated signigicantly with positive 14-3-3ζexpression in stage T1 NSCLC. It seems that overexpression of 14-3-3ζmight block apoptosis of NSCLC cells through some molecular mechanism.
14-3-3蛋白是一种高度保守的可溶性酸性蛋白家族。研究表明,14-3-3家族可以和许多信号蛋白,包括激酶、磷酸酶和跨膜受体等结合,在信号传导、细胞周期以及细胞凋亡的调控等方面发挥重要作用。哺乳动物中,14-3-3蛋白有7种亚型(β,γ,ε，η,σ,τ和ζ)。研究显示,在正常肺组织中只能检测到两种亚型：即ε和ζ,而在肺癌中β,γ,σ,τ,ε,ζ均有表达。14-3-3蛋白的亚型与肿瘤的发生密切相关,并被作为潜在的肿瘤抑制基因或致癌基因。因此,深入研究14-3-3蛋白的不同亚型可以为抗癌治疗提供新的思路。
14-3-3ζ是14-3-3家族中一个亚型。目前,14-3-3ζ的确切功能还不明了,对14-3-3ζ的研究大多集中在对凋亡的调控上。细胞凋亡程序失控与恶性肿瘤的发生、发展及预后密不可分。Bcl-2家族是调控凋亡过程最重要的蛋白家族之一其中的促凋亡因子Bad在肿瘤中的表达具有重要意义。Ichinose等[6]发现,Bad蛋白磷酸化后失活,失活的Bad加剧胶质母细胞瘤和前列腺癌的恶性转化。研究表明,14-3-3ζ可以通过不同机制对凋亡产生抑制效应,其中之一就是对Bcl-2家族的调控。14-3-3ζ可与磷酸化的Bad (p-Bad)结合,将其隔离在胞浆,从而阻止Bad与Bcl-2和Bcl-XL的结合,抑制凋亡。Akt是一个丝-苏氨酸激酶,位于磷脂酰肌醇-3-激酶(PI3-K)的下游,可被许多促细胞生存信号蛋白所激活。Taso等的研究提示Akt的活化参与癌变的早期阶段,是NSCLC(Non- small Cell LungCancer,非小细胞肺癌)发生中的重要事件。Powell等在体外及实体细胞中均发现Akt可以使14-3-3ζ的Ser-58磷酸化。14-3-3ζ对细胞凋亡的调控也可能在一定程度上对肿瘤的发生、发展、侵袭和转移进行调控,因此研究非小细胞肺癌中凋亡相关因子的表达及其与14-3-3ζ的相互关系具有重要意义。
恶性肿瘤往往伴有对邻近组织的浸润和远处转移。肿瘤的侵袭和转移是一个多步骤、多因素参与的极其复杂的过程,而细胞间粘附功能丧失则是其先决条件。研究表明,上皮型钙粘素(E-cad)与细胞间粘附功能异常、上皮肿瘤的增殖、浸润和转移关系最为密切[11]。E-cad的细胞粘附作用与其细胞内的连环蛋白有关,其中β-连环蛋白(β-catenin)对E-cad的功能调节尤为重要。近年来有关14-3-3ζ与粘附关系的研究开始出现。由于粘附在肿瘤侵袭、转移过程中的作用重要而且复杂,因而深入了解14-3-3ζ与粘附之间的关系,可以进一步加深我们对肺癌侵袭、转移机制的认识。
本课题应用免疫组织化学技术、免疫双标、激光共聚焦显微镜技术、Western Blotting技术及RT-PCR技术,并结合完整的临床资料,探讨14-3-3ζ在T1期非小细胞肺癌中的表达及与肺癌侵袭、转移、预后和凋亡的关系。了解14-3-3ζ蛋白与肺癌的关系并揭示其作用机制,将有可能为人类战胜这种疾病开辟一条新的途径。
实验方法
110例T1期非小细胞肺癌及癌旁组织均来自辽宁医学院2000～2006年手术切除存档蜡块。所有病例均有完整的随访记录,随访截尾日期为2008年4月。生存期的计算从手术日期到随访截尾日期,或由于复发、转移而死亡的日期为止。50例新鲜T1期非小细胞肺癌及癌旁正常肺组织来自中于国医科大学附属第一临床医院胸外科2007年11月～2008年11月手术切除标本。上述所有患者术前均未接受过放、化疗。
应用免疫组织化学技术及免疫印迹技术检测14-3-3ζ、β-catenin、E-cad以及p-Bad和p-Akt在T1期NSCLC及正常肺组织中的表达；应用RT-PCR技术检测14-3-3ζmRNA在T1期NSCLC及正常肺组织中含量的变化；应用免疫双标、共聚焦激光扫描显微镜技术观察T1期NSCLC中14-3-3ζ与β-catenin及14-3-3ζ与E-cad的表达和共存。
实验结果
一、14-3-3ζ在T1期NSCLC中的表达
我们通过免疫组化、Western Blotting及RT-PCR技术检测14-3-3ζ蛋白及14-3-3ζmRNA在T1期NSCLC及正常肺组织中的表达。结果显示,T1期NSCLC中14-3-3ζ表达较正常肺组织明显增强。14-3-3ζ的表达与患者的年龄、性别及病理学分型无关。但随着肺癌分化程度的降低,14-3-3ζ的表达逐渐增强。在淋巴结转移阳性的癌组织中,14-3-3ζ表达高于淋巴结转移阴性的癌组织。生存曲线分析显示14-3-3ζ表达阴性患者的5年生存率及平均生存时间均高于表达阳性者。
二、P-catenin在T1期NSCLC中的表达及与14-3-3ζ的关系
我们通过免疫组化、Western Blotting、免疫荧光双标及激光扫描共聚焦显微镜技术检测β-catenin蛋白在T1期NSCLC及正常肺组织中的表达。结果显示,T1期NSCLC中β-catenin的异常表达率明显增高。β-catenin的异常表达与患者的年龄、性别及病理学分型无关,而与癌组织分化程度相关。在高分化的癌组织中,β-catenin异常表达率较低；在低分化癌组织中,β-catenin异常表达率较高。在淋巴结转移阳性的癌组织中,β-catenin异常表达率明显高于淋巴结转移阴性的癌组织。统计学分析显示β-catenin异常表达与14-3-3ζ阳性表达呈显著负相关。
三、E-cad在T1期NSCLC中的表达及与14-3-3ζ的关系
我们通过免疫组化、Western Blotting、免疫荧光双标及激光扫描共聚焦显微镜技术检测E-cad蛋白在T1期NSCLC及正常肺组织中的表达。结果显示,在NSCLC中E-cad多表现为膜表达减弱或缺失。E-cad的异常表达与患者的年龄、性别及病理学分型无关,而与癌组织分化程度相关。在高分化癌组织中,E-cad异常表达率较低；在低分化癌组织中,E-cad异常表达率较高。在淋巴结转移阳性的癌组织中E-cad异常表达率明显高于淋巴结转移阴性的癌组织。统计学分析显不E-cad的异常表达与14-3-3ζ阳性表达呈显著负相关。
四、p-Bad在T1期NSCLC中的表达及与14-3-3ζ的关系
我们通过免疫组化及Western Blotting技术检测p-Bad蛋白在T1期NSCLC及正常肺组织中的表达。结果显示,在正常肺组织中p-Bad轻微表达,而在NSCLC中表达显著增强。p-Bad的表达与患者的年龄、性别及病理学分型无关,但与癌组织分化程度、淋巴结转移相关。随着肺癌分化程度的降低,p-Bad的表达逐渐增强；在淋巴结转移阳性的癌组织中,p-Bad表达高于淋巴结转移阴性的癌组织。统计学分析显示p-Bad蛋白与14-3-3ζ在T1期NSCLC中的表达明显正相关。
五、p-Akt在T1期NSCLC中的表达及与14-3-3ζ的关系
我们通过免疫组化及Western Blotting技术检测p-Akt蛋白在T1期NSCLC及正常肺组织中的表达。结果显示,在正常肺组织中p-Akt无表达,而在NSCLC中表达显著增强。p-Akt的表达与患者的年龄、性别及病理学分型无关,但与癌组织分化程度、淋巴结转移相关。随着肺癌分化程度的降低,p-Akt的表达逐渐增强；而在淋巴结转移阳性的癌组织中,p-Akt表达高于淋巴结转移阴性的癌组织。统计学分析显示p-Akt蛋白与14-3-3ζ在T1期NSCLC中的表达明显正相关。
结论
1.14-3-3ζ在T1期NSCLC中高表达,且其表达与患者的年龄、性别及病理学分型无关,而与癌组织分化程度、淋巴结转移及患者预后密切相关,提示14-3-3∈有可能作为判断T1期NSCLC发展与预后的生物学指标之一。
2.β-catenin、E-cad在T1期NSCLC表达明显低于正常肺组织。β-catenin、E-cad异常表达与患者的年龄、性别及病理学分型无关,而与肺癌分化程度及淋巴结转移密切相关,提示β-catenin、E-cad可能参与了NSCLC的发展和转移。
3.β-catenin、E-cad在T1期NSCLC中的异常表达与14-3-3ζ阳性表达呈明显负相关,提示14-3-3ζ的高表达可能导致了E-cad、β-catenin异常表达,从而对非小细胞肺癌的侵袭和转移产生影响。
4. p-Bad、p-Akt在T1期NSCLC中高表达,且与癌组织分化程度及淋巴结转移密切相关,提示p-Bad、p-Akt可能参与了NSCLC的发展和转移。
5.14-3-3ζ与p-Bad、p-Akt在T1期NSCLC中表达呈正相关,提示14-3-3ζ可能与p-Bad和p-Akt相互作用,共同参与NSCLC凋亡的调控。
Lung cancer is a malignant tumor and does harm to human health seriously.lt has been one of the leading causes of cancer death for urban residents in China.So it is important to detect the molecular mechanism of lung cancer invation and metastasis. Better understanding of the biological mechanisms in non-small cell lung cancer (NSCLC) is still needed to clarify the patients’progression,prognosis and achieve the most effective treatments. Research on relationship between 14-3-3 protein and lung cancer has become the focus now.
14-3-3 proteins are highly conserved small acidic proteins.Study shows 14-3-3 proteins can bind many signal proteins,such as kinase,phosphatase and transmembrane receptor et al. The 14-3-3 proteins play crucial roles in many biological processes including regulation of signaling,cell cycle and apoptosis.There are seven isoforms in mammalian genes:ζ,β,γ,ε，σ，ηandτ. Isoform s andζcan be found in normal lung tissue butβ,γ,σ,ε,ζandτare expressed in lung cancer tissue all.14-3-3 protein isoforms are relevant to tumorgenesis and regarded as potential tumor suppressor gene or oncogene. Thus intensive understanding the function of different 14-3-3 isoforms may offer us a new idea in aiticancer therapies.
14-3-3ζis one of the isoform of 14-3-3 proteins and its exact function is unclear now. Research of 14-3-3ζfocus on its regulation function in apoptosis. Incontrollable of apoptosis program is associated with the occurrence, development and prognosis in malignant tumor. Bcl-2 family is one of the most important protein family controlling apoptosis. Bad is one of the promoting apoptosis factor and its expression is of important significance in tumor. Ichinose reported that phosphorylated Bad lost its activity and inactive Bad(p-Bad) could enhance vicious transformation of glioblastoma and prostatic cancer.Research shows that 14-3-3ζ, can inhibit apoptosis through different mechanisms,such as by regulating the Bcl-2 family.14-3-3ζbinds the phosphorylated BAD and insulates it in the cytoplasm, prevents its binding with Bcl-2 and Bcl-XL and so as to inhibiting cell apoptosis. Akt is a serine-threonine kinase located in the downstream of phosphatidylinositol-3 - kinase (PI3-K) and can be activated by many cell survival promoting signal protein.Taso found that activation of Akt involved in early stage of canceration and may be the important event in NSCLC origination. Powell found that Akt phosphorylated Ser-58 on 14-3-3zeta both in vitro and in intact cells.Apoptosis regulated by 14-3-3ζmight control tumor occurrence, progression, invation and matastasis to some extent. So it is important for us to study the expression of apoptosis facters and its association with 14-3-3ζin NSCLC.
Malignancy often followed by invation and matastasia. Tumor invation and matastasia is a comolicated process involves many multiple steps and facters and its precondition is the loss of cell adherency. Study showed that E-cadherin had the most close relationship with the abnormal cell-cell adhesion and the proliferation,invation and matastasis of epithelial tumors.Function of cell-cell adhesion mediated by E-cadherin is associated with catenins andβ-catenin plays a key role in the regulation of cell adherency.Research on the relationship between 14-3-3ζand adhension occurs recently and thorough understanding of this relationship may intensify our knowledge and research on the invation and matastasis in NSCLC.
To test 14-3-3ζexpression and its relationship with invation, matastasis, prognosis and apoptosi in stage T1NSCLC tissues, western blotting, double labeling immunofluorescence and confocal laser scanning microscopy, immunohistochemical and RT-PCR analysis were carried out. Understanding the relationship between 14-3-3ζ protein and lung cancer and revealing the molecular mechanism will offer us a new idea to beat the disease.
MATERIALS AND METHODS
The study population included 110 patients who underwent resection of tumor for pathological stage T1 NSCLC between 2000 and 2006 at Department of Thoracic Surgery, First Affiliated Hospital of Liaoning Medical colledge. All cases were full of follow-up and cut-off date for April 2008, calculation of surial from surgery to cut-off date or to date dead of recurrence and metastasis.50 patients who underwent resection of tumor for pathological stage T1 NSCLC between November,2007 and November, 2008 at Department of Thoracic Surgery, First Affiliated Hospital of China Medical University. None of the patients received chemotherapy or radiotherapy before the operation.
To test 14-3-3ζ,β-catenin, E-cadherin, p-Bad and p-Akt expression in stage T1 NSCLC and normal lung tissues, western blotting, immunohistochemical analysis were carried out. To test 14-3-3ζmRNA content in NSCLC and normal lung tissues,RT-PCR analysis were carried out. Double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to test the coexistence of 14-3-3ζwithβ-catenin and E-cadherin.
RESULTS
一、Expression of 14-3-3ζin stage Ti NSCLC
Immunohistochemical, western blotting and RT-PCR analysis were carried out to test 14-3-3ζprotein and 14-3-3ζmRNA expression in stage T1 NSCLC and normal lung tissues. Result shows that more strong expression of 14-3-3ζwas observed in stage T1 NSCLC tissues than in normal lung cancer tissue. Overexpression of 14-3-3ζprotein did not show any correlation with age, gender and histological types,but was significantly correlated with histological grades and lymph node metastasis. Companied with lower lung cancer differentiation,14-3-3ζexpression increased. The 14-3-3ζ expression was higher in the lymph node metastasis positive cancer tissues than in the negatie cases. The survival curves analysis showed that 14-3-3ζexpression is closely related to the prognosis of patients.
二、Expression ofβ-catenin in stage T1 NSCLC and Relationship between 14-3-3ζand P-catenin
Immunohistochemical, western blotting, double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to testβ-catenin protein expression in stage T1 NSCLC and normal lung tissues. Result shows most NSCLC samples showed reduced membranous staining or increased nuclear or cytoplasmic staining. Abnormal expression ofβ-catenin protein did not show any correlation with age, gender and histological types but showed significantly correlation with poor differentiation and lymph node metastasis. Abnormalβ-catenin expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negatie cases. Statistical analysis showed significantly negative correlation between P-catenin protein and 14-3-3ζexpression.
三、Expression of E-cad in stage T1 NSCLC and Relationship between 14-3-3ζand E-cad
Immunohistochemical,western blotting, double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to test E-cad protein expression in stage T1 NSCLC and normal lung tissues. Result indicated most NSCLC samples showed reduced or deletion membranous staining. Abnormal expression of E-cad protein did not show any correlation with age, gender and histological types,but showed significantly correlation with poor differentiation and lymph node metastasis. Abnormal E-cad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negatie cases. Statistical analysis showed significantly negative correlation betweenβ-catenin protein and 14-3-3ζexpression in stage T, NSCLC.
四、Expression of p-Bad in stage T1 NSCLC and Relationship between 14-3-3ζand p-Bad
Immunohistochemical,western blotting analysis were carried out to test p-Bad protein expression in stage T1 NSCLC and normal lung tissues. Result showed increased expression of p-Bad in stage T1 NSCLC tissues. Expression of p-Bad protein did not show any correlation with age, gender and histological types but showed significantly correlation with poor differentiation and lymph node metastasis. Increased p-Bad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negative cases. Statistical analysis showed significantly positive correlation between p-Bad protein and 14-3-3ζexpression in stage T1 NSCLC.
五、Expression of p-Akt in stage T1 NSCLC and Relationship between 14-3-3ζand p-Akt
Immunohistochemical,western blotting analysis were carried out to test p-Akt protein expression in stage T1 NSCLC and normal lung tissues. Result showed no expression of p-Akt was observed in normal lung tissue but increased expression of p-Akt was observed in stage T1 NSCLC tissues. Expression of p-Akt protein did not show any correlation with age, gender and histological types,but showed significantly correlated with poor differentiation and lymph node metastasis. Increased p-Bad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negative cases. Statistical analysis showed significantly positive correlation between p-Akt protein and 14-3-3ζexpression in stage T1 NSCLC.
CONCLUSION
1. The upregulated expression of 14-3-3∫in stage T1 NSCLC indicate its possibility of promoting NSCLC dovelopment. Overexpression of 14-3-3ζcorrelated with histological grades, lymph node metastasis and poor clinical outcome but not with age, gender and histological types in stage T1NSCLC.lt seems that 14-3-3ζmight be used as develpomental and prognostic biomarkers for NSCLC.
2. Expression ofβ-catenin and E-cadherin is increased obviously in stage T1 NSCLC than in normel lung tissue.Abnormal expression ofβ-catenin and E-cadherin correlated with histological grades, lymph node metastasis but not with age, gender and histological types. It seems thatβ-catenin and E-cadherin might be involoved in development and metastasis of NSCLC.
3. Abnormalβ-catenin and E-cadherin expression was associated signigicantly with 14-3-3ζexpression in stage T1 NSCLC. It seems that overexpression of 14-3-3ζmight induce abnormal expression ofβ-catenin and E-cadherin so as to affect invation and metastasis of NSCLC.
4. Expression of p-Bad and p-Akt is increased obviously in stage T1 NSCLC and is correlated with histological grades, lymph node metastasis signigicantly. It seems that p-Bad and p-Akt might be involoved in development and metastasis of NSCLC.
5. Upregulated p-Bad and p-Akt expression was associated signigicantly with positive 14-3-3ζexpression in stage T1 NSCLC. It seems that overexpression of 14-3-3ζmight block apoptosis of NSCLC cells through some molecular mechanism.
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