Twist在肺癌中高表达并与肺癌的分化有关
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摘要
前言
转录因子Twist是上皮-间质转变(EMT)过程中的重要调控因子,在肿瘤进展中发挥重要作用。上皮间质化是肿瘤进展的重要过程,Twist被认为通过调节E-cadherin的表达通过EMT途径调控肿瘤的转移。然而Twist在肿瘤进展中的具体作用仍不清。本研究采用肺癌组织标本,检测Twist在肺癌组织中的表达水平,及其与临床病理因素的关系,同时检测Twist蛋白和Twist1、Twist2的mRNA在多种肺癌细胞系中的表达,深入研究Twist与肺癌发生发展的关系。
材料和方法
一、病例和材料
收集中国医科大学附属第一医院2000年11月至2006年4月间手术切除并经病理确诊的68例肺腺癌和肺鳞癌标本,其中男性40例,女性28例,患者平均年龄59岁;所有病例术前均未接受过放化疗及免疫治疗,取例癌旁正常肺组织作为对照。细胞系用含15%胎牛血清的RPMI-1640培养基,其它细胞都用含10%FBS的RPMI-1640培养基,pH值保持在7.2～7.4之间,于含5%CO_2的37℃孵箱内培养。
二、试剂
Twist兔抗人多克隆抗体Twist(H-81)购自美国Santa Cruz生物技术公司,SP超敏免疫组织化学试剂盒(KIT-9710)和DAB显色试剂盒(DAB-0031)购自福州迈新生物技术公司,Trizol试剂购自美国Invitrogen公司,RT-PCR试剂盒TaKaRa RNA PCR Kit(AMV)Ver.3.0购自大连宝生物(TaKaRa)公司,RPMI-1640购自GIBCO公司。
三、方法
应用免疫组化SP法检测肺癌组织及癌旁正常肺组织中Twist的表达。染色方法按SP免疫组化试剂盒说明书进行,经柠檬酸(pH=6.0)微波抗原修复。每张切片在光学显微镜(×400)下随机选取10个视野,每个视野计数100个肿瘤细胞,计数其中的阳性细胞数,然后取平均值。染色强度分级为:阴性为0,淡黄色为1,黄棕色为2,深棕色为3。以阳性肿瘤细胞数分级:＜5%为0,5%～25%为1,25%～50%为2,＞50%为3。利用染色强度与阳性肿瘤细胞数乘积来评定结果,0为阴性,1为弱阳性,2～4为阳性,6～9为强阳性。电泳成像后进行分析光密度值。将Twist1、Twist2的光密度值与GAPDH光密度值的比值作为Twist1、Twist2的相对表达量进行比较分析。
四、统计分析方法
统计学分析采用SPSS for Windows 13.0统计分析软件进行分析,对Twist的表达与临床病理特征的关系采用两组资料非参数检验和多组资料非参数秩和检验,Twist在细胞系中的表达采用方差分析,P＜0.05为有统计学意义。
实验结果
一、免疫组化
68例肺鳞癌和腺癌组织中,有8例为Twist阴性(11.76%),9例为Twist弱表达(13.24%),28例Twist表达(41.18%),有23例Twist高表达(33.82%)(图1)。Twist在8例癌旁肺组织中弱表达。Twist的表达情况与肺癌的分化程度(P=0.002)和肺癌患者的年龄(P=0.012)相关。Twist的表达水平与患者的性别(P=0.820)、组织学类型(P=0.426)、TNM分期(P=0.200)和有无淋巴结转移(P=0.392)关系并不显著。
二、RT-PCR
Twist1和Twist2的mRNA在HBE和8种肺癌细胞系中的表达并不一致(图2)。Twist1在HBE表达,在不同组织学类型的癌细胞系之间比较,Twist1在在肺腺癌A549表达最高,在肺鳞癌SK细胞系中表达较A549少;在肺大细胞癌460和661和肺巨细胞癌LH7和BE1细胞系中的表达水平基本相同,表达水平较低;在肺邢赴?46细胞系中表达水平较A549低。Twist2在HBE表达,但较Twist1表达水平低,在不同组织学类型的癌细胞系之间比较,Twist2在肺鳞癌细胞系SK中表达较高,肺腺癌A549和LTE中表达较SK少;在肺大细胞癌460和661和肺巨细胞癌LH7和BE1细胞系中的表达水平基本相同,表达水平较低;在肺小细胞癌446细胞系中表达水平较高。其次在同种组织学类型的不同细胞系中比较,Twist2 mRNA在A549的表达低于LTE;在460、661两种细胞系中表达水平也较为一致;在LH7和BE1细胞系表达水平也无明显差别。
三、免疫荧光
在人支气管上皮HBE细胞系中,Twist弱表达,Twist蛋白在不同肺癌细胞系中表达水平不同(图3～图8),在肺腺癌LTE和A549细胞系和肺鳞癌SK中表达较强,大细胞癌460和661荧光强度比腺癌和鳞癌弱,Twist蛋白表达定位以细胞浆为主。
结论
在癌旁肺组织中Twist弱表达,肺鳞癌和肺腺癌组织中Twist较高表达。Twist的表达情况与肺癌的分化程度相关,低分化的肺癌Twist高表达,中高分化的肺癌其阳性表达,这表明Twist可能在肺癌发生中起重要作用。Twist的mRNA表达在肺鳞癌和腺癌细胞系中均高于HBE。在蛋白水平,Twist的表达在肺鳞癌和腺癌细胞系中均高于HBE,表达于细胞浆。我们的研究提示Twist表达上调可能引起肺泡上皮和支气管粘膜上皮向肿瘤转化,Twist表达水平与肺癌的分化程度密切相关,可作为肺癌进展的生物学标志。
Introduction
Twist has been identified as a promoting factor for epithelial-mesenchymal transition(EMT),which enhances the metastatic potential of cancer.The aim of this study is to detect the expression level of Twist in lung cancer tissues and cell lines,and analyze its relationship with clinicopathologic characteristics and biological behavior of lung cancers.
Methods
Twist expression was examined in 68 lung cancer specimens and 8 normal lung specimens using immunohistochcmistry(S-P method).Expression levels of Twist1 and Twist2 mRNA were detected using RT-PCR(transcription-polymerase chain reaction, RT-PCR) in HBE((human normal bronchi epithelium,HBE) and 8 lung cancer cell lines.Immunofluorescence was used to detect the Twist protein expression level and subcellular localization in lung cancer cells and HBE cells.
Results
Among 68 lung cancer specimens,9 samples showed weak expression of Twist 13.24%(9 of 68),75.00%(51 of 68) lung cancer specimens showed moderate to strong Twist staining whereas 8 corresponding normal lung specimens showed weak staining extent.Twist expression level was positively correlated with differentiation(P=0.002) and age(P=0.012).Twist1 and Twist2 mRNA expression levels were not the same in different histology types.The fluorescence signal of Twist protein was conspicuous in lung squamous cell carcinoma cells and adenocarcinoma cells,primarily in cytoplasm, but low in HBE.
Conclusion
High expression of Twist in lung cancer was associated with differentiation.Twist expressed primarily in cytoplasm.Twist could be used as a valuable biomarker to evaluate the progression of lung cancer.
转录因子Twist是上皮-间质转变(EMT)过程中的重要调控因子,在肿瘤进展中发挥重要作用。上皮间质化是肿瘤进展的重要过程,Twist被认为通过调节E-cadherin的表达通过EMT途径调控肿瘤的转移。然而Twist在肿瘤进展中的具体作用仍不清。本研究采用肺癌组织标本,检测Twist在肺癌组织中的表达水平,及其与临床病理因素的关系,同时检测Twist蛋白和Twist1、Twist2的mRNA在多种肺癌细胞系中的表达,深入研究Twist与肺癌发生发展的关系。
材料和方法
一、病例和材料
收集中国医科大学附属第一医院2000年11月至2006年4月间手术切除并经病理确诊的68例肺腺癌和肺鳞癌标本,其中男性40例,女性28例,患者平均年龄59岁;所有病例术前均未接受过放化疗及免疫治疗,取例癌旁正常肺组织作为对照。细胞系用含15%胎牛血清的RPMI-1640培养基,其它细胞都用含10%FBS的RPMI-1640培养基,pH值保持在7.2～7.4之间,于含5%CO_2的37℃孵箱内培养。
二、试剂
Twist兔抗人多克隆抗体Twist(H-81)购自美国Santa Cruz生物技术公司,SP超敏免疫组织化学试剂盒(KIT-9710)和DAB显色试剂盒(DAB-0031)购自福州迈新生物技术公司,Trizol试剂购自美国Invitrogen公司,RT-PCR试剂盒TaKaRa RNA PCR Kit(AMV)Ver.3.0购自大连宝生物(TaKaRa)公司,RPMI-1640购自GIBCO公司。
三、方法
应用免疫组化SP法检测肺癌组织及癌旁正常肺组织中Twist的表达。染色方法按SP免疫组化试剂盒说明书进行,经柠檬酸(pH=6.0)微波抗原修复。每张切片在光学显微镜(×400)下随机选取10个视野,每个视野计数100个肿瘤细胞,计数其中的阳性细胞数,然后取平均值。染色强度分级为:阴性为0,淡黄色为1,黄棕色为2,深棕色为3。以阳性肿瘤细胞数分级:＜5%为0,5%～25%为1,25%～50%为2,＞50%为3。利用染色强度与阳性肿瘤细胞数乘积来评定结果,0为阴性,1为弱阳性,2～4为阳性,6～9为强阳性。电泳成像后进行分析光密度值。将Twist1、Twist2的光密度值与GAPDH光密度值的比值作为Twist1、Twist2的相对表达量进行比较分析。
四、统计分析方法
统计学分析采用SPSS for Windows 13.0统计分析软件进行分析,对Twist的表达与临床病理特征的关系采用两组资料非参数检验和多组资料非参数秩和检验,Twist在细胞系中的表达采用方差分析,P＜0.05为有统计学意义。
实验结果
一、免疫组化
68例肺鳞癌和腺癌组织中,有8例为Twist阴性(11.76%),9例为Twist弱表达(13.24%),28例Twist表达(41.18%),有23例Twist高表达(33.82%)(图1)。Twist在8例癌旁肺组织中弱表达。Twist的表达情况与肺癌的分化程度(P=0.002)和肺癌患者的年龄(P=0.012)相关。Twist的表达水平与患者的性别(P=0.820)、组织学类型(P=0.426)、TNM分期(P=0.200)和有无淋巴结转移(P=0.392)关系并不显著。
二、RT-PCR
Twist1和Twist2的mRNA在HBE和8种肺癌细胞系中的表达并不一致(图2)。Twist1在HBE表达,在不同组织学类型的癌细胞系之间比较,Twist1在在肺腺癌A549表达最高,在肺鳞癌SK细胞系中表达较A549少;在肺大细胞癌460和661和肺巨细胞癌LH7和BE1细胞系中的表达水平基本相同,表达水平较低;在肺邢赴?46细胞系中表达水平较A549低。Twist2在HBE表达,但较Twist1表达水平低,在不同组织学类型的癌细胞系之间比较,Twist2在肺鳞癌细胞系SK中表达较高,肺腺癌A549和LTE中表达较SK少;在肺大细胞癌460和661和肺巨细胞癌LH7和BE1细胞系中的表达水平基本相同,表达水平较低;在肺小细胞癌446细胞系中表达水平较高。其次在同种组织学类型的不同细胞系中比较,Twist2 mRNA在A549的表达低于LTE;在460、661两种细胞系中表达水平也较为一致;在LH7和BE1细胞系表达水平也无明显差别。
三、免疫荧光
在人支气管上皮HBE细胞系中,Twist弱表达,Twist蛋白在不同肺癌细胞系中表达水平不同(图3～图8),在肺腺癌LTE和A549细胞系和肺鳞癌SK中表达较强,大细胞癌460和661荧光强度比腺癌和鳞癌弱,Twist蛋白表达定位以细胞浆为主。
结论
在癌旁肺组织中Twist弱表达,肺鳞癌和肺腺癌组织中Twist较高表达。Twist的表达情况与肺癌的分化程度相关,低分化的肺癌Twist高表达,中高分化的肺癌其阳性表达,这表明Twist可能在肺癌发生中起重要作用。Twist的mRNA表达在肺鳞癌和腺癌细胞系中均高于HBE。在蛋白水平,Twist的表达在肺鳞癌和腺癌细胞系中均高于HBE,表达于细胞浆。我们的研究提示Twist表达上调可能引起肺泡上皮和支气管粘膜上皮向肿瘤转化,Twist表达水平与肺癌的分化程度密切相关,可作为肺癌进展的生物学标志。
Introduction
Twist has been identified as a promoting factor for epithelial-mesenchymal transition(EMT),which enhances the metastatic potential of cancer.The aim of this study is to detect the expression level of Twist in lung cancer tissues and cell lines,and analyze its relationship with clinicopathologic characteristics and biological behavior of lung cancers.
Methods
Twist expression was examined in 68 lung cancer specimens and 8 normal lung specimens using immunohistochcmistry(S-P method).Expression levels of Twist1 and Twist2 mRNA were detected using RT-PCR(transcription-polymerase chain reaction, RT-PCR) in HBE((human normal bronchi epithelium,HBE) and 8 lung cancer cell lines.Immunofluorescence was used to detect the Twist protein expression level and subcellular localization in lung cancer cells and HBE cells.
Results
Among 68 lung cancer specimens,9 samples showed weak expression of Twist 13.24%(9 of 68),75.00%(51 of 68) lung cancer specimens showed moderate to strong Twist staining whereas 8 corresponding normal lung specimens showed weak staining extent.Twist expression level was positively correlated with differentiation(P=0.002) and age(P=0.012).Twist1 and Twist2 mRNA expression levels were not the same in different histology types.The fluorescence signal of Twist protein was conspicuous in lung squamous cell carcinoma cells and adenocarcinoma cells,primarily in cytoplasm, but low in HBE.
Conclusion
High expression of Twist in lung cancer was associated with differentiation.Twist expressed primarily in cytoplasm.Twist could be used as a valuable biomarker to evaluate the progression of lung cancer.
引文
1 Kang Y,Massague J.Epithelial-mesenchymal transitions: Twist in development and metastasis.Cell,2004,118 (3): 277-279.
2 Howe L R,Watanabe O,Leonard J,et al.Twist is up-regulated in response to Wntl and inhibits mouse mammary cell differentiation.Cancer Res,2003,63 (8): 1906-1913.
3 Stasiopoulos IA,Mironchik Y,Raman A,et al.HOXA5-Twist interaction alters p53 homeostasis in breast cancer cells.J Bio Chem,2005,280 (3): 2294-2299.
4 Hamamori Y,SartorelliV,Ogrysko V,et al.Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein Twist and adenoviral oncoprotein El A.Cell,1999,96 (3): 405-413.
5 KwokW K,LingM T,Lee TW,et al.Up-regulation of Twist in prostate cancer and its implication as a therapeutic target.Cancer Res,2005,65 (12): 5153-5162.
6 Hosono S,Kajiyama H,Terauchi M,et al.Expression of Twist increases the risk for recurrence and for poor survival in epithelial ovarian carcinoma patients.Br J Cancer.2007,96(2):314-20.
7 EliasM C,Tozer K R,Silber J R,et al.Twist expressed in human gliomas and promotes invasion.Neoplasia,2005,7(9): 824-837.
8 Yang J,Mani S A,Donaher J L,et al.Twist,a master regulator of morphogenesis,plays an essential role in tumor metastasis.Cell,2004,117 (7): 927-939.
9 Mironchik Y,Winnard P T,Vesuna F,et al.Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer.Cancer Res,2005,65 (23): 10801-10809.
10 Horikawa T,Yang J,Kondo S,et al.Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma.Cancer Res,2007,l;67(5):1970-8.
11 Zhang Z,Xie D,Li X,et al.Significance of Twist expression and its association with E-cadherin in bladder cancer.Hum Pathol.2007,38(4):598-606.
12 Hoek K,Rimm D L,Williams K R,et al.Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas.Cancer Res,2004,64 (15): 5270-5282
13 Yuen HF,Chan YP,Lok-Yee Wong M ,et al.Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis.J Clin Pathol.2007,60(5):510-4.
14 Ste' phane Ansieau,Jeremy Bastid,Agne' s Doreau,et al.Induction of EMT by Twist Proteins as a Collateral Effect of Tumor-Promoting Inactivation of Premature Senescence.Cancer Cell.2008,14:79-89.
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9 Howe L R,Watanabe O,Leonard J,et al.Twist is up-regulated in response to Wntl and inhibits mouse mammary cell differentiation.Cancer Res,2003,63 (8): 1906-1913.
10 Stasiopoulos LA,Mironchik Y,Raman A,et al.HOXA5-twist interaction alters p53 homeostasis in breast cancer cells.J BiolChem,2005,280 (3): 2294-2299.
11 Hamamori Y,SartorelliV,Ogrysko V,et al.Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein El A .Cell,1999,96 (3): 405-413.
12 KwokW K,LingM T,Lee TW,et al.Up-regulation of Twist in prostate cancer and its implication as a therapeutic target .Cancer Res,2005,65 (12): 5153-5162.
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16 Yang J,Mani S A,Donaher J L,et al.Twist,a master regulator of morphogenesis,plays an essential role in tumor metastasis.Cell,2004,117(7): 927-939.
17 Mironchik Y,Winnard P T,Vesuna F,et al.Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer.Cancer Res,2005,65(23):10801-10809.
18 EliasM C,Tozer K R,Silber J R,et al.Twist is expressed in human gliomas and promotes invasion.Neoplasia,2005,7(9):824-837.
19 Horikawa T,Yang J,Kondo S,et al.Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma.Cancer Res,2007,1;67(5):1970-8.
20 Zhang Z,Xie D,Li X,et al.Significance of Twist expression and its association with E-cadherin in bladder cancer.Hum Pathol.2007,38(4):598-606.
21 Hosono S,Kajiyama H,Terauchi M,et al.Expression of Twist increases the risk for recurrence and for poor survival in epithelial ovarian carcinoma patients.Br J Cancer.2007,96(2):314-20.
22 Hoek K,Rimm D L,Williams K R,et al.Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas.Cancer Res,2004,64(15):5270-5282
23 Yuen HF,Chan YP,Lok-Yee Wong M,et al.Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis.J Clin Pathol.2007,60(5):510-4
24 卓文磊等.针对Twist的短发夹RNA抑制A549细胞上皮-间充质转分化及体外侵袭.中国癌症杂志,2006,16(5):325-328
25 Wang X,LingM T,Guan X Y,et al.Identification of a novel function of Twist,a bHLH protein,in the development of acquired taxol resistance in human cancer cells.Oncogene,2004,23(2):474-482.
26 Puisieux A,Valsesia-Wittmann S,Ansieau S,et al.A twist for survival and cancer progression.Br J Cancer,2006,94(1):13-17
2 Howe L R,Watanabe O,Leonard J,et al.Twist is up-regulated in response to Wntl and inhibits mouse mammary cell differentiation.Cancer Res,2003,63 (8): 1906-1913.
3 Stasiopoulos IA,Mironchik Y,Raman A,et al.HOXA5-Twist interaction alters p53 homeostasis in breast cancer cells.J Bio Chem,2005,280 (3): 2294-2299.
4 Hamamori Y,SartorelliV,Ogrysko V,et al.Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein Twist and adenoviral oncoprotein El A.Cell,1999,96 (3): 405-413.
5 KwokW K,LingM T,Lee TW,et al.Up-regulation of Twist in prostate cancer and its implication as a therapeutic target.Cancer Res,2005,65 (12): 5153-5162.
6 Hosono S,Kajiyama H,Terauchi M,et al.Expression of Twist increases the risk for recurrence and for poor survival in epithelial ovarian carcinoma patients.Br J Cancer.2007,96(2):314-20.
7 EliasM C,Tozer K R,Silber J R,et al.Twist expressed in human gliomas and promotes invasion.Neoplasia,2005,7(9): 824-837.
8 Yang J,Mani S A,Donaher J L,et al.Twist,a master regulator of morphogenesis,plays an essential role in tumor metastasis.Cell,2004,117 (7): 927-939.
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