p120-catenin及其亚型在肺癌中的表达和意义
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摘要
目的
p120-catenin(p120ctn)作为catenin家族的重要成员,不仅可以通过与E-cadherin(E-cad)直接结合形成E-cadherin/catenin复合体,调节细胞间的粘附,还可以通过对small GTP酶(例如RhoA、Cdc42、Rac1)活性的调节来影响细胞间的粘附。但是,p120ctn对细胞粘附究竟是促进作用还是抑制作用,目前尚存较大争议,导致这些争议的原因之一就是由于p120ctn亚型的存在。理论上,p120ctn由于剪切方式的不同可以产生多种功能各异的亚型,并且这种亚型的剪切方式是具有组织和细胞学特异性的,因而在不同类型的组织和细胞中p120ctn的不同亚型可能会对细胞粘附起到不同的调节作用,但目前尚缺乏实验加以证实。那么在肺癌组织中是否存在p120ctn及其亚型的表达?它们对细胞粘附又会起到怎样的调节作用呢?目前仍未见相关报道,为此本研究检测了p120ctn及其亚型在肺癌中的表达情况及其与临床病理因素的关系,并初步探讨其对肺癌细胞粘附功能的调节作用及其可能的调控机制。
方法
1、材料
原发性肺鳞癌、腺癌标本来自中国医科大学第一附属医院行外科手术切除的标本。所有患者术前均未接受放化疗,术后常规化疗。标本均经4%中性甲醛固定,石蜡包埋,HE常规染色后明确诊断。其中部分标本取出后立即放入-70℃冰箱保存备用。
2、细胞培养
人类正常支气管上皮细胞系HBE,人类肺癌细胞系SPC,LTE,460,A549,BE1和LH7分别培养于含10%灭活胎牛血清的RPMI 1640培养液中,贴壁生长。
3、SP免疫组织化学染色及结果判定
单克隆抗体p120ctn、E-cad、RhoA、Cdc42和Rac1 4℃孵育过夜。以正常支气管粘膜上皮细胞着色强度和定位为阳性对照,PBS缓冲液代替一抗为阴性对照。每张切片随机选取5个高倍视野,每高倍视野计数100个瘤细胞,无着色为(-),阳性细胞数＜25%为(+),26～75%为(++),≥76%为(+++)。
4、Western Blot
将含80μg总蛋白的裂解产物进行SDS-PAGE电泳后,转印至PVDF膜,单克隆抗体p120ctn,E-cad,β-cat,RhoA,Cdc42和Rac1 4℃孵育过夜,辣根过氧化物酶标记二抗37℃孵育1h后DAB或ECL显色。
5、RT-PCR
采用Trizol试剂提取肺癌组织中或培养细胞中的总RNA,利用RNA PCR Kit(AMV)Ver.3.0试剂盒进行RT-PCR。RT-PCR产物于1.5%琼脂糖凝胶电泳。
6、质粒构建与转染
将分别含有目的基因的载体导入感受态宿主细菌DH5α中扩增、纯化,测序验证后,使用Lipofect 2000转染试剂将质粒分别转染至肺癌细胞系中,并以G418筛选出阳性细胞克隆,通过Western Blot和RT-PCR鉴定转染效果后,挑取转染成功的单克隆细胞在含有G418的RPMI 1640培养液中继续培养。
7、RhoA、Cdc42和Rac1的蛋白活性分析
分别采用RhoA activity G-lisa和EZ-detect cdc42/Rac1 activation kit试剂盒进行分析,与GTP结合的RhoA以分光光度计检测其吸光度值,与GTP结合的Cdc42或Rac1用Western Blot方法检测。
8、Transwell法检测细胞侵袭能力
按照BD公司说明操作,取100μl细胞悬液(5×10~6个/ml)滴入上室内,下室加入含有10%胎牛血清的RPMI 1640培养液,上下室之间用孔径为8μm的聚碳酸酯微孔滤膜分开,将小室置37℃,5%CO_2条件下放置后,弃去上室内液体,擦尽膜上Matrigel胶,100%甲醇固定30分钟,常规苏木素染色,光镜下计数细胞数。
9、流式细胞仪检测细胞周期
收集对数生长期细胞制成1×10~7/ml细胞悬液。取1ml细胞悬液75%冷乙醇于4℃固定、离心后制成500μl的细胞悬液,加碘化丙啶染液于4℃孵育45min后上机检测,ModFit LT 3.0软件分析细胞周期。
10、动物实验
BALB/c裸小鼠由本校实验动物部提供。取0.3ml细胞(8×10~6个/ml)于裸鼠背部皮下注射。自注射当日起连续观察6周后统一处死,记录成瘤及转移情况,取所有脏器经10%中性福尔马林固定,石蜡包埋后进行连续切片,HE染色。
11、统计分析
各组资料利用SPSS for Window 11.5进行统计分析。p＜0.05有统计学意义。
结果
(1)在正常支气管粘膜细胞中,p120ctn、E-cad呈现连续的胞膜阳性表达,而在肺癌组织中二者的蛋白和mRNA表达水平均明显低于正常肺组织,呈现胞膜表达减弱、胞浆异位等异常表达现象。RhoA、Cdc42和Rac1在肺癌组织中其蛋白及mRNA均呈过表达,明显强于正常肺组织。并且p120ctn的异常表达与E-cad的异常表达和RhoA、Cdc42和Rac1的过表达有较好的相关性。肺癌组织中p120ctn、E-cad的异常表达和三种small GTP酶的过表达与肺癌的低分化,高分期,淋巴结转移和不良预后明显相关。
(2)在p120ctn高表达的肺癌细胞系中p120ctn的基因“沉默”在明显减弱E-cad和β-cat蛋白表达的同时,还可以抑制β-cat的mRNA表达。p120ctn的基因缺失能激活Cdc42和Rac1,失活RhoA,并且促进肺癌细胞的增殖、侵袭和转移能力。将稳定转染了p120siRNA和转染空质粒的细胞克隆分别移植至裸鼠皮下,发现整合了p120siRNA的荷瘤鼠的肿瘤生长迅速、侵袭和转移能力增强。
(3)正常肺组织中p120ctn蛋白以亚型1(120KD)和亚型3(100KD)的表达为主,而在肺癌组织和PG细胞系中则出现1、3亚型(特别是1亚型)表达缺失或减弱。正常肺组织中p120ctn mRNA可见1.2、1.3、2.3、3.1及3.3五条亚型带,而在肺癌组织和PG细胞系中各亚型出现不同缺失或减弱。并且这种亚型减弱或缺失的现象与肺癌淋巴结转移有不同程度的相关性:p120ctn亚型1与肺癌的淋巴结转移呈负相关,而亚型3与肺癌淋巴结转移呈正相关。
(4)在p120ctn低表达的肺癌细胞系中,p120ctn亚型1A的高表达通过促进E-cad、β-cat的表达,和下调Rac1的活性,进而明显抑制肺癌细胞的侵袭能力,而p120ctn亚型3A的高表达虽然可以失活Cdc42,激活RhoA,但是对于肺癌细胞侵袭能力的抑制作用却明显弱于亚型1A。同时,流式细胞测试结果表明,p120ctn亚型3A抑制细胞周期的强度明显强于亚型1A,裸鼠皮下接种结果也表明二者均有抑制肿瘤细胞生长作用,但亚型3A的移植瘤重量明显小于p120ctn亚型1A的移植瘤重量。
结论
(1)肺癌组织中p120ctn的异常表达与E-cad的异常表达和small GTP酶的过表达具有较好一致性,并且它们的协同表达与肺癌的恶性程度显著相关。
(2)p120ctn的缺失可促进肺癌细胞的侵袭和转移能力,其机制可能通过下调E-cad、β-cat的表达,和激活Cdc42/Rac1、失活RhoA等调节small GTP酶活性来实现的。
(3)在正常肺组织中p120ctn的表达以亚型1和亚型3为主,而在肺癌组织中这两种亚型表达减弱或缺失,并且它们的异常表达在肺癌进展过程中可能起到了不同的作用。
(4)p120ctn亚型1和亚型3均可抑制肺癌细胞的增殖和侵袭,但二者的抑制程度不同,其调控的机制也不尽相同。
(5)在上述实验中,我们还意外地发现p120ctn的表达可在mRNA水平上调节β-cat的表达,但具体机制还有待与深入研究。
Introduction
p120-catenin(p120ctn)belongs to the Armadillo family of proteins.It influences cell-cell adhesion by directly interacting with E-cadherin and also can regulate cell motility through the actin cytoskeleton via Rho family GTPases.However,the role of p120ctn is currently controversial since evidence suggests that p120ctn can both positively and negatively regulate adhesive activity of cancer cells.One reason for this contradiction may lie in the large number of p120ctn isoforms expressed in different cells and tissues.Owing to alternative splicing and multiple translation initiation codons,several p120ctn isoforms can be expressed,which are tissue and cell specific. These p120ctn isoforms contribute differently to cancer cell adhesion and migration. The present study examined the expression pattern of p120ctn isoforms in lung cancer and their correlation to clinical parameters,the mechanism of p120ctn regulating cancer cell adhesion was also explored.
Materials and Methods
1.Patients and specimens
The primary tumors specimens were from patients with lung SCC and adenocarcinoma who underwent complete resection in the First Affiliated Hospital of China Medical University None of the patients had received radiotherapy or chemotherapy before surgical resection,and all were treated with routine chemotherapy after the operation.Among these samples,some fresh specimens and corresponding normal tissue samples were stored at -70℃immediately after resection until the extraction of protein and RNA.
2.Cell culture
BE1,LH7,SPC,LYE,A549,H460 cells were cultured in RPMI 1640 medium, containing 10%fetal calf serum,100IU/ml penicillin,and 100μg/ml streptomycin. Cells were grown on sterilized culture dishes glass and were passaged every 2 days with 0.25%trypsin.
3.Immunohistochemical assessment
Immunostaining was performed by the avidin-biotin-peroxidase complex method. They were then incubated with p120ctn,E-cadherin,RhoA,Cdc42,Rac1 antibody overnight.Five views were examined per slide,and 100 cells were observed per view, at 400x magnification.Labeling scores were determined by the percentage of positive cells per slide.We define no positive cells as(-),positive cells≤25%as(+),26～75% as(++),≥76%as(+++).
4.Western Blot
50μg proteins were separated by SDS-PAGE.After transferring to polyvinylidene fluoride membrane,the membrane was incubated overnight at 4℃with either the mouse monoclonal antibody against p120ctn,E-cadherin,β-catenin,RhoA,Cdc42 and Rac1.After incubation with peroxidase-coupled anti-mouse IgG at 37℃for 2 hours, the proteins were visualized using DAB/ECL
5.RT-PCR
RT-PCR was performed with the RNA PCR Kit(AMV)Version 3.0,according to the manufacturer's instructions.
6.Plasmid construction and transfection
The cells were stably transfected with the p120ctn-siRNA plasmids or p120ctn plasmids using Lipofectamine 2000,following the manufacturer's instructions.The empty plasmid was used as a negative control.Selection was accomplished with G418. Transfected cells were cultured in RPMI 1640 medium containing 10%fetal calf serum and G418.
7.RhoA,Cdc42,and Rac1 activity assay
RhoA activity was measured using colorimetric-based RhoA G-LISA~(TM)activation assay according to the manufacturer's instructions.The resulting samples were analyzed by spectrophotometer.Cdc42 and Rac1 activity was measured using a pull-down assay.Cdc42-GTP/Rac1-GTP bound proteins were separated by 12% SDS-PAGE and transferred to PVDF membrane.The membrane was incubated in the anti-Cdc42/Rac1 antibody solutionat 4℃overnight,and detected with the BioImaging System.
8.Matrigel invasion assay
Following the manufacturer's instructions,in the upper chambers,5x10~5 BE1 cells were grown in serum-free medium on 8μm porous polycarbonate membranes, which were coated with Matrigel basement membrane matrix.The lower chambers were filled with RPMI 1640 medium containing 10%fetal calf serum.After incubation for 6,16,or 24 hours at 37℃in a humid atmosphere of 5%CO2 and 95%air,the cells that had migrated through the pores were fixed with methanol for 30 minutes and stained with hematoxylin.Then the number of cells counted visually using Nikon E200 microscope in five different fields under 200×magnifications per filter.
9.Flow cytometry(FCM)
After 48 hours of culture,cells from each experimental group were collected and digested with trypsin and fixed with 75%ice-cold ethanol at 4℃overnight.Cells (1×10~6)were centrifuged at 1500 rpm for 5 minutes,and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis.The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.
10.Xenograft to nude mice
Four-week-old male BALB/c nude mice were obtained from the animal facility approved by the China Medical University.Each mouse was inoculated subcutaneously in the back with 2.4x10~6 tumor cells.Mice were sacrificed after 6 weeks.
11.Statistical analysis
All statistical calculations were performed by SPSS for Windows software,p values less than 0.05 were considered statistically significant.
Results
(1)p120ctn and E-cad was expressed mainly in the cell membrane of the normal bronchial epithelium.Of 138 lung cancers,p120ctn or E-cad abnormal expression, including reduced or absent membranous expression and cytoplasmic expression. RhoA,Cdc42,and Rac1 expression is significantly higher in lung cancers in comparison with corresponding normal lung tissues.There was a correlation between abnormal p120ctn expression and overexpression of RhoA,Rac1 and Cdc42,the combination of which associate with poor differentiation,high TNM stage,and lymph node metastasis.
(2)We knocked down p120ctn using siRNA in BE1,SPC,LTE cells.Ablation of p120ctn reduced the levels of E-cadherin andβ-catenin proteins,as well as the mRNA ofβ-catenin.Furthermore,p120ctn ablation inactivated RhoA,but increased the activity of Cdc42 and Rac1,and promoted proliferation and the invasive ability of lung cancer cells both in vitro and in vivo.
(3)We observed reduced expression or even the absence of p120ctn isoform 1 and 3 in tumor cell compared to normal tissues.The loss of p120ctn isofoms,especially isoform 1 may be responsible for cancer cell development.
(4)Different p120ctn isoforms serve in distinct biological functions in lung cancer.Expression of p120ctn isoform 1A upregulated E-cadherin andβ-catenin and downregulated Rac1 activity thus inhibited metastatic ability of lung cancer cell. p120ctn isoform 3A expression resulted in the inactivation of Cdc42 and activation of RhoA,and did not influence metastasis significantly.Flow cytometry showed that p120ctn isoform 3A inhibit cell cycle.In vivo test showed that in nude mice,the mean tumor weight in the p120ctn-3A group was significantly lower than that in p120ctn-1A groups.
Conclusions
(1)We demonstrate a positive association between increased RhoA,Rac1,Cdc42 expression and abnormal p120ctn expression.In addition,the combination of abnormal p120ctn and Rho family over-expression is associated with high TNM stage and poor differentiation.
(2)In lung cancer,p120ctn loss reduced the levels of E-cadherin andβ-catenin, inactivated RhoA,but increased the activity of Cdc42 and Rac1,and promoted proliferation and the invasive ability cancer cells.
(3)In normal bronchial epithelial cells,p120ctn isoform 1A and 3A were expressed,which is reduced in lung cancer cells.These p120ctn isoforms contribute differently to cancer cell adhesion and migration.
(4)Expression of p120ctn isoform 1A upregulated E-cadherin andβ-catenin and downregulated Rac1 activity thus inhibited metastatic ability of lung cancer cell. p120ctn isoform 3A expression resulted in the inactivation of Cdc42 and activation of RhoA,and did not influence metastasis significantly,p120ctn also induceβ-catenin in lung cancer cell lines,the underlying mechanism is under investigation.
p120-catenin(p120ctn)作为catenin家族的重要成员,不仅可以通过与E-cadherin(E-cad)直接结合形成E-cadherin/catenin复合体,调节细胞间的粘附,还可以通过对small GTP酶(例如RhoA、Cdc42、Rac1)活性的调节来影响细胞间的粘附。但是,p120ctn对细胞粘附究竟是促进作用还是抑制作用,目前尚存较大争议,导致这些争议的原因之一就是由于p120ctn亚型的存在。理论上,p120ctn由于剪切方式的不同可以产生多种功能各异的亚型,并且这种亚型的剪切方式是具有组织和细胞学特异性的,因而在不同类型的组织和细胞中p120ctn的不同亚型可能会对细胞粘附起到不同的调节作用,但目前尚缺乏实验加以证实。那么在肺癌组织中是否存在p120ctn及其亚型的表达?它们对细胞粘附又会起到怎样的调节作用呢?目前仍未见相关报道,为此本研究检测了p120ctn及其亚型在肺癌中的表达情况及其与临床病理因素的关系,并初步探讨其对肺癌细胞粘附功能的调节作用及其可能的调控机制。
方法
1、材料
原发性肺鳞癌、腺癌标本来自中国医科大学第一附属医院行外科手术切除的标本。所有患者术前均未接受放化疗,术后常规化疗。标本均经4%中性甲醛固定,石蜡包埋,HE常规染色后明确诊断。其中部分标本取出后立即放入-70℃冰箱保存备用。
2、细胞培养
人类正常支气管上皮细胞系HBE,人类肺癌细胞系SPC,LTE,460,A549,BE1和LH7分别培养于含10%灭活胎牛血清的RPMI 1640培养液中,贴壁生长。
3、SP免疫组织化学染色及结果判定
单克隆抗体p120ctn、E-cad、RhoA、Cdc42和Rac1 4℃孵育过夜。以正常支气管粘膜上皮细胞着色强度和定位为阳性对照,PBS缓冲液代替一抗为阴性对照。每张切片随机选取5个高倍视野,每高倍视野计数100个瘤细胞,无着色为(-),阳性细胞数＜25%为(+),26～75%为(++),≥76%为(+++)。
4、Western Blot
将含80μg总蛋白的裂解产物进行SDS-PAGE电泳后,转印至PVDF膜,单克隆抗体p120ctn,E-cad,β-cat,RhoA,Cdc42和Rac1 4℃孵育过夜,辣根过氧化物酶标记二抗37℃孵育1h后DAB或ECL显色。
5、RT-PCR
采用Trizol试剂提取肺癌组织中或培养细胞中的总RNA,利用RNA PCR Kit(AMV)Ver.3.0试剂盒进行RT-PCR。RT-PCR产物于1.5%琼脂糖凝胶电泳。
6、质粒构建与转染
将分别含有目的基因的载体导入感受态宿主细菌DH5α中扩增、纯化,测序验证后,使用Lipofect 2000转染试剂将质粒分别转染至肺癌细胞系中,并以G418筛选出阳性细胞克隆,通过Western Blot和RT-PCR鉴定转染效果后,挑取转染成功的单克隆细胞在含有G418的RPMI 1640培养液中继续培养。
7、RhoA、Cdc42和Rac1的蛋白活性分析
分别采用RhoA activity G-lisa和EZ-detect cdc42/Rac1 activation kit试剂盒进行分析,与GTP结合的RhoA以分光光度计检测其吸光度值,与GTP结合的Cdc42或Rac1用Western Blot方法检测。
8、Transwell法检测细胞侵袭能力
按照BD公司说明操作,取100μl细胞悬液(5×10~6个/ml)滴入上室内,下室加入含有10%胎牛血清的RPMI 1640培养液,上下室之间用孔径为8μm的聚碳酸酯微孔滤膜分开,将小室置37℃,5%CO_2条件下放置后,弃去上室内液体,擦尽膜上Matrigel胶,100%甲醇固定30分钟,常规苏木素染色,光镜下计数细胞数。
9、流式细胞仪检测细胞周期
收集对数生长期细胞制成1×10~7/ml细胞悬液。取1ml细胞悬液75%冷乙醇于4℃固定、离心后制成500μl的细胞悬液,加碘化丙啶染液于4℃孵育45min后上机检测,ModFit LT 3.0软件分析细胞周期。
10、动物实验
BALB/c裸小鼠由本校实验动物部提供。取0.3ml细胞(8×10~6个/ml)于裸鼠背部皮下注射。自注射当日起连续观察6周后统一处死,记录成瘤及转移情况,取所有脏器经10%中性福尔马林固定,石蜡包埋后进行连续切片,HE染色。
11、统计分析
各组资料利用SPSS for Window 11.5进行统计分析。p＜0.05有统计学意义。
结果
(1)在正常支气管粘膜细胞中,p120ctn、E-cad呈现连续的胞膜阳性表达,而在肺癌组织中二者的蛋白和mRNA表达水平均明显低于正常肺组织,呈现胞膜表达减弱、胞浆异位等异常表达现象。RhoA、Cdc42和Rac1在肺癌组织中其蛋白及mRNA均呈过表达,明显强于正常肺组织。并且p120ctn的异常表达与E-cad的异常表达和RhoA、Cdc42和Rac1的过表达有较好的相关性。肺癌组织中p120ctn、E-cad的异常表达和三种small GTP酶的过表达与肺癌的低分化,高分期,淋巴结转移和不良预后明显相关。
(2)在p120ctn高表达的肺癌细胞系中p120ctn的基因“沉默”在明显减弱E-cad和β-cat蛋白表达的同时,还可以抑制β-cat的mRNA表达。p120ctn的基因缺失能激活Cdc42和Rac1,失活RhoA,并且促进肺癌细胞的增殖、侵袭和转移能力。将稳定转染了p120siRNA和转染空质粒的细胞克隆分别移植至裸鼠皮下,发现整合了p120siRNA的荷瘤鼠的肿瘤生长迅速、侵袭和转移能力增强。
(3)正常肺组织中p120ctn蛋白以亚型1(120KD)和亚型3(100KD)的表达为主,而在肺癌组织和PG细胞系中则出现1、3亚型(特别是1亚型)表达缺失或减弱。正常肺组织中p120ctn mRNA可见1.2、1.3、2.3、3.1及3.3五条亚型带,而在肺癌组织和PG细胞系中各亚型出现不同缺失或减弱。并且这种亚型减弱或缺失的现象与肺癌淋巴结转移有不同程度的相关性:p120ctn亚型1与肺癌的淋巴结转移呈负相关,而亚型3与肺癌淋巴结转移呈正相关。
(4)在p120ctn低表达的肺癌细胞系中,p120ctn亚型1A的高表达通过促进E-cad、β-cat的表达,和下调Rac1的活性,进而明显抑制肺癌细胞的侵袭能力,而p120ctn亚型3A的高表达虽然可以失活Cdc42,激活RhoA,但是对于肺癌细胞侵袭能力的抑制作用却明显弱于亚型1A。同时,流式细胞测试结果表明,p120ctn亚型3A抑制细胞周期的强度明显强于亚型1A,裸鼠皮下接种结果也表明二者均有抑制肿瘤细胞生长作用,但亚型3A的移植瘤重量明显小于p120ctn亚型1A的移植瘤重量。
结论
(1)肺癌组织中p120ctn的异常表达与E-cad的异常表达和small GTP酶的过表达具有较好一致性,并且它们的协同表达与肺癌的恶性程度显著相关。
(2)p120ctn的缺失可促进肺癌细胞的侵袭和转移能力,其机制可能通过下调E-cad、β-cat的表达,和激活Cdc42/Rac1、失活RhoA等调节small GTP酶活性来实现的。
(3)在正常肺组织中p120ctn的表达以亚型1和亚型3为主,而在肺癌组织中这两种亚型表达减弱或缺失,并且它们的异常表达在肺癌进展过程中可能起到了不同的作用。
(4)p120ctn亚型1和亚型3均可抑制肺癌细胞的增殖和侵袭,但二者的抑制程度不同,其调控的机制也不尽相同。
(5)在上述实验中,我们还意外地发现p120ctn的表达可在mRNA水平上调节β-cat的表达,但具体机制还有待与深入研究。
Introduction
p120-catenin(p120ctn)belongs to the Armadillo family of proteins.It influences cell-cell adhesion by directly interacting with E-cadherin and also can regulate cell motility through the actin cytoskeleton via Rho family GTPases.However,the role of p120ctn is currently controversial since evidence suggests that p120ctn can both positively and negatively regulate adhesive activity of cancer cells.One reason for this contradiction may lie in the large number of p120ctn isoforms expressed in different cells and tissues.Owing to alternative splicing and multiple translation initiation codons,several p120ctn isoforms can be expressed,which are tissue and cell specific. These p120ctn isoforms contribute differently to cancer cell adhesion and migration. The present study examined the expression pattern of p120ctn isoforms in lung cancer and their correlation to clinical parameters,the mechanism of p120ctn regulating cancer cell adhesion was also explored.
Materials and Methods
1.Patients and specimens
The primary tumors specimens were from patients with lung SCC and adenocarcinoma who underwent complete resection in the First Affiliated Hospital of China Medical University None of the patients had received radiotherapy or chemotherapy before surgical resection,and all were treated with routine chemotherapy after the operation.Among these samples,some fresh specimens and corresponding normal tissue samples were stored at -70℃immediately after resection until the extraction of protein and RNA.
2.Cell culture
BE1,LH7,SPC,LYE,A549,H460 cells were cultured in RPMI 1640 medium, containing 10%fetal calf serum,100IU/ml penicillin,and 100μg/ml streptomycin. Cells were grown on sterilized culture dishes glass and were passaged every 2 days with 0.25%trypsin.
3.Immunohistochemical assessment
Immunostaining was performed by the avidin-biotin-peroxidase complex method. They were then incubated with p120ctn,E-cadherin,RhoA,Cdc42,Rac1 antibody overnight.Five views were examined per slide,and 100 cells were observed per view, at 400x magnification.Labeling scores were determined by the percentage of positive cells per slide.We define no positive cells as(-),positive cells≤25%as(+),26～75% as(++),≥76%as(+++).
4.Western Blot
50μg proteins were separated by SDS-PAGE.After transferring to polyvinylidene fluoride membrane,the membrane was incubated overnight at 4℃with either the mouse monoclonal antibody against p120ctn,E-cadherin,β-catenin,RhoA,Cdc42 and Rac1.After incubation with peroxidase-coupled anti-mouse IgG at 37℃for 2 hours, the proteins were visualized using DAB/ECL
5.RT-PCR
RT-PCR was performed with the RNA PCR Kit(AMV)Version 3.0,according to the manufacturer's instructions.
6.Plasmid construction and transfection
The cells were stably transfected with the p120ctn-siRNA plasmids or p120ctn plasmids using Lipofectamine 2000,following the manufacturer's instructions.The empty plasmid was used as a negative control.Selection was accomplished with G418. Transfected cells were cultured in RPMI 1640 medium containing 10%fetal calf serum and G418.
7.RhoA,Cdc42,and Rac1 activity assay
RhoA activity was measured using colorimetric-based RhoA G-LISA~(TM)activation assay according to the manufacturer's instructions.The resulting samples were analyzed by spectrophotometer.Cdc42 and Rac1 activity was measured using a pull-down assay.Cdc42-GTP/Rac1-GTP bound proteins were separated by 12% SDS-PAGE and transferred to PVDF membrane.The membrane was incubated in the anti-Cdc42/Rac1 antibody solutionat 4℃overnight,and detected with the BioImaging System.
8.Matrigel invasion assay
Following the manufacturer's instructions,in the upper chambers,5x10~5 BE1 cells were grown in serum-free medium on 8μm porous polycarbonate membranes, which were coated with Matrigel basement membrane matrix.The lower chambers were filled with RPMI 1640 medium containing 10%fetal calf serum.After incubation for 6,16,or 24 hours at 37℃in a humid atmosphere of 5%CO2 and 95%air,the cells that had migrated through the pores were fixed with methanol for 30 minutes and stained with hematoxylin.Then the number of cells counted visually using Nikon E200 microscope in five different fields under 200×magnifications per filter.
9.Flow cytometry(FCM)
After 48 hours of culture,cells from each experimental group were collected and digested with trypsin and fixed with 75%ice-cold ethanol at 4℃overnight.Cells (1×10~6)were centrifuged at 1500 rpm for 5 minutes,and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis.The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.
10.Xenograft to nude mice
Four-week-old male BALB/c nude mice were obtained from the animal facility approved by the China Medical University.Each mouse was inoculated subcutaneously in the back with 2.4x10~6 tumor cells.Mice were sacrificed after 6 weeks.
11.Statistical analysis
All statistical calculations were performed by SPSS for Windows software,p values less than 0.05 were considered statistically significant.
Results
(1)p120ctn and E-cad was expressed mainly in the cell membrane of the normal bronchial epithelium.Of 138 lung cancers,p120ctn or E-cad abnormal expression, including reduced or absent membranous expression and cytoplasmic expression. RhoA,Cdc42,and Rac1 expression is significantly higher in lung cancers in comparison with corresponding normal lung tissues.There was a correlation between abnormal p120ctn expression and overexpression of RhoA,Rac1 and Cdc42,the combination of which associate with poor differentiation,high TNM stage,and lymph node metastasis.
(2)We knocked down p120ctn using siRNA in BE1,SPC,LTE cells.Ablation of p120ctn reduced the levels of E-cadherin andβ-catenin proteins,as well as the mRNA ofβ-catenin.Furthermore,p120ctn ablation inactivated RhoA,but increased the activity of Cdc42 and Rac1,and promoted proliferation and the invasive ability of lung cancer cells both in vitro and in vivo.
(3)We observed reduced expression or even the absence of p120ctn isoform 1 and 3 in tumor cell compared to normal tissues.The loss of p120ctn isofoms,especially isoform 1 may be responsible for cancer cell development.
(4)Different p120ctn isoforms serve in distinct biological functions in lung cancer.Expression of p120ctn isoform 1A upregulated E-cadherin andβ-catenin and downregulated Rac1 activity thus inhibited metastatic ability of lung cancer cell. p120ctn isoform 3A expression resulted in the inactivation of Cdc42 and activation of RhoA,and did not influence metastasis significantly.Flow cytometry showed that p120ctn isoform 3A inhibit cell cycle.In vivo test showed that in nude mice,the mean tumor weight in the p120ctn-3A group was significantly lower than that in p120ctn-1A groups.
Conclusions
(1)We demonstrate a positive association between increased RhoA,Rac1,Cdc42 expression and abnormal p120ctn expression.In addition,the combination of abnormal p120ctn and Rho family over-expression is associated with high TNM stage and poor differentiation.
(2)In lung cancer,p120ctn loss reduced the levels of E-cadherin andβ-catenin, inactivated RhoA,but increased the activity of Cdc42 and Rac1,and promoted proliferation and the invasive ability cancer cells.
(3)In normal bronchial epithelial cells,p120ctn isoform 1A and 3A were expressed,which is reduced in lung cancer cells.These p120ctn isoforms contribute differently to cancer cell adhesion and migration.
(4)Expression of p120ctn isoform 1A upregulated E-cadherin andβ-catenin and downregulated Rac1 activity thus inhibited metastatic ability of lung cancer cell. p120ctn isoform 3A expression resulted in the inactivation of Cdc42 and activation of RhoA,and did not influence metastasis significantly,p120ctn also induceβ-catenin in lung cancer cell lines,the underlying mechanism is under investigation.
引文
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