非小细胞肺癌中Ecadherin、MUC1及PINCH的表达及其临床意义

doi:10.3969/j.issn.1000⁃484X.2019.01.018非小细胞肺癌中E⁃cadherin ㊁MUC1及PINCH 的表达及其临床意义①胡兆鹏　郭丽霞　李慧明　徐　妍　梁　贺　(萍乡市人民医院病理科,萍乡337000) 中图分类号　R365 文献标志码　A 文章编号　1000⁃484X (2019)01⁃0088⁃04①本文为江西省卫生计生委科技计划项目(20167177)㊂作者简介:胡兆鹏,男,副主任医师,主要从事非小细胞肺癌方面的研究,Email:1197368995@㊂[摘　要]　目的:分析与细胞黏附相关的E⁃cadherin㊁MUC1及PINCH 在非小细胞肺癌(NSCLC)中表达水平,并探讨三者之间的相关性,探寻NSCLC 病变进展更具代表性的指标㊂方法:通过免疫组化法检测70例非小细胞肺癌患者E⁃cadherin㊁MUC1及PINCH 的表达情况并结合临床分期及有无淋巴结转移进行统计学分析,并探讨三者之间的相关性㊂结果:NSCLC 肿瘤组织中E⁃cadherin㊁MUC1及PINCH 蛋白的阳性表达率分别是25.7%㊁57.1%和62.9%,与对照组相比差异具有统计学意义(P <0.01);E⁃cadherin 阳性表达率随着临床分期的升高而下降(P <0.01),而MUC1和PINCH 阳性表达率随着分期的升高而升高(P <0.01);有淋巴结转移的肺癌组织中E⁃cadherin 阳性表达率较无淋巴结转移者显著降低(P <0.05),而有淋巴结转移的肺癌组织中MUC1和PINCH 阳性表达率较无淋巴结转移者显著升高(P <0.05);NSCLC 肿瘤组织中MUC1与PINCH 的表达水平呈正相关(r =0.589,P <0.01),MUC1㊁PINCH 的表达水平与E⁃cadherin 呈负相关(相关系数r 值分别为-0.562,-0.415,P 值均<0.01)㊂结论:E⁃cadherin,MUC1及PINCH 三者可能互相联系共同影响细胞间的黏附性进而促进NSCLC 的进展;同时检测三者在NSCLC 癌组织中的表达,对于探寻NSCLC 病变进展更具代表性的指标可能有重要意义㊂[关键词]　E⁃cadherin;MUC1;PINCH;非小细胞肺癌Expression of E⁃cadherin ,MUC1and PINCH in NSCLC and their clinical signif⁃icanceHU Zhao⁃Peng ,GUO Li⁃Xia ,LI Hui⁃Ming ,XU Yan ,LIANG He.Department of Pathology ,Pingxiang people′s Hospital ,Pingxiang 337000,China[Abstract ]　Objective :To examine the expression of E⁃cadherin,Mucins1(MUC1)and PINCH in non⁃small cell lung cancer(NSCLC),and to analyze their correlation.Methods :The expression of E⁃cadherin Mucins1(MUC1)and PINCH were detected by in 70primary NSCLC samples by immunohistochemistry method,and their correlation then analyzed.Results :The expression of E⁃cadherin (25.7%)was remarkably decreased in NSCLC tissues,while the expression of MUC1and PINCH (57.1%and 62.9%re⁃spectively)was remarkably increased (P <0.01).E⁃cadherin,MUC1and PINCH expression in NSCLC were related to clinical stageand lymphatic metastasis (P <0.05).The expression of MUC1and PINCH were positively correlated with the expression of MUC1and PINCH (r =0.589,P <0.01),and E⁃cadherin was negative correlation with them (r was -0.562and -0.415respectively,P <0.01).Conclusion :E⁃cadherin,MUC1and PINCH may interrelated mutually on cell⁃adhesion,and further promote the progress ofNSCLC.Detecting this three protein in NSCLC cancer,may have important significance for the invasion and metastasis in NSCLC.[Key words ]　E⁃cadherin;MUC1;PINCH;Non⁃small⁃cell lung cancer 非小细胞肺癌(Non⁃small⁃cell lung cancer,NSCLC)是常见肺部肿瘤病变类型,远期预后较差,5年存活率较低[1,2]㊂目前通常以临床分期作为评估NSCLC 患者病变程度及预后的方法,而肿瘤病情迁延进展是一个漫长而复杂的过程,临床分期也处于动态变化中,因此提升NSCLC 病变进展评测的准确性,需要更具有代表性的评测指标㊂肿瘤的侵袭转移与细胞间的黏附性密切相关,本研究目的在于分析与细胞黏附相关的E⁃cadherin㊁MUC1及PINCH 在NSCLC 中表达水平,并探讨三者之间的相关性,探寻NSCLC 病变进展更具代表性的指标㊂1　资料与方法1.1　临床资料　抽选2001年1月至2013年1月本院收治的70例NSCLC 患者石蜡包埋标本,其中男性患者38例,女性患者32例,年龄42~65岁,平均(51.4±3.5)岁,均进行手术治疗,患者手术治疗前未接受其他治疗干预,并且取得患者监护人或患者本人的书面知情同意㊂70例NSCLC 患者手术标本均为石蜡包埋,肺癌组织标本为观察组,癌旁正常㊃88㊃中国免疫学杂志2019年第35卷组织标本记为对照组㊂癌变组织高㊁中㊁低分化者分别为14例㊁30例㊁26例;分期为Ⅰ期至Ⅳ期,分别16例㊁20例㊁18例㊁16例㊂根据术后病理报告详细记录组织学类型㊁是否伴有淋巴转移等情况㊂1.1.2　主要试剂　E⁃cadherin鼠抗人单克隆抗体(美国Santa Cruz公司),MUC1IgG鼠抗人单克隆抗体(美国Santa Cruz公司),PINCH蛋白兔抗人多克隆抗体(博士德公司),免疫组化SP法试剂盒(福建迈新生物技术公司)㊂1.2　方法1.2.1　实验方法　病例标本均来源于手术切除标本,均用10%中性缓冲福尔马林固定液固定24h,常规取材㊁脱水㊁石蜡包埋㊁4μm连续切片,置于56℃烤箱中烘烤12h,用于免疫组织化学染色㊂免疫组化染色采用SP法,并结合抗体说明书进行操作㊂用已知的阳性组织作阳性对照,用PBS液代替一抗作空白对照,其他步骤相同㊂1.2.2　结果判断　以细胞膜或细胞浆出现棕黄色颗粒分布为阳性细胞,随机观察10个不同高倍视野(10×40)的阳性细胞数及总细胞数,计算阳性细胞率(阳性细胞数/总细胞数),阳性细胞率小于10%为阴性,大于等于10%为阳性㊂1.3　统计学处理　用SPSS17.0统计软件分析数据,采用四格表χ2检验㊁Spearman相关检验,以0.05为显著性检验水准,即P<0.05有统计学意义㊂2　结果2.1.1　E⁃cadherin㊁MUC1及PINCH蛋白在非小细胞肺癌组织(观察组)及癌旁正常组织(对照组)中的表达情况　E⁃cadherin㊁MUC1及PINCH蛋白在观察组及对照组中的表达情况详见表1,三者在观察组与对照组组织的阳性表达率均有显著统计学意义(P<0.05),提示非小细胞肺癌组织中MUC1㊁PINCH 蛋白的表达增加,而E⁃cadherin蛋白的表达下调㊂2.1.2　E⁃cadherin㊁MUC1及PINCH蛋白表达与表1　E⁃cadherin㊁MUC1及PINCH蛋白表达情况Tab.1　Expression of E⁃cadherin,MUC1and PINCH in NSCLCGroups n E⁃cadherin positiveexpression MUC1positive expressionPINCH positiveexpressionControl7053(75.7%)4(5.7%)6(8.6%) Observation7018(25.7%)44(62.9%)40(57.1%)χ2value-35.00750.72537.428 P value-0.0000.0000.000NSCLC临床病理特征间的关系　E⁃cadherin㊁MUC1及PINCH蛋白在非小细胞肺癌中的表达情况详见表2㊂不同临床分期中,E⁃cadherin阳性表达率随着分期的升高而下降(P<0.05),而MUC1和PINCH阳性表达率随着分期的升高而升高(P< 0.05);有淋巴结转移的肺癌组织中E⁃cadherin阳性表达率较无淋巴结转移的肺癌组织显著降低(P<0.01),而有淋巴结转移的肺癌组织中MUC1和PINCH阳性表达率较无淋巴结转移的肺癌组织显著升高(P<0.01)㊂2.2　非小细胞肺癌中E⁃cadherin㊁MUC1及PINCH 蛋白表达的相关性2.2.1　MUC1和PINCH蛋白表达的相关性分析　MUC1和PINCH蛋白在非小细胞肺癌组织中共同阳性表达例数为35例㊂MUC1阳性组中有9例PINCH呈阴性表达(9/44),MUC1阴性组中有5例PINCH呈阳性表达(5/26),以P<0.05为检验水准,结果表明两者之间呈正相关(r=0.589,P=0.000)㊂见表3㊂2.2.2　E⁃cadherin和MUC1蛋白表达的相关性分析　E⁃cadherin和MUC1蛋白在非小细胞肺癌组织中共同阳性表达例数为3例㊂MUC1阳性组中有41例E⁃cadherin呈阴性表达(41/44),MUC1阴性组有15例E⁃cadherin呈阳性表达(15/26),两者之间呈负相关(r=-0.562,P=0.000)㊂见表4㊂表2　E⁃cadherin㊁MUC1及PINCH蛋白表达与NSCLC临床病理特征间关系Tab.2　Expression of E⁃cadherin,MUC1and PINCH in NSCLC of different clinical stage andlymphatic metastasisVariable Item n E⁃cadherinpositive casesMUC1positive casesPINCHpositive cases StagesⅠ-Ⅱ3613(36.1%)18(50.0%)14(38.9%)Ⅲ-Ⅳ345(14.7%)26(76.5%)26(76.5%) Lymphatic Metastasis463(6.5%)40(86.9%)34(73.9%) Non⁃metastasis2415(62.5%)4(16.7%)6(25%)表3　非小细胞肺癌中MUC1和PINCH蛋白表达的关系Tab.3　Correlation of MUC1and PINCH expression in NSCLCMUC1Positive cases Negative cases Total PINCH Positive cases35540 Negative cases92130 Total442670㊃98㊃胡兆鹏等　非小细胞肺癌中E⁃cadherin㊁MUC1及PINCH的表达及其临床意义　第1期表4　非小细胞肺癌中E⁃cadherin和MUC1蛋白表达的关系Tab.4　Correlation of E⁃cadherin and MUC1expression in NSCLCE⁃cadherinPositive cases Negative cases Total MUC1Positive cases34144 Negative cases151126 Total185270表5　非小细胞肺癌中E⁃cadherin和PINCH蛋白表达的关系Tab.5　Correlation of E⁃cadherin and PINCH expression in NSCLCE⁃cadherinPositive cases Negative cases Total PINCH Positive cases43640 Negative cases141630 Total1852702.2.3　E⁃cadherin和PINCH蛋白表达的相关性分析　E⁃cadherin和PINCH蛋白在非小细胞肺癌组织中共同阳性表达例数为4例㊂PINCH阳性组中有36例E⁃cadherin呈阴性表达(36/40),PINCH阴性组有14例呈E⁃cadherin阳性表达(14/30),两者之间呈负相关(r=-0.415,P=0.000)㊂见表5㊂3　讨论肿瘤的侵袭转移与细胞间的黏附性密切相关,细胞间黏附性的下降与癌细胞的去分化㊁侵袭性及转移倾向有关㊂E⁃cadherin是一种钙依赖的细胞黏附分子,它介导细胞间结合,抑制癌细胞转移㊂研究发现在乳腺癌[3]㊁膀胱癌[4]和胰腺癌[5]等多种肿瘤中E⁃cadherin不同程度表达下降或不表达,其表达水平与肿瘤的侵袭转移和复发率密切相关㊂有研究发现[6⁃7],E⁃cadherin缺失或低表达可以成为预测非小细胞肺癌疾病进展㊁淋巴结转移及预后评估的有效指标㊂在本研究中,NSCLC肿瘤组织中仅有18例E⁃cadherin阳性表达(25.7%),与正常组织相比,阳性表达强度明显减弱;随着临床分期升高,其阳性表达率显著降低;有淋巴结转移的肿瘤组织中其阳性表达率较无淋巴结转移的肿瘤组织显著降低,与Jiang等[8]研究结果基本一致,提示E⁃cadherin可能参与了NSCLC肿瘤侵袭转移过程㊂MUC1是Mucins黏蛋白家族成员,主要表达于正常组织的上皮细胞[9]㊂研究表明当肿瘤细胞转染MUC1基因后,肿瘤细胞获得了更高的移动性和侵袭性[10];以MUC1为靶点研制抗肿瘤疫苗也是目前的研究热点[11,12]㊂大量研究提示MUC1可在卵巢肿瘤等多种肿瘤中异常表达[13⁃15],与肿瘤细胞转移密切相关㊂本研究中NSCLC肿瘤组织MUC1的阳性表达率分别为62.9%(44/70),与对照组比较具有显著统计学意义(P<0.01);其阳性表达率随着pTNM分期升高而降低;且有淋巴结转移的肺癌组织中MUC1阳性表达率较无淋巴结转移的肺癌组织显著升高,提示MUC1蛋白可能与NSCLC的肿瘤进展密切相关,与Kuemmel等[15]的研究结果基本一致㊂PINCH是黏着斑蛋白,对细胞黏附㊁迁移过程进行调节,在正常组织中仅少量表达,但在肺癌㊁乳腺癌㊁前列腺癌等多种恶性肿瘤间质及肿瘤浸润的边缘中异常高表达[16,17],提示参与肿瘤发生发展㊁侵袭转移的过程㊂本研究中NSCLC患者肿瘤组织中PINCH的阳性表达率为57.1%(40/70),与对照组比较差异明显(P<0.01);随着临床分期升高,其阳性表达率显著降低;而且有淋巴结转移的肺癌组织中PINCH阳性表达率较无淋巴结转移的肺癌组织显著升高,提示PINCH蛋白的阳性表达可能是NSCLC浸润和转移的重要标志物,具有一定的代表性㊂E⁃cadherin是细胞黏附分子,MUC1是黏蛋白家族成员,PINCH是黏着斑蛋白,三者均与细胞间的黏附性能密切相关,对细胞黏附㊁迁移过程进行调节㊂有学者认为[18],MUC1的异常高表达,可使E⁃cadherin表达水平下调㊂与此结果一致,本研究显示NSCLC中E⁃cadherin和MUC1蛋白表达呈负相关,E⁃cadherin和PINCH蛋白表达呈负相关㊂另外,本研究分析了MUC1和PINCH蛋白之间的相关性,结果显示二者之间呈正相关关系,提示E⁃cadherin㊁MUC1及PINCH三者可能互相联系共同促进NSCLC的侵袭转移;更为具体的分子机制有待于进一步研究㊂综上,我们推测E⁃cadherin㊁MUC1及PINCH三者可能互相联系共同影响细胞间的黏附性进而促进NSCLC侵袭与转移;同时检测E⁃cadherin和MUC1和PINCH蛋白在NSCLC癌组织中的表达,对于潜在病变组织以及转移组织的检测可能有重要意义㊂在此研究的基础上,我们将追踪分析NSCLC患者的五年存活率,以进一步确定E⁃cadherin和MUC1和PINCH蛋白在NSCLC预后中的作用㊂(下转第95页)㊃09㊃中国免疫学杂志2019年第35卷2017,152(2):313⁃321.e2.[4]　Ananthakrishnan 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