大肠癌中Ezrin蛋白的表达及其与临床病理特征的关系

　　　　作者：范婕　史威 李秀娟 郭颖 白美玲

【摘要】 目的：探讨Ezrin在大肠癌中的表达特点及其与临床病理参数的关系。方法：应用免疫组织化学SP法检测100例大肠癌组织及11例癌旁正常黏膜中Ezrin的表达情况。结果：Ezrin阳性表达率在大肠正常粘膜、大肠癌组织中分别为36.4%、75%，差异有统计学意义(P&<0.05);淋巴结转移组Ezrin阳性率(89.6%)显著高于无淋巴结转移组(61.5%)(P&<0.05);在大肠癌中，Ezrin异常表达与大肠癌患者的年龄、性别、肿瘤的发生部位以及分化程度无关(P&>0.05)。结论：Ezrin表达水平提高提示癌组织具有更高的浸润、侵袭能力和淋巴结转移风险，检测 Ezrin有望成为判断大肠癌患者预后的新指标或治疗肿瘤转移的一个靶点。

【关键词】 肠肿瘤;肿瘤转移;免疫组织化学;膜细胞骨架连接蛋白

Ezrin，a member of the ezrinradixinmoesin(ERM)family in speciesconserved protein，is a membrane cytoskeleton linker and is involved in cellular functions，including epithelial cell morphogenesis and adhesion[1].It is encode by vil2(6q25.2～q26)and its functional domain is FERM domain.The inactivation of Ezrin causes a massive cell retraction and leads to the destruction of both cellcell and cellsubstrate adhesion，whereas the overexpression of Ezrin in insect cells results in enhanced cell adhesion[2，3].The expression of Ezrin in a series of patients with colorectal carcinoma was studied，using immunohistochemistry to elucidate its functional importance and clinical significance.

　　1 Materials and methods

　　1.1 Materials

　　Between March 2005 and May 2006，100 colorectal carcinoma samples were obtained from the archives of the First Affiliated Hospital，Hebei North University Zhangjiakou，which were all surgically resected without chemotherapy and radiotherapy before operation.There were 41 men(41%)and 59 women(59%)with a median age of 58.3 years(range，30～78 years).Fortysix patients with colon tumors and fiftyfour patients in rectum.Fortyeight patients(48%)had histologically confirmed lymph node metastasis，whereas the remaining 52 patients(52%)were found to have no clinical or histopathologic evidence of lymph node involvement.According to current World Health Organization classification，52 patients were well differentiated，26 patients were moderately differentiated，11 patients were poorly differentiated and 11 patients were Mucous.Immunohistochemistry.

　　1.2 Methods

　　The samples were fixed in 10% neutrally buffered formalin and paraffin embedded，processed and stained with HE.All H&&E sections were reviewed to confirm the diagosis.One paraffin block with the maximum bulk of tumor was chosen from each case for immunohistochemical studies.All slides showed the non neoplastic colorectal tissuecarcinoma junction.Serial sections of 4 μm thickness were cut from the paraffin blocks.The sections were deparaffinized with xylene and rehydrated with ethanol.Nonenzymatic antigen retrieval was performed on each slide and washed with phosphatebuffered saline(PBS).Heat induced epitope retrieval techniques were used for antigen retrieval as follow:citrate buffer(pH6.0)and a water bath at 95～98 ℃ for 30 min.Sections were incubated for 10 min in 3% hydrogen peroxide to quench endogenous tissue peroxidase.The sections were immunostained for mono clonal antibody against Ezrin(mouse mAbIgG1，3C12;Lab Vision Neomarker Corp)at a dilution of 1:50 and incubated at 4 ℃ whole night.This antibody didnt crossreact with other ERM family proteins.After washing with phosphatebuffered saline，a secondary antibody was used in 37℃ for 30min.And also after washing with phosphatebuffered saline，SP were used in 37℃ for 30 min.Then，DAB was used.

　　1.3 Evaluation of immunohistochemical staining

　　Histological and immunohistochemical evaluation was performed by one pathologist.Sometimes，Ezrin expression was located in cytoplasm，and the other was in membrane [4].Each stained slide was assessed and given a score，in which the intensity of the staining(no staining=0，weak staining=1，medium staining=2，and strong staining=3)and the percent of stained cells(0%=0，1%～10%=1，11%～50%=2，50%～74%=3 and greater than 75%=4)were multiplied.With the applied system，the maxi mum score≥3 was positive staining，the maximum score&<3 was negative staining.

　　1.4 Statistical analysis

　　SPSS for Windows version 11.0 was used for statistical analysis.The correlateion of each score according to the intensity and percentage of labeled cells or intercellular substance with relevant clinical data were statistically analyzed.Generally，P&<0.05 was regarded as significant.

　　2 Results

　　2.1 Ezrin expression in adjacent normal colorectal mucosa and colorectal carcinoma

　　Ezrin staining was mainly located in cytoplasm of tumor cells in colorectal carcinoma.The abnomal positive expressing rates of Ezrin in groups of colorectal adjacent normal mucosa and colorectal carcinoma were separately 36.4% and 75%，the differences had significant statistical significance(P&<0.05，Table 1).

　　Table 1 The expression of Ezrin in colorectal carcinoma and normalcolorectal mucosa n(%)

　　GroupnEzrin expression+-P valueCC10075(75.0)25(25.0)NM114(36.4)7(63.6)&<0.05

　　CC：colorectal carcinoma，NM：normalcolorectal mucosa

　　2.2 Ezrin expression in metastatic group and nonmetastatic group of colorectal carcinoma

　　The percentage of Ezrin expression in metastatic group was 89.6%(43/48)，and the percentage in nonmetastatic group was 61.5%(32/52).Respectively so the expression rate in metastatic group was significant higher than that in nonmetastatic group(P&<0.05，Table 2).

　　2.3 Correlation between Ezrin expression and clinical pathological characteristics

　　The percentage of expression in colorectal carcinoma was compared in patients age，sexal，tumor location and degree of differentiation.Statistical significance was not observed(P&>0.05;Table 2)　Table 2 The ralationship between clinical pathological parameters of CC and Ezrin expression n

　　3 Discussion

　　To metastasize successfully into a clinically relevant mass，tumor cells must overcome a series of challenges.These include invasion into the surrounding tissue，extravasation into the lymphovascular space，arriving at a distant side，and intravasation into a new environment.This study aims were to assess staining patterns in metastatic and nonmetastatic colorectal carcinoma.At the same time，to investigate possible correlations between Ezrin expression in colorectal carcinoma and histopathological and prognostic data were also important in this study.

　　The expression rate of Ezrin was significantly higher in colorectal carcinoma tissues than that in colorectal normal mucosa，and significantly correlated with lymph node metastasis.The higher expression of Ezrin contributed to the metastasis of colorectal carcinoma，and might be useful for colorectal carcinoma prognosis estimation.The results showed gradual increase in Ezrin expression following tumor carcinogenesis，progression and metastasis.No significant correlation was found between Ezrin expression with patients age，sexal，location and degree of differentiation.

　　Ezrin is a member of ERM(Ezrinradixinmoesin)family.The proteins Ezrin，moesin，and radixin act as linkers between the plasma membrane and the actin cytoskeleton.The carboxyl termini of Ezrin binds to actin filaments，whereas the amino termini binds to plasma membranes via a binding partner，such as CD43，CD44 or ICAM1.CD44 was regarded to be closely related to tumor metastasis，especially in SCRC [5].CD44 and Ezrin and their respective complex have properties suggesting that they may be important in the process of tumorendothelium interactions，cell migrations，cell adhesion，tumor progression and metastasis.One of the functions of ezrin is to participate in the formation of cellsurface complexes，such as Ecadherin，integrin that mediates cellcell and cellextracellular matrix attachments [69].

　　In conclusion，Ezrin may play an important role in the carcinogenesis，progression and metastasis of colorectal carcinoma.Ezrin protein has hopes to become a new therapy target to evaluate the tumor prognosis.

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