胡偉 ,王磊 ,孫磊 ,高泰龍
(1.江蘇省連云港市第一人民醫(yī)院,江蘇 連云港 222002;2.湖南省人民醫(yī)院 肝膽外科,湖南 長(zhǎng)沙410005)
IQ結(jié)構(gòu)域GTP酶激活蛋白1在胰腺癌中表達(dá)及與預(yù)后的相關(guān)性研究*
胡偉 1,王磊 1,孫磊 1,高泰龍 2
(1.江蘇省連云港市第一人民醫(yī)院,江蘇 連云港 222002;2.湖南省人民醫(yī)院 肝膽外科,湖南 長(zhǎng)沙410005)
目的檢測(cè)胰腺癌組織中IQ結(jié)構(gòu)域GTP酶激活蛋白1(IQGAP1)的表達(dá)與臨床病理特征及與預(yù)后的關(guān)系。方法采用免疫組織化學(xué)法檢測(cè)66例胰腺癌組織和49例癌旁組織中IQGAP1的表達(dá)情況,分析IQGAP1與胰腺癌臨床病理特征及其與預(yù)后的相關(guān)性。結(jié)果胰腺癌組織和癌旁組織中IQGAP1的陽(yáng)性表達(dá)率分別為63.636%和12.245%,胰腺癌組織高于癌旁組織(P<0.05);IQGAP1的表達(dá)與腫瘤分化程度、淋巴結(jié)轉(zhuǎn)移有關(guān)(P<0.05),與患者年齡、性別、腫瘤部位、大小、神經(jīng)浸潤(rùn)及臨床分期差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。IQGAP1陽(yáng)性患者的總生存期短于IQGAP1陰性患者(P=0.002),Cox多因素分析顯示IQGAP1陽(yáng)性表達(dá)是獨(dú)立預(yù)后危險(xiǎn)因素結(jié)論胰腺癌組織中高表達(dá)的IQGAP1可能參與胰腺癌的發(fā)生、發(fā)展及轉(zhuǎn)移過程,并可作為判斷胰腺癌預(yù)后的重要指標(biāo)。
IQ結(jié)構(gòu)域GTP酶激活蛋白1;胰腺癌;免疫組織化學(xué);生存分析
IQ結(jié)構(gòu)域GTP酶激活蛋白1(IQ-domain GTPase-activating protein 1,IQGAP1)作為一種進(jìn)化保守的蛋白家族成員,具有多個(gè)介導(dǎo)蛋白質(zhì)相互作用結(jié)構(gòu)域,調(diào)節(jié)多種細(xì)胞活動(dòng)功能,包括胞質(zhì)分裂,細(xì)胞遷移、增殖和信號(hào)轉(zhuǎn)導(dǎo),囊泡轉(zhuǎn)運(yùn)及細(xì)胞骨架結(jié)構(gòu)調(diào)節(jié)[1]。研究已證實(shí),IQGAP1作為一種致癌基因,在多種惡性腫瘤生長(zhǎng)、侵襲和轉(zhuǎn)移過程中發(fā)揮重要作用[2-3]。目前IQGAP1在胰腺癌中的研究甚少,尚未見報(bào)道其與胰腺癌生存預(yù)后的關(guān)系。本文通過免疫組織化學(xué)方法檢測(cè)IQGAP1在胰腺癌組織中的表達(dá)情況,分析其與胰腺癌臨床病理特征及預(yù)后的關(guān)系,以期判斷其作為胰腺癌臨床預(yù)后指標(biāo)的可能性。
選取2010年3月胰腺癌標(biāo)本來源于連云港市第一人民醫(yī)院行胰腺癌切除的患者,66例患者具有完整的臨床病理及隨訪資料。其中,男性43例,女性23例;年齡34~85歲,中位年齡60.5歲。采用電話或門診隨訪,隨訪截止日期為2016年12月。隨訪時(shí)間為0.6~81.0個(gè)月,死亡42例、存活14例。所有標(biāo)本術(shù)后病理均證實(shí)為胰腺導(dǎo)管腺癌,高中分化47例,低分化19例。按照國(guó)際抗癌聯(lián)盟(UICC)TNM分期標(biāo)準(zhǔn):Ⅰ、Ⅱ期64例,Ⅲ、Ⅳ期2例。同時(shí)取配對(duì)胰腺癌旁組織49例,由病理學(xué)檢查證實(shí)無(wú)癌細(xì)胞。所有入組患者術(shù)前均未接受任何針對(duì)腫瘤的治療。
兔抗人IQGAP1多克隆抗體(美國(guó)Abcam公司),SP免疫組織化學(xué)試劑盒(北京中衫金橋生物技術(shù)有限公司)及DAB顯色試劑盒(武漢博士德生物技術(shù)有限公司)。
免疫組織化學(xué)染色法(SP法)主要操作步驟如下:手術(shù)所取標(biāo)本經(jīng)10%甲醛固定,常規(guī)石蠟包埋,4 μm厚切片,烤箱60℃烤片60 min,二甲苯脫蠟,梯度酒精水化,滅活內(nèi)源性過氧化物酶,抗原修復(fù),血清封閉,一抗4℃過夜(稀釋1∶400),二抗孵育,DAB顯色,蘇木素復(fù)染,脫水、透明、干燥、封片,鏡檢。PBS代替一抗作陰性對(duì)照。
IQGAP1陽(yáng)性反應(yīng)主要為細(xì)胞漿或細(xì)胞膜呈黃色或棕黃色。高倍鏡下(×400)隨機(jī)觀察5個(gè)視野,根據(jù)陽(yáng)性細(xì)胞比率:陽(yáng)性細(xì)胞數(shù)<10%為陰性,>10%為陽(yáng)性表達(dá),其中,10%~25%為1分,25%~50%為2分,50%~75%為3分,>75%為4分。根據(jù)染色強(qiáng)度分為0~3分。陽(yáng)性細(xì)胞率與染色強(qiáng)度之積0~4分為IQGAP1陰性表達(dá),5~12分為陽(yáng)性表達(dá);閱片采用雙盲法,每張切片均由2位病理醫(yī)師獨(dú)立觀察,分別記錄。
數(shù)據(jù)分析采用SPSS 23.0統(tǒng)計(jì)軟件,計(jì)數(shù)資料以率(%)表示,采用χ2檢驗(yàn)或Fisher確切概率法,Kaplan-Meier法繪制生存率曲線,采用Log-rank檢驗(yàn),影響因素分析采用Cox比例風(fēng)險(xiǎn)回歸模型,P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
IQGAP1主要表達(dá)于胰腺癌細(xì)胞的胞漿或胞膜。在66例胰腺癌標(biāo)本中,IQGAP1蛋白的陽(yáng)性表達(dá)率為63.636%(42/66);而在癌旁組織中IQGAP1陽(yáng)性表達(dá)率僅為12.245%(6/49),低于胰腺癌組織(χ2=30.542,P=0.001)。見圖 1。
IQGAP1表達(dá)與胰腺癌分化程度相關(guān)(P=0.046),低分化胰腺癌組織中IQGAP1的陽(yáng)性表達(dá)率最高,同時(shí)IQGAP1陽(yáng)性組中淋巴結(jié)轉(zhuǎn)移的患者較IQGAP1陰性組增多(P=0.019),而IQGAP1表達(dá)與胰腺癌患者的年齡、性別、腫瘤大小、部位、臨床分期及神經(jīng)浸潤(rùn)均差異無(wú)統(tǒng)計(jì)學(xué)意義,癌旁組織IQGAP1的表達(dá)與患者年齡、性別、腫瘤大小、部位、分化程度、臨床分期、神經(jīng)浸潤(rùn)及淋巴結(jié)轉(zhuǎn)移均無(wú)關(guān)。見表1。
圖1 IQGAP1在胰腺癌旁和癌組織中的表達(dá)(×200)
圖2 IQGAP1表達(dá)、淋巴結(jié)轉(zhuǎn)移與胰腺癌患者預(yù)后關(guān)系的Kaplan-Meier生存率曲線
Kaplan-Meier法繪制生存率曲線并用Log-rank檢驗(yàn)差異,結(jié)果顯示,IQGAP1陽(yáng)性組患者的總生存期較陰性組縮短,淋巴結(jié)轉(zhuǎn)移組患者總生存期較無(wú)轉(zhuǎn)移組縮短,差異有統(tǒng)計(jì)學(xué)意義(PIQGAP1=0.002,P淋巴結(jié)轉(zhuǎn)移=0.001)(見圖2)。Cox單因素分析結(jié)果顯示胰腺癌組織IQGAP1表達(dá)、淋巴結(jié)有無(wú)轉(zhuǎn)移均與胰腺癌預(yù)后相關(guān),而腫瘤部位、大小、臨床分期、分化程度及神經(jīng)浸潤(rùn)等因素與預(yù)后無(wú)關(guān)。Cox多因素回歸分析(α入=0.05,α出=0.10)結(jié)果示IQGAP1在胰腺癌組織中的陽(yáng)性表達(dá)為胰腺癌獨(dú)立的預(yù)后指標(biāo)。見表2。
表2 66例胰腺癌患者預(yù)后的Cox回歸模型分析
細(xì)胞支架蛋白IQGAP1含有4個(gè)IQ基序和類似于GAP相關(guān)結(jié)構(gòu)域,這些結(jié)構(gòu)域可與多種蛋白結(jié)合,通過調(diào)節(jié)細(xì)胞間黏附、細(xì)胞極性及遷移而參與腫瘤的進(jìn)程[2]。IQGAP1直接結(jié)合E-cadherin調(diào)節(jié)細(xì)胞間黏附[1],直接結(jié)合F-actin[4]或經(jīng)透明質(zhì)酸介導(dǎo)[5]增強(qiáng)細(xì)胞運(yùn)動(dòng),通過actin、Cdc42/Rac1、MAPK信號(hào)通路促進(jìn)細(xì)胞侵襲、增殖和分化[6-7]。此外,IQGAP1還通過VEGFR-2信號(hào)通路調(diào)節(jié)內(nèi)皮細(xì)胞的增殖及血管生成[8],并在小鼠乳腺癌腫瘤模型中促進(jìn)腫瘤血管生成,同時(shí)證實(shí)IQGAP1能夠促進(jìn)乳腺M(fèi)CF-7細(xì)胞增殖及腫瘤形成[6]。反之,通過可滲透肽類破壞IQGAP1與ERK1/2的相互作用能夠抑制Ras驅(qū)動(dòng)的小鼠腫瘤形成及侵襲能力[9]。已證實(shí)IQGAP1在肝癌[2]、肺癌[10]及食管癌等[11]多種實(shí)體腫瘤組織中高表達(dá),并與病理分化級(jí)別相關(guān)[12]。本研究顯示,胰腺癌組織IQGAP1蛋白表達(dá)高于癌旁組織,在低分化組表達(dá)高于高中分化組,表明IQGAP1與胰腺癌分化程度相關(guān),提示IQGAP1的高表達(dá)與胰腺癌的侵襲、轉(zhuǎn)移有關(guān)。與上述IQGAP1的調(diào)節(jié)功能一致。
IQGAP1與β-catenin相互作用可提高肝癌細(xì)胞的轉(zhuǎn)移能力從而促進(jìn)腫瘤進(jìn)展[13]。過表達(dá)IQGAP1能促進(jìn)胰腺癌細(xì)胞SW1990的增殖,而沉默IQGAP1通過下調(diào)活化性Cdc42/Rac1降低細(xì)胞增殖率,抑制腫瘤細(xì)胞遷移[14]。子宮內(nèi)膜癌細(xì)胞IQGAP1高表達(dá)可促進(jìn)細(xì)胞上皮間質(zhì)轉(zhuǎn)化及增殖、侵襲[15]。IQGAP1在腫瘤組織中高表達(dá)及在腫瘤侵襲前緣表達(dá)[16]、彌漫型表達(dá)[17]均與預(yù)后不良密切相關(guān)。而且IQGAP1高表達(dá)可作為判斷惡性膠質(zhì)瘤的診斷指標(biāo)[18]。本研究顯示,IQGAP1表達(dá)與淋巴結(jié)轉(zhuǎn)移有關(guān),提示其可作為預(yù)測(cè)胰腺癌侵襲與轉(zhuǎn)移的有效指標(biāo)。表明IQGAP1可能作為胰腺癌治療的潛在靶點(diǎn)。本研究中發(fā)現(xiàn)IQGAP1表達(dá)與臨床分期無(wú)關(guān),分析原因可能與胰腺癌晚期樣本量少有關(guān)。
單因素分析IQGAP1與胰腺癌預(yù)后的關(guān)系發(fā)現(xiàn),其陽(yáng)性表達(dá)較陰性表達(dá)患者生存期縮短,預(yù)后相對(duì)差,這可能與IQGAP1參與胰腺癌細(xì)胞黏附、遷移、轉(zhuǎn)移及血管生成有關(guān)。這種關(guān)聯(lián)的科學(xué)依據(jù)是腫瘤侵襲、轉(zhuǎn)移過程中需要細(xì)胞之間黏附的動(dòng)態(tài)重排。通過多因素分析發(fā)現(xiàn),IQGAP1陽(yáng)性表達(dá)可作為判斷胰腺癌患者預(yù)后的獨(dú)立危險(xiǎn)因素。
綜上所述,IQGAP1上調(diào)在胰腺癌的發(fā)生發(fā)展、轉(zhuǎn)移過程中發(fā)揮重要作用,且與胰腺癌的惡性程度、淋巴結(jié)轉(zhuǎn)移及預(yù)后不良有關(guān),可能作為胰腺癌預(yù)后評(píng)估的參考指標(biāo),但I(xiàn)QGAP1在胰腺癌的具體機(jī)制,比如是否參與胰腺癌組織的血管形成尚不清楚,有待于進(jìn)一步研究。
[1]HEDMAN A C,SMITH J M,SACKS D B.The biology of IQGAP proteins:beyond the cytoskeleton[J].EMBO Rep,2015,16(4):427-446.
[2]XIA F D,WANG Z L,CHEN H X,et al.Differential expression of IQGAP1/2 in hepatocellular carcinoma and its relationship with clinical outcomes[J].Asian Pac J Cancer Prev,2014,15(12):4951-4956.
[3]WHITE C D,ERDEMIR H H,SACKS D B.IQGAP1 and its binding proteins control diverse biological functions[J].Cell Signal,2012,24(4):826-834.
[4]MATARAZA J M,LI Z,JEONG H,et al.Multiple proteins mediate IQGAP1-stimulated cell migration[J].CellularSignalling,2007,19(9):1857-1865.
[5]KOZLOVA I,RUUSALA A,VOYTYUK O,et al.IQGAP1 regulates hyaluronan-mediated fibroblast motility and proliferation[J].Cellular Signalling,2012,24(9):1856-1862.
[6]JADESKI L,MATARAZA J M,JEONG H,et al.IQGAP1 stimulates proliferation and enhances tumorigenesis of human breast epithelial cells[J].Journal of Biological Chemistry,2008,283(2):1008-1017.
[7]BROWN M D,SACKS D B.IQGAP1 in cellular signaling:bridging the GAP[J].Trends in Cell Biology,2006,16(5):242-249.
[8]MEYER R D,SACKS D B,RAHIMI N.IQGAP1-dependent signaling pathway regulates endothelial cell proliferation and angiogenesis[J].PLoS One,2008,3(12):e3848.
[9]JAMESON K L,MAZUR P K,ZEHNDER A M,et al.IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors[J].Nature Medicine,2013,19(5):626-630.
[10]LI G,GAO Y,CUI Y,et al.Overexpression of CD44 is associated with the occurrence and migration of non-small cell lung cancer[J].Molecular Medicine Reports,2016,14(4):3159-3167.
[11]WANG X,WANG K,LI X,et al.Targeted knockdown of IQGAP1 inhibits the progression of esophageal squamous cell carcinoma in vitro and in vivo[J].PloS One,2014,9(5):e965015.
[12]WANG X,LI X,ZHAI L,et al.Overexpression of IQGAP1 in human pancreatic cancer[J].Hepatobiliary&Pancreatic Diseases International,2013,12(5):540-545.
[13]JIN X,LIU Y,LIU J,et al.The overexpression of IQGAP1 and beta-catenin is associated with tumor progression in hepatocellular carcinoma in vitro and in vivo[J].PLoS One,2015,10(8):e133770.
[14]JIN Y,LV X,ZHOU J,et al.Potential involvement of IQGAP1 in proliferation and metastasis of human pancreatic cancer[J].Front Biosci(Landmark Ed),2016,21:1076-1083.
[15]DONG P,IHIRA K,XIONG Y,et al.Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression[J].Oncotarget,2016,7(15):20260-20270.
[16]DONG P,NABESHIMA K,NISHIMURA N,et al.Overexpression and diffuseexpression pattern ofIQGAP1 atinvasion fronts are independent prognostic parameters in ovarian carcinomas[J].Cancer Lett,2006,243(1):120-127.
[17]HAYASHI H,NABESHIMA K,AOKI M,et al.Overexpression of IQGAP1 in advanced colorectal cancer correlates with poor prognosis-critical role in tumor invasion[J].Int J Cancer,2010,126(11):2563-2574.
[18]MCDONALD K L,O'SULLIVAN M G,PARKINSON J F,et al.IQGAP1 and IGFBP2:valuable biomarkersfordetermining prognosis in glioma patients[J].J Neuropathol Exp Neurol,2007,66(5):405-417.
Expression and prognostic role of IQGAP1 in patients with pancreatic adenocarcinoma*
Wei Hu1,Lei Wang1,Lei Sun1,Tai-long Gao2
(1.The First People's Hospital of Lianyungang,Lianyungang,Jiangsu 222002,China;2.Department of hepatobiliary Surgery,Hunan Provincial People's Hospital,Changsha,Hunan 410005,China)
ObjectiveTo investigate expression the level of IQ-domain GTPase-activating protein 1(IQGAP1)and its prognostic significance in pancreatic adenocarcinoma.MethodsA total of 66 cases of patients diagnosed with pancreatic adenocarcinoma were included in this study.A total of 66 cancer samples as well as 49 paracancerous normal tissues were harvested,and the expression level of IQGAP1 was measured by Immunohistochemistry.The correlation analysis of IQGAP1 expression and clinical manifestations were performed.ResultsIQGAP1 was upregulated significantly in cancer tissue when compared with normal tissue(63.636%vs 12.245%,P<0.05).Expression of IQGAP1 was closely associated with clinical features including histological grading and lymph node metastasis (P<0.05).No significant correlation was observed between IQGAP1 and age,gender,size or position of tumor,neural invasion or clinical stage of patients (P>0.05).Patients with positive expression of IQGAP1 experienced shorter overall survival time as compared with those without IQGAP1 expression (P=0.002).Multivariate Cox regression analysis demonstrated that IQGAP1 overexpression was an independent prognostic factor in pancreatic adenocarcinoma(^HR=2.128,95%CI=1.127,4.019,P=0.020).ConclusionsUp-regulation of IQGAP1 may be involved in tumor progression,and is potentially a prognostic biomarker.
IQ-domain GTPase-activating protein 1;pancreatic ductal adenocarcinoma;Immunohistochemistry;prognosis
R735.9
A
10.3969/j.issn.1005-8982.2017.30.007
1005-8982(2017)30-0041-05
2017-04-10
連云港市第一人民醫(yī)院青年英才豪森基金資助項(xiàng)目(No:QN150102);連云港市科技局項(xiàng)目(No:SH1218)
高泰龍,E-mail:docgtl@163.com
(王榮兵 編輯)
中國(guó)現(xiàn)代醫(yī)學(xué)雜志2017年30期