RGC-32和E-cadherin在胰腺癌中的表達及其臨床病理學意義

朱亮 趙慧貞 龐慧芳 覃華 黎培員 李德民 趙秋

·論著·

RGC-32和E-cadherin在胰腺癌中的表達及其臨床病理學意義

朱亮 趙慧貞 龐慧芳 覃華 黎培員 李德民 趙秋

目的探討RGC-32和E-cadherin蛋白在胰腺癌組織中的表達，分析其臨床病理學意義以及兩種蛋白表達之間的相關性。方法采用免疫組織化學SP法檢測42例胰腺癌、12例慢性胰腺炎和8例正常胰腺組織中RGC-32和E-cadherin蛋白的表達。結果RGC-32主要表達于胰腺腺泡細胞胞質(zhì)內(nèi)；E-cadherin主要表達于正常胰腺和慢性胰腺炎組織胰腺腺泡細胞胞膜，而在胰腺癌中則可出現(xiàn)胞質(zhì)表達和(或)表達減弱等異常表達。胰腺癌組織中RGC-32的陽性表達率以及E-cadherin異常表達率分別為78.6%(33/42)和54.8%(23/42)，均顯著高于正常胰腺組織的37.5%(3/8)和0以及慢性胰腺炎組織41.7%(5/12)和8.3%(1/12)(P值均<0.05)。胰腺癌組織RGC-32表達與腫瘤淋巴結轉(zhuǎn)移和TNM分期有關(P值分別為0.016、0.025)，而與患者的年齡、性別及腫瘤分化程度無關(P值分別為0.831、1.000、0.629)；E-cadherin異常表達與胰腺癌分化程度、淋巴結轉(zhuǎn)移和TNM分期有關(P值分別為0.024、0.004、0.004)，而與患者年齡、性別無關(P值分別為0.970、1.000)。RGC-32的陽性表達率與E-cadherin異常表達率呈正相關(r=0.458，P<0.01)。結論胰腺癌組織中RGC-32陽性表達率及E-cadherin異常表達率均顯著升高，RGC-32可能通過調(diào)控上皮間質(zhì)轉(zhuǎn)化過程參與胰腺癌的侵襲和轉(zhuǎn)移。

胰腺腫瘤； 免疫組織化學； RGC-32； 上皮-鈣黏素； 上皮間質(zhì)轉(zhuǎn)化

RGC-32(response gene to complement-32)是一種新發(fā)現(xiàn)的補體激活基因，在包括胰腺在內(nèi)的多種正常組織表達，參與細胞周期以及細胞分化的調(diào)節(jié)等[1-3]。有研究表明，RGC-32在人類多種腫瘤中異常表達[4-7]，但其在胰腺癌中的表達及與胰腺癌發(fā)生、發(fā)展的關系尚未見報道。上皮-鈣黏素(E-cadherin)是上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT)的重要標志分子之一，并與腫瘤的侵襲轉(zhuǎn)移有關。本研究檢測RGC-32、E-cadherin在正常胰腺、慢性胰腺炎及胰腺癌組織中的表達，分析二者與胰腺癌臨床病理特征之間的關系及兩者間的相互關系。

材料和方法

一、臨床資料

收集華中科技大學同濟醫(yī)學院附屬同濟醫(yī)院2005年至2010年手術切除的胰腺癌標本42例，其中男性21例，女性21例，年齡22～74歲，平均55歲。另取 12 例慢性胰腺炎(CP)和 8 例正常胰腺組織標本作為對照。

二、RGC-32和E-cadherin蛋白檢測

采用免疫組織化學染色法，即用型鏈霉親和素-過氧化物酶(SP)試劑盒購自北京中杉金橋公司，嚴格按照說明書操作。兔抗人RGC-32多抗購自美國Santa Cruz公司，工作濃度1∶50；兔抗人E-cadherin多抗購自美國Proteintech公司，工作濃度1∶100。采用已知陽性(結腸癌)片為陽性對照，PBS代替一抗為陰性對照。

每張切片隨機選取 5個高倍鏡視野(×400)，每視野計數(shù)500個細胞。RGC-32陽性表達為胞質(zhì)內(nèi)出現(xiàn)棕黃色染色顆粒。結果判斷標準：無染色為0分，淺黃色1分，棕黃色2分，棕褐色3分；無陽性細胞為0 分，陽性細胞<10%為1分，10%～50%為2分，>50%為3分。兩分乘積≥2為免疫組化陽性，否則計為陰性。E-cadherin陽性表達為胞膜上出現(xiàn)棕黃色染色顆粒。正常胰腺和CP組織中主要表達于腺泡細胞胞膜，胰腺癌中則可出現(xiàn)胞質(zhì)表達和(或)表達減弱。結果判斷參照Jawhari等[8]的方法，以正常胰腺或胰腺癌組織周圍殘存的正常胰腺組織E-cadherin表達為陽性對照，癌細胞完全不表達為0分，癌細胞胞質(zhì)表達為1分，癌細胞染色強度降低為2分，癌細胞染色部位及強度與正常胰腺組織相同為3分?！?分為異常表達，3分為正常表達。

三、統(tǒng)計學處理

采用SPSS17.0統(tǒng)計軟件，應用χ2檢驗、Fisher精確檢驗和Spearman等級相關檢驗分析數(shù)值間的差異和相關性。P<0.05為差異具有統(tǒng)計學意義。

結 果

一、RGC-32、E-cadherin蛋白的表達

42例胰腺癌組織的RGC-32陽性表達率及E-cadherin異常表達率分別為78.6%(33/42)和54.8%(23/42)，均高于正常胰腺組織的37.5%(3/8)和0以及CP組織的41.7%(5/12)和8.3%(1/12)，差異均具有統(tǒng)計學意義(圖1、2；χ2值分別為5.623、8.113、6.097、8.148，P值分別為0.030、0.005、0.028、0.004)；正常胰腺組織和CP組織中兩者差異無統(tǒng)計學意義(χ2值分別為0.035、0.702，P=1.000)。

圖1胰腺癌(a)、慢性胰腺炎(b)及正常胰腺組織(c)中RGC-32蛋白表達(SP ×400)

圖2胰腺癌(a)、慢性胰腺炎(b)及正常胰腺組織(c)中E-cadherin蛋白表達(SP ×400)

二、RGC-32、E-cadherin蛋白表達與胰腺癌臨床病理學特征的關系

RGC-32蛋白的陽性表達與胰腺癌的淋巴結轉(zhuǎn)移和TNM分期有關(P值均<0.05)，而與患者的年齡、性別及胰腺癌的分化程度無關(P值均>0.05)。E-cadherin蛋白的異常表達與胰腺癌的分化程度、淋巴結轉(zhuǎn)移和TNM分期有關(P值均<0.05)，而與患者的年齡、性別無關(表1)。

表1RGC-32及E-cadherin蛋白表達與腫瘤臨床病理學特征的關系

病理特征例數(shù)RGC-32陽性表達[例,率(%)]χ2值P值E-cadherin異常表達[例,率(%)]χ2值P值年齡(歲)0.3710.8310.0210.990 <4575(71.4)4(57.1) 45～592218(81.8)12(54.5) ≥601310(76.9)7(53.8)性別0.1411.0000.0961.000 男2117(81.0)11(52.4) 女2116(76.2)12(57.1)分化程度0.9280.6297.4270.024 高分化1612(75.0)5(31.3) 中分化118(72.7)6(54.5) 低分化1513(86.7)12(80.0)淋巴結轉(zhuǎn)移7.6490.0169.2410.004 無169(56.3)4(25.0) 有2624(92.3)19(73.1)TNM分期5.7040.0259.2590.004 Ⅰ～Ⅱ1811(61.1)5(27.8) Ⅲ～Ⅳ2422(91.7)18(75.0)

三、胰腺癌組織RGC-32陽性表達與E-cadherin異常表達之間的關系

42例胰腺癌組織中，RGC-32陽性表達且E-cadherin異常表達者22例， RGC-32陰性表達且E-cadherin正常表達者8例，RGC-32陽性表達率與E-cadherin異常表達率之間呈正相關(r=0.458，P<0.01)。

討 論

RGC-32基因1998年由Badea等[1]首次克隆獲得。作為一種重要的補體應答基因，RGC-32參與許多生物學功能，如細胞增殖、細胞分化等[2-3]。近年來，RGC-32在腫瘤中的作用受人關注。現(xiàn)已發(fā)現(xiàn)，在人類多種腫瘤中存在RGC-32異常表達。Fosbrink等[4]研究發(fā)現(xiàn)，肺癌、胃癌、卵巢癌、乳癌及前列腺癌等腫瘤組織中均有不同程度的RGC-32表達上調(diào)，然而也有報道在原發(fā)性星狀細胞瘤的進展期，RGC-32 mRNA表達水平下調(diào)[5]。另外，在各種轉(zhuǎn)移性癌中RGC-32表達的研究結果也不盡相同。在伴有溶骨性轉(zhuǎn)移的乳腺癌中RGC-32表達上調(diào)[6]，而在前列腺轉(zhuǎn)移癌中其表達下調(diào)[7]。因此，RGC-32在腫瘤中的作用比較復雜，在不同腫瘤中其表達強度、作用可能不盡相同。RGC-32在胰腺癌中的表達目前國內(nèi)外尚無報道。

本研究結果顯示，胰腺癌中RGC-32的陽性表達率及表達強度顯著高于慢性胰腺炎及正常胰腺組織，且與患者淋巴結轉(zhuǎn)移和TNM分期有關，提示RGC-32表達上調(diào)可能與胰腺癌的轉(zhuǎn)移有關。

多項研究表明，EMT是促進胰腺癌侵襲和轉(zhuǎn)移的重要因素之一[9-11]。EMT是指上皮細胞在特定的生理或病理情況下從上皮細胞特性向間質(zhì)細胞特性轉(zhuǎn)變的現(xiàn)象，表現(xiàn)為細胞黏附性下降或消失，極性喪失，細胞運動和侵襲能力增強[12]。EMT過程伴有一系列分子水平的變化，其中上皮表型標記分子E-cadherin表達下調(diào)是EMT的重要標志之一[13]。

E-cadherin是鈣黏素(cadherin)家族成員之一，為上皮細胞之間及細胞與細胞外基質(zhì)黏附的重要黏附分子。在正常情況下，E-cadherin與連接蛋白β-catenin形成復合物共同維持組織結構的完整性和極性。當E-cadherin的表達下降或者E-cadherin-β-catenin基因突變而致其結構異常時可導致腫瘤細胞間的黏附性下降，細胞間的連接松散，極性喪失，腫瘤細胞易于從原發(fā)灶脫離而向外浸潤性生長乃至遠處轉(zhuǎn)移。有研究表明[14-15]，E-cadherin的表達異常和(或)功能喪失在胰腺癌侵襲轉(zhuǎn)移過程中起著重要作用。本研究結果也顯示，胰腺癌中E-cadherin的異常表達率顯著升高，并與胰腺癌的分化程度、淋巴結轉(zhuǎn)移及TNM分期有關。

本實驗還證實，胰腺癌中RGC-32的陽性表達與E-cadherin的異常表達呈正相關。這提示RGC-32可能是胰腺癌EMT新的標志物。近期的一項研究表明，RGC-32參與TGF-β誘導的近端腎小管上皮細胞EMT過程[16]。結合本實驗的結果，我們推測RGC-32可能通過調(diào)控EMT過程參與胰腺癌的侵襲和轉(zhuǎn)移，其具體作用及機制有待進一步研究。

[1] Badea TC, Niculescu FI, Soane L, et al. Molecular cloning and characterization of RGC-32, a novel gene induced by complement activation in oligodendrocytes. J Biol Chem, 1998, 273: 26977-26981.

[2] Badea T, Niculescu F, Soane L, et al. RGC-32 increases p34CDC2 kinase activity and entry of aortic smooth muscle cells into S-phase. J Biol Chem, 2002, 277: 502-508.

[3] Li F, Luo Z, Huang W, et al. Response gene to complement 32, a novel regulator for transforming growth factor-beta-induced smooth muscle differentiation of neural crest cells. J Biol Chem, 2007, 282: 10133-10137.

[4] Fosbrink M, Cudrici C, Niculescu F, et al. Overexpression of RGC-32 in colon cancer and other tumors. Exp Mol Pathol, 2005, 78: 116-122.

[5] Saigusa K, Imoto I, Tanikawa C, et al. RGC32, a novel p53-inducible gene, is located on centrosomes during mitosis and results in G2/M arrest. Oncogene, 2007, 26: 1110-1121.

[6] Kang Y, Siegel PM, Shu W, et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell, 2003, 3: 537-549.

[7] Chandran UR, Ma C, Dhir R, et al. Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process. BMC Cancer, 2007, 7: 64.

[8] Jawhari A, Jordan S, Poole S, et al. Abnormal immunoreactivity of the E-cadherin-catenin complex in gastric carcinoma: relationship with patient survival. Gastroenterology, 1997, 112: 46-54.

[9] Ellenrieder V, Hendler SF, Boeck W, et al. Transforming growth factor beta1 treatment leads to an epithelial-mesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation. Cancer Res, 2001, 61: 4222-4228.

[10] Zhao S, Venkatasubbarao K, Lazor JW, et al. Inhibition of STAT3 Tyr705 phosphorylation by Smad4 suppresses transforming growth factor beta-mediated invasion and metastasis in pancreatic cancer cells. Cancer Res, 2008, 68: 4221-4228.

[11] Levy L, Hill CS. Smad4 dependency defines two classes of transforming growth factor {beta} (TGF-{beta}) target genes and distinguishes TGF-{beta}-induced epithelial-mesenchymal transition from its antiproliferative and migratory responses. Mol Cell Biol, 2005, 25: 8108-8125.

[12] Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer, 2009, 9:265-273.

[13] Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions in development and disease. Cell, 2009, 139: 871-890.

[14] Von Burstin J, Eser S, Paul MC, et al. E-cadherin regulates metastasis of pancreatic cancer in vivo and is suppressed by a SNAIL/HDAC1/HDAC2 repressor complex. Gastroenterology, 2009, 137: 361-371.

[15] Pryczynicz A, Guzińska-Ustymowicz K, Kemona A, et al. Expression of the E-cadherin-catenin complex in patients with pancreatic ductal adenocarcinoma. Folia Histochem Cytobiol, 2010, 48: 128-133.

[16] Huang WY, Li ZG, Rus H, et al. RGC-32 mediates transforming growth factor-beta-induced epithelial-mesenchymal transition in human renal proximal tubular cells. J Biol Chem, 2009, 284: 9426-9432.

ExpressionsofRGC-32andE-cadherininpancreaticcancerandtheirclinicopathologicalsignificance

ZHULiang,ZHAOHui-zhen,PANGHui-fang,QINHua,LIPei-yuan,LIDe-min,ZHAOQiu.

DepartmentofGastroenterology,TongjiHospital,TongjiMedicalcollege,HuazhongUniversityofScienceandTechnology,Wuhan430030,China

ZHAOQiu,Email:zhaoqiu@medmail.com.cn

ObjectiveTo investigate the expressions of RGC-32 and E-cadherin in pancreatic cancer and analyze their clinicopathological significance and the correlation with each other.MethodsSP immunohistochemistry was used to detect the expressions of RGC-32 and E-cadherin in 42 cases of pancreatic cancer tissues, 12 cases of chronic pancreatitis tissues and 8 cases of normal pancreatic tissues.ResultsThe positive staining for RGC-32 was predominantly observed in the cytoplasm of pancreatic acinar cells. The positive staining for E-cadherin was mainly observed in the cytomembrane of normal pancreatic and chronic pancreatitis acinar cells, but aberrant expression (cytoplasm expression and (or) weaker expression) could be found in pancreatic cancer cells. The positive expression rate of RGC-32 and aberrant expression rate of E-cadherin were 78.6%(33/42) and 54.8%(23/42), respectively, in pancreatic cancer tissues, which were significantly higher than those in normal pancreatic tissues [37.5%(3/8) and 0]and chronic pancreatitis [41.7%(5/12)and8.3%(1/12) with statisticai significance,P<0.05]. The expression of RGC-32 in pancreatic cancer was associated with lymph node metastasis and TNM staging (P=0.016, 0.025, respectively),but not with age, gender and differentiation degree (P=0.831, 1.000, 0.629, respectively). The aberrant expression of E-cadherin was associated with differentiation degree, lymph node metastasis and TNM staging(P=0.024,0.004, 0.004, respectively), but not with age and gender (P=0.970,1.000, respectively). A significantly positive correlation was found between positive expression rate of RGC-32 and aberrant expression rate of E-cadherin (r=0.458,P<0.01).ConclusionsBoth positive expression rate of RGC-32 and aberrant expression rate of E-cadherin are up-regulated significantly in pancreatic cancer tissues and RGC-32 may be involved in the invasion and metastasis of pancreatic cancer by regulating epithelial mesenchymal transition.

Pancreatic neoplasmas; Immunohistochemistry; Response gene to complement-32; Epithelial-cadherin; Epithelial-mesenchymal transition

10.3760/cma.j.issn.1674-1935.2012.03.010

教育部高等學校博士學科點專項科研基金(20110142110013)

430030 武漢，華中科技大學同濟醫(yī)學院附屬同濟醫(yī)院消化內(nèi)科(朱亮、趙慧貞、龐慧芳、覃華、黎培員、李德民、趙秋)；南昌大學第一附屬醫(yī)院消化內(nèi)科(朱亮)

趙秋，Email: zhaoqiu@medmail.com.cn

2011-07-20)

(本文編輯：呂芳萍)