杜冀暉 張厚德 高燕 謝宏民

·論著·

Dysadherin基因沉默對(duì)胰腺癌細(xì)胞侵襲移行能力的影響

杜冀暉 張厚德 高燕 謝宏民

目的探討靶向封閉dysadherin基因?qū)σ认侔┘?xì)胞PANC1、BxPC3體外侵襲移行能力的影響。方法應(yīng)用脂質(zhì)體轉(zhuǎn)染方法將小分子RNA(siRNA)轉(zhuǎn)染細(xì)胞。實(shí)驗(yàn)分為dysadherin-siRNA轉(zhuǎn)染(dysa)組、陰性對(duì)照siRNA轉(zhuǎn)染(HK)組、脂質(zhì)體對(duì)照(對(duì)照)組、。采用RT-PCR、免疫組化方法檢測(cè)轉(zhuǎn)染細(xì)胞的dysadherin mRNA及蛋白表達(dá)；采用Transwell侵襲小室檢測(cè)轉(zhuǎn)染細(xì)胞體外侵襲移行能力。結(jié)果轉(zhuǎn)染dysadherin-siRNA(5 nmol/L)的PANC1和BxPC3細(xì)胞的dysadherin mRNA表達(dá)較HK組細(xì)胞分別下降95.4%、52.1%(P<0.05)；dysadherin蛋白表達(dá)亦分別降低91.2%、83.6%(P<0.01)。PANC1細(xì)胞的對(duì)照組、HK組和dysa組穿膜細(xì)胞數(shù)分別為163.2±15.5、154.4±17.3和53.6±7.9；BxPC3細(xì)胞的對(duì)照組、HK組和dysa組穿膜細(xì)胞數(shù)分別為30.7±3.2、27.5±2.8和4.7±2.4。dysa組顯著低于HK組和對(duì)照組(P值均<0.01)。結(jié)論應(yīng)用RNA干擾技術(shù)沉默人胰腺癌細(xì)胞株P(guān)ANC1、BxPC3的dysadherin基因可使細(xì)胞的侵襲移行能力下降。

胰腺腫瘤； RNA干擾； 癌，移行細(xì)胞； Dysadherin

近年來的研究表明，癌相關(guān)細(xì)胞膜糖蛋白dysadherin在腫瘤細(xì)胞的過表達(dá)能降低細(xì)胞間黏附、促進(jìn)細(xì)胞運(yùn)動(dòng)和轉(zhuǎn)移[1-2]。本研究應(yīng)用RNA干擾技術(shù)沉默人胰腺癌細(xì)胞株P(guān)ANC1、BxPC3的dysadherin基因，探討其對(duì)胰腺癌細(xì)胞侵襲移行能力的影響，為臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。

材料與方法

一、實(shí)驗(yàn)分組

胰腺癌細(xì)胞株P(guān)ANC1、BxPC3 購自中國(guó)科學(xué)院上海生科院細(xì)胞資源中心，常規(guī)培養(yǎng)傳代后收集對(duì)數(shù)生長(zhǎng)期PANC1和BxPC細(xì)胞，分別接種于6孔板，每孔(0.8～3.0)×105個(gè)細(xì)胞，分為脂質(zhì)體對(duì)照組、陰性對(duì)照siRNA轉(zhuǎn)染(HK)組和dysadherin-siRNA轉(zhuǎn)染(dysa)組。當(dāng)培養(yǎng)細(xì)胞達(dá)到50%～80%融合時(shí)，HK組和dysa組分別應(yīng)用脂質(zhì)體轉(zhuǎn)染HK siRNA和dysadherin 141 siRNA，dysadherin 141 siRNA(序列為GCGCCTGCACCCCGGAGCG)及通用陰性對(duì)照HK siRNA(序列為GACTTCATAAGGCGCATGC)由武漢晶賽生物工程技術(shù)有限公司提供。對(duì)照組僅加脂質(zhì)體。每組設(shè)3個(gè)復(fù)孔。轉(zhuǎn)染后繼續(xù)培養(yǎng)24 h，收集各組細(xì)胞。

二、RT-PCR 檢測(cè)細(xì)胞dysadherin mRNA表達(dá)

采用Trizol試劑抽提各組細(xì)胞總RNA，常規(guī)進(jìn)行RT-PCR。dysadherin正義引物為5′-CCTGTGTCTTCTCACCATCG-3′,反義引物為5′-AGGAGGTTGTCAGCTCCTGT-3′，擴(kuò)增產(chǎn)物為551 bp；內(nèi)參GAPDH正義引物為5′-AACGTGTCAGTGGTGGACCT-3′，反義引物為5′-AGGGGAGATTCAGTGTGGTG-3′, 擴(kuò)增產(chǎn)物為400 bp。PCR擴(kuò)增條件：94℃ 3 min，94℃ 45 s、54℃ 45 s、72℃ 1 min,30個(gè)循環(huán)，最后72℃ 10 min。產(chǎn)物經(jīng)瓊脂糖凝膠電泳分離，采用凝膠成像系統(tǒng)(法國(guó)Vilber Lourma Bio-vision1000)測(cè)定條帶灰度。以目的條帶與GAPDH灰度值之比作為dysadherin mRNA相對(duì)表達(dá)量，表達(dá)抑制率=(1- 轉(zhuǎn)染組表達(dá)量/ 對(duì)照組表達(dá)量)×100%。

三、免疫組化檢測(cè)細(xì)胞dysadherin蛋白表達(dá)

制備細(xì)胞爬片，同上述方法轉(zhuǎn)染細(xì)胞，繼續(xù)培養(yǎng)24 h后，進(jìn)行免疫組化檢測(cè)。由病理科醫(yī)師在高倍鏡下隨機(jī)觀察5個(gè)視野，計(jì)數(shù)細(xì)胞并對(duì)每個(gè)細(xì)胞進(jìn)行評(píng)分。細(xì)胞不著色為0分，淡黃色為1分，棕黃色為2分，深棕黃色為3分。將評(píng)分之和除以細(xì)胞數(shù)作為dysadherin蛋白表達(dá)強(qiáng)度，表達(dá)抑制率= (1-轉(zhuǎn)染組表達(dá)強(qiáng)度/對(duì)照組表達(dá)強(qiáng)度) ×100%。

四、Transwell轉(zhuǎn)移小室測(cè)定細(xì)胞侵襲移行能力

將Transwell小室放入24孔培養(yǎng)板中，上室加300 μl預(yù)溫的無血清培養(yǎng)基，室溫下靜置1 h使基質(zhì)膠再水化，吸去剩余培養(yǎng)液，分別接種300 μl用無血清培養(yǎng)基制備的各組細(xì)胞懸液[(0.5～1.0)×106/ml]，下室加500 μl含有20%胎牛血清的培養(yǎng)基，常規(guī)培養(yǎng)24 h后用棉頭拭子擦去基質(zhì)和聚碳酸酯膜上方的細(xì)胞，加入500 μl結(jié)晶紫染液浸泡小室20 min，用蒸餾水洗去多余染液，晾干。在顯微鏡下隨機(jī)觀察10個(gè)視野，計(jì)數(shù)每個(gè)視野內(nèi)穿過膜的細(xì)胞數(shù)，表示腫瘤細(xì)胞的侵襲移行能力。

五、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、腫瘤細(xì)胞dysadherin mRNA及蛋白的表達(dá)

PANC1的dysa組和HK組dysadherin mRNA表達(dá)量分別為8.5和183，BxPC分別為102和213，dysa組兩株細(xì)胞dysadherin mRNA表達(dá)抑制率分別為95.4%、52.1%(P<0.05)。兩細(xì)胞對(duì)照組的dysadherin mRNA表達(dá)量分別為187和213，與HK組無顯著性差異(圖1)。

PANC1和BxPC3的dysa組的dysadherin蛋白表達(dá)均較HK組明顯降低，蛋白表達(dá)抑制率分別為91.2%、83.6%(P<0.01)；HK組與對(duì)照組比較，其抑制率僅為1.8%、0.7% (圖2)。

二、轉(zhuǎn)染細(xì)胞體外侵襲移行能力的變化

dysa組穿過膜的PANC1、BxPC3細(xì)胞明顯減少(圖3)。PANC1的對(duì)照組、HK組和dysa組穿膜細(xì)胞數(shù)分別為163.2±15.5、154.4±17.3和53.6±7.9；BxPC3分別為30.7±3.2、27.5±2.8和4.7±2.4。dysa組顯著低于HK組和對(duì)照組(P值均<0.01)。

l.DNA marker；2.對(duì)照組；3.dysa組；4.HK組

圖2各組細(xì)胞dysadherin蛋白表達(dá)的變化(免疫組化 ×400)

圖3 兩組細(xì)胞的穿膜細(xì)胞量( ×100)

討 論

Dysadherin是位于細(xì)胞膜的一種糖蛋白，是一種腫瘤相關(guān)性抗原[3]。近年研究發(fā)現(xiàn)，dysadherin可能在癌細(xì)胞的浸潤(rùn)及轉(zhuǎn)移方面發(fā)揮重要作用。Dysadherin主要表達(dá)于包括胰腺癌在內(nèi)的各種不同類型的腫瘤細(xì)胞，僅在少數(shù)幾種正常人體細(xì)胞表達(dá)[4-6]。Shimamara等[4]報(bào)道，dysadherin的表達(dá)與胰腺癌的遠(yuǎn)處轉(zhuǎn)移及不良預(yù)后明顯相關(guān)，且表達(dá)dysadherin的胰腺癌細(xì)胞在癌巢中所占比例與胰腺癌的組織病理分級(jí)明顯相關(guān)。Ino等[1]報(bào)道，dysadherin在人胰腺癌細(xì)胞株Capan-1和HPAF-Ⅱ低表達(dá)，在PANC1和Miapaca-2中等量表達(dá)，在Mpanc-96和BxPC3高表達(dá)。將轉(zhuǎn)染dysadherin基因的Capan-1細(xì)胞種植于裸鼠胰腺，35 d后裸鼠肝轉(zhuǎn)移明顯較對(duì)照組增多。

PANC1、BxPC3細(xì)胞dysadherin基因沉默后，通過Transwell侵襲小室的細(xì)胞數(shù)明顯減少，提示下調(diào)dysadherin的表達(dá)能明顯抑制胰腺癌細(xì)胞的侵襲能力，表明dysadherin 表達(dá)可能與胰腺癌細(xì)胞的侵襲能力有一定的相關(guān)性，與Ino等[1]的研究相似。

腫瘤發(fā)生侵襲轉(zhuǎn)移是多因素作用的結(jié)果，包括促進(jìn)細(xì)胞運(yùn)動(dòng)及侵襲細(xì)胞外基質(zhì)。研究表明，dysadherin可通過抑制E-cadherin介導(dǎo)的細(xì)胞間黏附等多種機(jī)制降低細(xì)胞間黏附、促進(jìn)細(xì)胞運(yùn)動(dòng)和轉(zhuǎn)移及腫瘤進(jìn)展[1,4,7]。此外，dysadherin陽性細(xì)胞分泌趨化因子配體CCL2也可能是dysadherin促進(jìn)腫瘤發(fā)生、發(fā)展和轉(zhuǎn)移的機(jī)制之一[8]。因此，有關(guān)以dysadherin為靶點(diǎn)的基因治療在胰腺癌中的應(yīng)用及其機(jī)制尚需進(jìn)一步地深入研究。

[1] Ino Y,Gotoh M,Sakamoto M,et al.Dysadherin,a cancer-associated cell membrane glycoprotein,down-regulates E-cadherin and promotes metastasis.Proc Natl Acad Sci USA，2002，99:365-370.

[2] Shimamura T,Yasuda J,Ino Y,et al.Dysadherin expression facilitates cell motility and metastatic potential of human pancreatic cancer cells.Cancer Res，2004，64:6989-6995.

[3] Tsuiji H,Takasaki S,Sakamoto M,et al.Aberrant O-glycosylation inhibits stable expression of dysadherin,a carcinoma-associated antigen,and facilitates cell-cell adhesion.Glycobiology,2003,13:521-527.

[4] Shimamura T,Sakamoto M,Ino Y,et al.Dysadherin overexpression in pancreatic ductal adenocarcinoma reflects tumor aggressiveness: relationship to e-cadherin expression.J Clin Oncol,2003,21:659-667.

[5] Shimada Y,Yamasaki S,Hashimoto Y,et al.Clinical significance of dysadherin expression in gastric cancer patients.Clin Cancer Res,2004,10:2818-2823.

[6] Kyzas PA,Stefanou D,Batistatou A,et al.Dysadherin expression in head and neck squamous cell carcinoma:association with lymphangiogenesis and prognostic significance.Am J Surg Pathol,2006,30:185-193.

[7] Sato H,Ino Y,Miura A,et al.Dysadherin: expression and clinical significance in thyroid carcinoma.J Clin Endocrinol Metab,2003,88:4407-4412.

[8] Nam JS,Kang MJ,Suchar AM,et al.Chemokine (C-C motif) ligand 2 mediates the prometastatic effect of dysadherin in human breast cancer cells.Cancer Res,2006,66:7176-7184.

2009-05-08)

(本文編輯：屠振興)

EffectofdysadheringenesilencingmediatedbyRNAinterferenceonmetastasisandinvasionofpancreaticcancercells

DUJi-hui,ZHANGHou-de,GAOYan,XIEHong-min.

CentralLaboratory,NanshanHospital,GuangdongMedicalCollege,Shenzhen518052,China

ZHANGHou-de,Email:szkjk@126.com

ObjectiveTo investigate the effect of dysadherin gene silencing on metastasis and invasion in pancreatic cancer cell line PANC1, BxPC3 in vitro.MethodsPANC1 and BxPC3 cells were divided into dysa group, negative siRNA control group (HK), liposomes control group(control). dysa group and HK group were tranfected by dysadherin siRNA and Negative siRNA, respectively.The expression of dysadherin mRNA and protein were detected by RT-PCR and immunohistochemical method. Transwell test was used to evaluate the invasion ability of pancreatic cancer cells.ResultsAfter transfected by dysadherin siRNA, the dysadherin mRNA levels in PANC1 and Bxpc3 cells were decreased by 95.4% and 52.1%. The expression of dysadherin protein was also down-regulated by 91.2% and 83.6%, respectively, when compared with HK groups (P<0.05). The numbers of invasive cells migrated in Transwell in PANC1 cells control group, HK group and dysa group were 163.2±15.5, 154.4±17.3 and 53.6±7.9; the numbers of invasive cells in BxPC cells control group, HK group and dysa group were 30.7±3.2, 27.5±2.8 and 4.7±2.4, respectively. The numbers in dysa group were significantly lower than those of HK group and control group (P<0.01).ConclusionsSilencing the dysadherin gene of PANC1, BxPC3 by RNA interference could significantly inhibit the invasive and migratory ability of canceroas cells.

Pancreatic neoplasms; RNA interference; Carcinoma,transitional cell; Dysadherin

10.3760/cma.j.issn.1674-1935.2010.01.009

深圳市科技計(jì)劃資助項(xiàng)目(JH200505260369A)

518052 深圳，廣東醫(yī)學(xué)院附屬南山醫(yī)院中心實(shí)驗(yàn)室(杜冀暉)，消化內(nèi)科(張厚德、高燕、謝宏民)

張厚德,Email: szkjk @126.com