沉默人胰腺癌細胞S100A4基因表達對腫瘤相關(guān)基因mRNA表達的影響

李鵬 劉江偉 韓振魁 朱淑萍 張瓊

·論著·

沉默人胰腺癌細胞S100A4基因表達對腫瘤相關(guān)基因mRNA表達的影響

李鵬 劉江偉 韓振魁 朱淑萍 張瓊

目的觀察沉默S100A4基因表達對人胰腺癌BxPC-3、AsPC-1細胞腫瘤相關(guān)基因COX-2、bcl-2、Surviving、MMP-9 mRNA表達的影響，探討它們之間的關(guān)系。方法應(yīng)用靶向S100A4的小干擾RNA(siRNA)轉(zhuǎn)染人胰腺癌BxPC-3、AsPC-1細胞，應(yīng)用無同源性的siRNA-C轉(zhuǎn)染細胞作為陰性對照，以未轉(zhuǎn)染細胞作為對照組。采用RT-PCR檢測干擾后細胞S100A4、COX-2、Survivin、MMP-9、bcl-2 mRNA的表達。結(jié)果人胰腺癌BxPC-3細胞的對照組、siRNA-C組、siRNA-S100A4組的S100A mRNA表達量分別為0.661±0.023、0.659±0.043、0.379±0.039；COX-2 mRNA表達量分別為0.760±0.026、0.830±0.017、0.443±0.006；Survivin mRNA表達量分別為0.948±0.049、0.909±0.081、0.068±0.006；bcl-2 mRNA表達量分別為0.462±0.018、0.421±0.049、0.184±0.025；MMP-9 mRNA表達量分別為0.813±0.008、0.908±0.063、0.246±0.027。AsPC-1細胞的對照組、siRNA-C組、siRNA-S100A4組的S100A4 mRNA表達量分別為0.641±0.042、0.626±0.053、0.320±0.081；COX-2 mRNA表達量分別為0.727±0.021、0.743±0.025、0.560±0.035；Survivin mRNA表達量分別為0.994±0.032、0.984±0.049、0.063±0.005；bcl-2 mRNA表達量分別為0.458±0.004、0.537±0.046、0.181±0.007；MMP-9 mRNA表達量分別為0.698±0.011、0.718±0.073、0.199±0.013。2株細胞的siRNA-S100A4組的S100A、COX-2、Survivin、bcl-2、MMP-9 mRNA表達量均較其相應(yīng)的siRNA-C組及對照組顯著減少(P值均<0.01)，而siRNA-C組與對照組間的表達量差異無統(tǒng)計學(xué)意義。結(jié)論S100A4通過上調(diào)COX-2、Survivin、bcl-2、MMP-9基因的表達在胰腺癌發(fā)生、發(fā)展中發(fā)揮作用。

胰腺腫瘤； RNA，小分子干擾； 癌基因； S100A4； COX-2； Survivin； MMP-9； bcl-2

胰腺癌發(fā)生、發(fā)展過程是多種基因聯(lián)合作用的結(jié)果。S100A4是S100鈣結(jié)合蛋白家族的成員，參與細胞增殖、凋亡、信號轉(zhuǎn)導(dǎo)、細胞黏附、胞外基質(zhì)重建、細胞運動等多種生命活動。近年來研究發(fā)現(xiàn)S100A4在胰腺癌細胞中過表達，與胰腺癌的發(fā)生、侵襲、轉(zhuǎn)移和預(yù)后密切相關(guān)[1]。COX-2、Survivin、bcl-2、MMP-9在多種腫瘤細胞中均有不同程度的表達，在胰腺癌組織中也有表達。本研究應(yīng)用靶向S100A4的小干擾RNA(siRNA)沉默胰腺癌細胞S100A4的表達，觀察干擾后細胞COX-2、Survivin、bcl-2、MMP-9表達的變化，探討S100A4的作用機制。

材料與方法

一、細胞培養(yǎng)及分組

人胰腺癌細胞株BxPC-3、AsPC-1購自中科院上海細胞庫，常規(guī)培養(yǎng)、傳代。取對數(shù)生長期的細胞，以5×103個密度接種于6孔板，分為對照組、靶向S100A4的siRNA轉(zhuǎn)染組(siRNA-S100A4組)和無同源siRNA轉(zhuǎn)染組(siRNA-C組)。siRNA-S100A4序列上游5′-GUGGACUUCCAAGAGUACUdTdT-3′，下游5′-AGUACUCUUGGAAGUCCACdTdT-3′；siRNA-C上游5′-UUCUCCGAACGUGUCACGUTT-3′，下游5′-ACGUGACACGUUCGGAGAATT-3′，均由Sigma公司設(shè)計并合成。采用DharmaFECT轉(zhuǎn)染試劑(Dharmacon公司)分別將siRNA-S100A4、siRNA-C轉(zhuǎn)染細胞，按說明書操作。未轉(zhuǎn)染的細胞作為對照組。

二、細胞S100A4、COX-2、Survivin、bcl-2、MMP-9 mRNA表達的檢測

收集各組培養(yǎng)48 h的細胞，采用Trizol(Invitrogen公司)抽提細胞總RNA。采用RT-PCR方法檢測S100A4、COX-2、Survivin、bcl-2、MMP-9 mRNA的表達。引物序列：S100A4(320 bp)上游5′-ATCCCGTGCCCTCTGGAGAA-3′，下游5′-TCATTTCTTCCTGGGCTGCT-3′；COX-2(440 bp)上游 5′-TCCAGATCACATTTGATTGACAG-3′，下游5′-TGTGGGAGGATACATCTCTCC-3′；Survivin(439 bp)上游5′-GGCATGGGTGCCCCGACGTT-3′，下游5′-AGAGGCCTCAATCCATGGCA-3′；MMP-9(400 bp)上游5′-CAACATCACCTATTGGATCC-3′，下游5′-CTGTAGAGTCTCTCGCT-3′；bcl-2(318 bp)上游5′-CGACGACTTCTCCCGCCGCTACCGC-3′，下游5′-CCGCATGCTGGGGCCGTACAGTTCC-3′；內(nèi)參β-actin(233 bp)上游5′-GGACTTCGAGCAGGAGATGG-3′，下游5′-GCACCGTGTTGGCGTAGAGG-3′。引物均由上海生工生物技術(shù)有限公司合成。RT-PCR試劑盒購自TaKaRa公司。反轉(zhuǎn)錄體系：5×PrimeScript Buffer 5 μl，PrimeScript Enzyme MixⅠ 1.25 μl，Oligo dT primer 1.25 μl，Random 6 mers 5 μl，RNA 500/檢測濃度，dd H2O(無RNase)加至25 μl。PCR反應(yīng)體系：TaKaRa Ex Taq 0.125 μl，10×Ex Taq Buffer 2.5 μl，Mgcl20.5，d NTP MIX 2 μl，cDNA 1 μl，Primer forward 1 μl，Primer reverse 1 μl，dd H2O加至25 μl。PCR擴增條件：94℃ 5 min，94℃ 30 s、58.1℃ 30 s、72℃ 1 min，35個循環(huán)，72℃ 5 min。擴增產(chǎn)物經(jīng)電泳分離、凝膠成像后Quantity One軟件行灰度掃描。以目的條帶與內(nèi)參條帶灰度值比值表示mRNA相對表達量。實驗重復(fù)3次，取均值。

三、統(tǒng)計學(xué)處理

結(jié) 果

一、siRNA-S100A4轉(zhuǎn)染對人胰腺癌BxPC-3、AsPC-1細胞S100A4 mRNA表達的抑制效率

人胰腺癌BxPC-3細胞對照組、siRNA-C組、siRNA-S100A4組的S100A4 mRNA表達量分別為0.661±0.023、0.659±0.043、0.379±0.039；AsPC-1細胞對照組、siRNA-C組、siRNA-S100A4組的S100A4 mRNA表達量分別為0.641±0.042、0.626±0.053、0.320±0.081。2株細胞siRNA-S100A4組的S100A mRNA表達均較其相應(yīng)的對照組及siRNA-C組的表達顯著減少(t值分別為21.564、30.602、10.403、9.216，P值均<0.01，圖1)，而siRNA-C組與對照組的差異無統(tǒng)計學(xué)意義。

圖1BxPC-3細胞對照組(1)、siRNA-c組(2)、siRNA-S100A4組(3)及AsPC-1細胞對照組(4)、siRNA-c組(5)、siRNA-S100A4組(6)的S100A4 mRNA表達

二、S100A4基因沉默對人胰腺癌細胞COX-2 mRNA表達的影響

人胰腺癌BxPC-3細胞對照組、siRNA-C組、siRNA-S100A4組的COX-2 mRNA表達量分別為0.760±0.026、0.830±0.017、0.443±0.006；AsPC-1細胞對照組、siRNA-C組、siRNA-S100A4組的COX-2 mRNA表達量分別為0.727±0.021、0.743±0.025、0.560±0.035。2株細胞的siRNA-S100A4組的COX mRNA表達均較其相應(yīng)的對照組及siRNA-C組的表達顯著減少(t值分別為20.254、36.682、7.143、7.416，P值均<0.01，圖2)，而siRNA-C組與對照組的差異無統(tǒng)計學(xué)意義。

三、S100A4基因沉默對人胰腺癌細胞Survivin mRNA表達的影響

人胰腺癌BxPC-3細胞對照組、siRNA-C組、siRNA-S100A4組Survivin mRNA的表達量分別為0.948±0.049、0.909±0.081、0.068±0.006；AsPC-1細胞對照組、siRNA-C組、siRNA-S100A4組Survivin mRNA的表達量分別為0.994±0.032、0.984±0.049、0.063±0.005。2株細胞的siRNA-S100A4組的Survinin mRNA表達均較其相應(yīng)的對照組及siRNA-C組的表達顯著減少(t值分別為30.991、17.971、49.848、32.578，P值均<0.01，圖2)，而siRNA-C組與對照組的差異無統(tǒng)計學(xué)意義。

四、S100A4基因沉默對人胰腺癌細胞bcl-2 mRNA表達的影響

人胰腺癌BxPC-3細胞對照組、siRNA-C組、siRNA-S100A4組的bcl-2 mRNA表達量分別為0.462±0.018、0.421±0.049、0.184±0.025；AsPC-1細胞對照組、siRNA-C組、siRNA-S100A4組的bcl-2 mRNA表達量分別為0.458±0.004、0.537±0.046、0.181±0.007。2株細胞的siRNA-S100A4組的bcl-2 mRNA表達均較其相應(yīng)的對照組及siRNA-C組的表達顯著減少(t值分別為15.653、7.457、60.154、13.269，P值均<0.01，圖2)，而siRNA-C組與對照組的差異無統(tǒng)計學(xué)意義。

五、S100A4基因沉默對人胰腺癌細胞MMP-9 mRNA表達的影響

人胰腺癌BxPC-3細胞對照組、siRNA-C組、siRNA-S100A4組的MMP-9 mRNA表達量分別為0.813±0.008、0.908±0.063、0.246±0.027；AsPC-1細胞對照組、siRNA-C組、siRNA-S100A4組的MMP-9 mRNA表達量分別為0.698±0.011、0.718±0.073、0.199±0.013。2株細胞的siRNA-S100A4組的MMP-9 mRNA表達均較其相應(yīng)的對照組及siRNA-C組的表達顯著減少(t值分別為35.859、14.831、49.848、12.018，P值均<0.01，圖2)，而siRNA-C組與對照組的差異無統(tǒng)計學(xué)意義。

圖2BxPC-3細胞對照組(1)、siRNA-c組(2)、siRNA-S100A4組(3)及AsPC-1細胞對照組(4)、siRNA-c組(5)、siRNA-S100A4組(6)的COX-2、Survivin、bcl-2、MMP-9 mRNA表達

討 論

Shi等[2]通過小發(fā)夾RNA沉默人甲狀腺癌細胞株S100A4基因的表達，結(jié)果細胞生長被明顯抑制，細胞阻滯在G2/M期，提示抑制S100A4的表達可能是治療腫瘤的潛在靶點。Hua等[3]運用RNA干擾技術(shù)沉默胃癌BGC823細胞S100A4的表達，結(jié)果細胞的增殖被抑制，細胞凋亡增加。在裸鼠瘤內(nèi)注射靶向S100A4的shRNA可明顯抑制腫瘤細胞增殖、侵襲及轉(zhuǎn)移。近年來研究發(fā)現(xiàn)S100A4在胰腺癌細胞中過表達[1]，與胰腺癌的發(fā)生、侵襲、轉(zhuǎn)移和預(yù)后密切相關(guān)，極有可能成為基因治療的有效靶點。

COX-2在人胰腺癌組織中呈現(xiàn)高表達[4]。研究證實[1]，COX-2與胰腺癌患者預(yù)后有關(guān)，是判斷胰腺癌患者預(yù)后的重要因子。Zhong等[5]應(yīng)用siRNA干擾COX-2基因表達可抑制胰腺癌細胞增殖，增加細胞凋亡。荷瘤裸鼠的體內(nèi)實驗也證實COX-2基因沉默后腫瘤生長受到抑制。Takahashi等[6]研究證明，穩(wěn)定的COX-2表達并伴隨PGE2的增加能促進貼壁細胞的生長。Bergmann等[7]報道，COX-2在胰腺癌中的表達率為93%。

Bcl-2家族是細胞凋亡通路中關(guān)鍵的調(diào)節(jié)者。Wang等[8]報道，抑制bcl-2的表達可抑制細胞增殖，誘導(dǎo)細胞凋亡。在胰腺癌組織中bcl-2及其家族高表達[9]。

Survivin，又名存活素，是凋亡抑制家族的新成員，在很多腫瘤中高表達，包括肺癌、直腸癌、胰腺癌、前列腺癌及乳腺癌。Satoh等[10]報道，胰腺導(dǎo)管癌Survivin表達率為76.9%，Survivin的表達增加伴隨細胞凋亡指數(shù)的下降。Sarela等[11]報道，胰腺癌Survivin高表達與細胞增殖和凋亡相關(guān)。Yang等[12]認為 Survivin表達的增加與胰腺導(dǎo)管癌進展有關(guān)。我們前期的臨床研究證實，胰腺癌Survivin的表達與臨床分期及淋巴結(jié)轉(zhuǎn)移相關(guān)。

MMP-9在很多腫瘤中表達上調(diào)，如食管癌、乳腺癌、胃癌、胰腺癌。Giannopoulos等[13]報道，胰腺癌中MMP-2、MMP-9均高表達。Im等[14]應(yīng)用MMPs抑制劑抑制MMP-2、MMP-9的表達，可減少肺癌細胞的轉(zhuǎn)移。Shin等[15]報道，降低MMP-9的活性可抑制前列腺癌細胞發(fā)生遠處轉(zhuǎn)移，而打破MMPs/TIMPs的平衡可激活MMP-9，促使腫瘤細胞發(fā)生浸潤和轉(zhuǎn)移。

本研究結(jié)果顯示，S100A4基因表達被抑制后，胰腺癌BxPC-3、AsPC-1細胞COX-2、bcl-2、Survivin、MMP-9的表達均下調(diào)，提示COX-2、bcl-2、Survivin、MMP-9均可能是S100A4的下游基因，但其具體機制仍需要進一步探究。

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EffectofS100A4silencingontumorrelatedgenemRNAexpression

LIPeng,LIUJiang-wei,HANZhen-kui,ZHUShu-ping,ZHANGQiong.

DepartmentofGastrointestinalSurgery,People′sHospitalofXinjiangUygurAutonomousRegion,Urumqi830001,China

Correspondingauthor:LIUJiang-wei,Email:ljw273@sohu.com

ObjectiveTo investigate the effect of S100A4 silencing on tumor related gene COX-2, bcl-2, Surviving, MMP-9 mRNA expressions of pancreatic cancer BxPC-3, AsPC-1 cells, and explore their relationship.MethodsSmall interfering RNA interfering S100A4 gene (siRNA-S100A4) was applied to transfect human pancreatic cancer BxPC-3, AsPC-1 cells, and nonhomologous siRNA-C was used as negative control, and cells without transfection were used as control group. The expressions of S100A4, COX-2, Survivin, MMP-9, bcl-2 mRNA after interference were detected by using RT-PCR.ResultsS100A mRNA expressions of BxPC-3′s control group, siRNA-C group, siRNA-S100A4 group were 0.661±0.023, 0.659±0.043, 0.379±0.039, and expressions of COX-2 mRNA were 0.760±0.026, 0.830±0.017, 0.443±0.006, and expressions of Survivin mRNA were 0.948±0.049, 0.909±0.081, 0.068±0.006, and expressions of bcl-2 mRNA were 0.462±0.018, 0.421±0.049, 0.184±0.025, and expressions of MMP-9 mRNA were 0.813±0.008, 0.908±0.063, 0.246±0.027. S100A mRNA expressions of AsPC-1′s control group, siRNA-C group, siRNA-S100A4 group were 0.641±0.042, 0.626±0.053, 0.320±0.081, and expressions of COX-2 mRNA were 0.727±0.021, 0.743±0.025, 0.560±0.035, and expressions of Survivin mRNA were 0.994±0.032, 0.984±0.049, 0.063±0.005, and expressions of bcl-2 mRNA were 0.458±0.004, 0.537±0.046, 0.181±0.007; and expressions of MMP-9 mRNA were 0.698±0.011, 0.718±0.073, 0.199±0.013. The expressions of S100A, COX-2, Survivin, bcl-2, MMP-9 mRNA in groups with siRNA-S100A4 transfection were significantly lower than those of siRNA-C group and control group (P<0.01), but the difference between siRNA-C group and control group was not statistically significant.ConclusionsS100A4 plays a role in the pathogenesis of pancreatic cancer through up-regulation of COX-2, Survivin, bcl-2, MMP-9 expressions.

Pancreatic neoplasms; RNA, small interfering; Oncogenes; S100A4; COX-2; Survivin; MMP-9; bcl-2

2013-03-06)

(本文編輯：屠振興)

10.3760/cma.j.issn.1674-1935.2013.04.006

中國博士后基金面上資助(20100481517)

830001 新疆烏魯木齊，新疆維吾爾自治區(qū)人民醫(yī)院胃腸外科(李鵬、韓振魁)；蘭州軍區(qū)烏魯木齊總醫(yī)院肝膽外科(劉江偉)，美容科(朱淑萍)，院長辦公室(張瓊)

劉江偉，Email: ljw273@sohu.com