李鵬 劉江偉 韓振魁 朱淑萍 張瓊

·論著·

沉默人胰腺癌細(xì)胞S100A4基因表達(dá)對(duì)腫瘤相關(guān)基因mRNA表達(dá)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(dá)對(duì)人胰腺癌BxPC-3、AsPC-1細(xì)胞腫瘤相關(guān)基因COX-2、bcl-2、Surviving、MMP-9 mRNA表達(dá)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(xì)胞，應(yīng)用無(wú)同源性的siRNA-C轉(zhuǎn)染細(xì)胞作為陰性對(duì)照，以未轉(zhuǎn)染細(xì)胞作為對(duì)照組。采用RT-PCR檢測(cè)干擾后細(xì)胞S100A4、COX-2、Survivin、MMP-9、bcl-2 mRNA的表達(dá)。結(jié)果人胰腺癌BxPC-3細(xì)胞的對(duì)照組、siRNA-C組、siRNA-S100A4組的S100A mRNA表達(dá)量分別為0.661±0.023、0.659±0.043、0.379±0.039；COX-2 mRNA表達(dá)量分別為0.760±0.026、0.830±0.017、0.443±0.006；Survivin mRNA表達(dá)量分別為0.948±0.049、0.909±0.081、0.068±0.006；bcl-2 mRNA表達(dá)量分別為0.462±0.018、0.421±0.049、0.184±0.025；MMP-9 mRNA表達(dá)量分別為0.813±0.008、0.908±0.063、0.246±0.027。AsPC-1細(xì)胞的對(duì)照組、siRNA-C組、siRNA-S100A4組的S100A4 mRNA表達(dá)量分別為0.641±0.042、0.626±0.053、0.320±0.081；COX-2 mRNA表達(dá)量分別為0.727±0.021、0.743±0.025、0.560±0.035；Survivin mRNA表達(dá)量分別為0.994±0.032、0.984±0.049、0.063±0.005；bcl-2 mRNA表達(dá)量分別為0.458±0.004、0.537±0.046、0.181±0.007；MMP-9 mRNA表達(dá)量分別為0.698±0.011、0.718±0.073、0.199±0.013。2株細(xì)胞的siRNA-S100A4組的S100A、COX-2、Survivin、bcl-2、MMP-9 mRNA表達(dá)量均較其相應(yīng)的siRNA-C組及對(duì)照組顯著減少(P值均<0.01)，而siRNA-C組與對(duì)照組間的表達(dá)量差異無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論S100A4通過(guò)上調(diào)COX-2、Survivin、bcl-2、MMP-9基因的表達(dá)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(guò)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(xì)胞增殖、凋亡、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞黏附、胞外基質(zhì)重建、細(xì)胞運(yùn)動(dòng)等多種生命活動(dòng)。近年來(lái)研究發(fā)現(xiàn)S100A4在胰腺癌細(xì)胞中過(guò)表達(dá)，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(xì)胞中均有不同程度的表達(dá)，在胰腺癌組織中也有表達(dá)。本研究應(yīng)用靶向S100A4的小干擾RNA(siRNA)沉默胰腺癌細(xì)胞S100A4的表達(dá)，觀察干擾后細(xì)胞COX-2、Survivin、bcl-2、MMP-9表達(dá)的變化，探討S100A4的作用機(jī)制。

材料與方法

一、細(xì)胞培養(yǎng)及分組

人胰腺癌細(xì)胞株BxPC-3、AsPC-1購(gòu)自中科院上海細(xì)胞庫(kù)，常規(guī)培養(yǎng)、傳代。取對(duì)數(shù)生長(zhǎng)期的細(xì)胞，以5×103個(gè)密度接種于6孔板，分為對(duì)照組、靶向S100A4的siRNA轉(zhuǎn)染組(siRNA-S100A4組)和無(wú)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(jì)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(xì)胞，按說(shuō)明書操作。未轉(zhuǎn)染的細(xì)胞作為對(duì)照組。

二、細(xì)胞S100A4、COX-2、Survivin、bcl-2、MMP-9 mRNA表達(dá)的檢測(cè)

收集各組培養(yǎng)48 h的細(xì)胞，采用Trizol(Invitrogen公司)抽提細(xì)胞總RNA。采用RT-PCR方法檢測(cè)S100A4、COX-2、Survivin、bcl-2、MMP-9 mRNA的表達(dá)。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(gòu)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(cè)濃度，dd H2O(無(wú)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(kuò)增條件：94℃ 5 min，94℃ 30 s、58.1℃ 30 s、72℃ 1 min，35個(gè)循環(huán)，72℃ 5 min。擴(kuò)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(duì)表達(dá)量。實(shí)驗(yàn)重復(fù)3次，取均值。

三、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、siRNA-S100A4轉(zhuǎn)染對(duì)人胰腺癌BxPC-3、AsPC-1細(xì)胞S100A4 mRNA表達(dá)的抑制效率

人胰腺癌BxPC-3細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組的S100A4 mRNA表達(dá)量分別為0.661±0.023、0.659±0.043、0.379±0.039；AsPC-1細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組的S100A4 mRNA表達(dá)量分別為0.641±0.042、0.626±0.053、0.320±0.081。2株細(xì)胞siRNA-S100A4組的S100A mRNA表達(dá)均較其相應(yīng)的對(duì)照組及siRNA-C組的表達(dá)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(duì)照組的差異無(wú)統(tǒng)計(jì)學(xué)意義。

圖1BxPC-3細(xì)胞對(duì)照組(1)、siRNA-c組(2)、siRNA-S100A4組(3)及AsPC-1細(xì)胞對(duì)照組(4)、siRNA-c組(5)、siRNA-S100A4組(6)的S100A4 mRNA表達(dá)

二、S100A4基因沉默對(duì)人胰腺癌細(xì)胞COX-2 mRNA表達(dá)的影響

人胰腺癌BxPC-3細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組的COX-2 mRNA表達(dá)量分別為0.760±0.026、0.830±0.017、0.443±0.006；AsPC-1細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組的COX-2 mRNA表達(dá)量分別為0.727±0.021、0.743±0.025、0.560±0.035。2株細(xì)胞的siRNA-S100A4組的COX mRNA表達(dá)均較其相應(yīng)的對(duì)照組及siRNA-C組的表達(dá)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(duì)照組的差異無(wú)統(tǒng)計(jì)學(xué)意義。

三、S100A4基因沉默對(duì)人胰腺癌細(xì)胞Survivin mRNA表達(dá)的影響

人胰腺癌BxPC-3細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組Survivin mRNA的表達(dá)量分別為0.948±0.049、0.909±0.081、0.068±0.006；AsPC-1細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組Survivin mRNA的表達(dá)量分別為0.994±0.032、0.984±0.049、0.063±0.005。2株細(xì)胞的siRNA-S100A4組的Survinin mRNA表達(dá)均較其相應(yīng)的對(duì)照組及siRNA-C組的表達(dá)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(duì)照組的差異無(wú)統(tǒng)計(jì)學(xué)意義。

四、S100A4基因沉默對(duì)人胰腺癌細(xì)胞bcl-2 mRNA表達(dá)的影響

人胰腺癌BxPC-3細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組的bcl-2 mRNA表達(dá)量分別為0.462±0.018、0.421±0.049、0.184±0.025；AsPC-1細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組的bcl-2 mRNA表達(dá)量分別為0.458±0.004、0.537±0.046、0.181±0.007。2株細(xì)胞的siRNA-S100A4組的bcl-2 mRNA表達(dá)均較其相應(yīng)的對(duì)照組及siRNA-C組的表達(dá)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(duì)照組的差異無(wú)統(tǒng)計(jì)學(xué)意義。

五、S100A4基因沉默對(duì)人胰腺癌細(xì)胞MMP-9 mRNA表達(dá)的影響

人胰腺癌BxPC-3細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組的MMP-9 mRNA表達(dá)量分別為0.813±0.008、0.908±0.063、0.246±0.027；AsPC-1細(xì)胞對(duì)照組、siRNA-C組、siRNA-S100A4組的MMP-9 mRNA表達(dá)量分別為0.698±0.011、0.718±0.073、0.199±0.013。2株細(xì)胞的siRNA-S100A4組的MMP-9 mRNA表達(dá)均較其相應(yīng)的對(duì)照組及siRNA-C組的表達(dá)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(duì)照組的差異無(wú)統(tǒng)計(jì)學(xué)意義。

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

討 論

Shi等[2]通過(guò)小發(fā)夾RNA沉默人甲狀腺癌細(xì)胞株S100A4基因的表達(dá)，結(jié)果細(xì)胞生長(zhǎng)被明顯抑制，細(xì)胞阻滯在G2/M期，提示抑制S100A4的表達(dá)可能是治療腫瘤的潛在靶點(diǎn)。Hua等[3]運(yùn)用RNA干擾技術(shù)沉默胃癌BGC823細(xì)胞S100A4的表達(dá)，結(jié)果細(xì)胞的增殖被抑制，細(xì)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(xì)胞增殖、侵襲及轉(zhuǎn)移。近年來(lái)研究發(fā)現(xiàn)S100A4在胰腺癌細(xì)胞中過(guò)表達(dá)[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(diǎn)。

COX-2在人胰腺癌組織中呈現(xiàn)高表達(dá)[4]。研究證實(shí)[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(dá)可抑制胰腺癌細(xì)胞增殖，增加細(xì)胞凋亡。荷瘤裸鼠的體內(nèi)實(shí)驗(yàn)也證實(shí)COX-2基因沉默后腫瘤生長(zhǎng)受到抑制。Takahashi等[6]研究證明，穩(wěn)定的COX-2表達(dá)并伴隨PGE2的增加能促進(jìn)貼壁細(xì)胞的生長(zhǎng)。Bergmann等[7]報(bào)道，COX-2在胰腺癌中的表達(dá)率為93%。

Bcl-2家族是細(xì)胞凋亡通路中關(guān)鍵的調(diào)節(jié)者。Wang等[8]報(bào)道，抑制bcl-2的表達(dá)可抑制細(xì)胞增殖，誘導(dǎo)細(xì)胞凋亡。在胰腺癌組織中bcl-2及其家族高表達(dá)[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(dá)，包括肺癌、直腸癌、胰腺癌、前列腺癌及乳腺癌。Satoh等[10]報(bào)道，胰腺導(dǎo)管癌Survivin表達(dá)率為76.9%，Survivin的表達(dá)增加伴隨細(xì)胞凋亡指數(shù)的下降。Sarela等[11]報(bào)道，胰腺癌Survivin高表達(dá)與細(xì)胞增殖和凋亡相關(guān)。Yang等[12]認(rèn)為 Survivin表達(dá)的增加與胰腺導(dǎo)管癌進(jìn)展有關(guān)。我們前期的臨床研究證實(shí)，胰腺癌Survivin的表達(dá)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(dá)上調(diào)，如食管癌、乳腺癌、胃癌、胰腺癌。Giannopoulos等[13]報(bào)道，胰腺癌中MMP-2、MMP-9均高表達(dá)。Im等[14]應(yīng)用MMPs抑制劑抑制MMP-2、MMP-9的表達(dá)，可減少肺癌細(xì)胞的轉(zhuǎn)移。Shin等[15]報(bào)道，降低MMP-9的活性可抑制前列腺癌細(xì)胞發(fā)生遠(yuǎn)處轉(zhuǎn)移，而打破MMPs/TIMPs的平衡可激活MMP-9，促使腫瘤細(xì)胞發(fā)生浸潤(rùn)和轉(zhuǎn)移。

本研究結(jié)果顯示，S100A4基因表達(dá)被抑制后，胰腺癌BxPC-3、AsPC-1細(xì)胞COX-2、bcl-2、Survivin、MMP-9的表達(dá)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(jī)制仍需要進(jìn)一步探究。

[1] 賈福鑫，劉江偉，張東，等.胰腺癌組織S100A4和MMP-9表達(dá)及其預(yù)后意義的探討.中華腫瘤防治雜志，2011,18：1105-1109.

[2] Shi Y, Zou M, Collison K, et al. Ribonucleic acid interference targeting S100A4 (Mts1) suppresses tumor growth and metastasis of anaplastic thyroid carcinoma in a mouse model. J Clin Endocrinol Metab, 2006,91:2373-2379.

[3] Hua J, Chen D, Fu H, et al. Short hairpin RNA-mediated inhibition of S100A4 promotes apoptosis and suppresses proliferation of BGC823 gastric cancer cells in vitro and in vivo. Cancer Lett, 2010,292:41-47.

[4] Kokawa A, Kondo H, Gotoda T, et al. Increased expression of cyclooxygenase-2 in human pancreatic neoplasms and potential for chemoprevention by inhibitors. Cancer, 2001,91, 333-338.

[5] Zhong Y, Xia Z, Liu J, et al. The effects of cyclooxygenase-2 gene silencing by siRNA on cell proliferation, cell apoptosis, cell cycle and tumorigenicity of Capan-2 human pancreatic cancer cells. Oncol Rep, 2012, 27:1003-1010.

[6] Takahashi H, Li A, Dawson DW, et al. Cyclooxygenase-2 confers growth advantage to syngeneic pancreatic cancer cells. Pancreas, 2011,40:453-459.

[7] Bergmann F, Moldenhauer G, Herpel E, et al. Expression of L1CAM, COX-2, EGFR, c-KIT and Her2/neu in anaplastic pancreatic cancer: putative therapeutic targets? Histopathology, 2010,56:440-448.

[8] Wang Z, Azmi AS, Ahmad A, et al. TW-37, a Small-Molecule Inhibitor of Bcl-2, Inhibits Cell Growth and Induces Apoptosis in Pancreatic Cancer: Involvement of Notch-1 Signaling Pathway. Cancer Res,2009,69:2757-2765.

[9] Mortenson MM, Galante JG, Gilad O, et al. BCL-2 functions as an activator of the AKT signaling pathway in pancreatic cancer. J Cell Biochem, 2007,102:1171-1179.

[10] Satoh K, Kaneko K, Hirota M, et al. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer, 2001, 92: 271-278.

[11] Sarela AI, Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J Cancer, 2002, 86: 886-892.

[12] Yang L, Cao Z, Lin Y, et al. Molecular beacon imaging of tumor marker gene expression in pancreatic cancer cells. Cancer Biol Ther, 2005, 4: 561-570.

[13] Giannopoulos G, Pavlakis K, Parasi A, et al. The expression of matrix metallo-proteinases-2 and-9 and their tissue inhibitor 2 in pancreatic ductal and ampullary carcinoma and their relation to angiogenesis and clinicopathological parameters. Anticancer Research, 2008,28: 1875-1882.

[14] Im I, Park KR, Kim SM. The butanol fraction of guava (Psidium cattleianum Sabine) leaf extract suppresses MMP-2 and MMP-9 expression and activity through the suppression of the ERK1/2 MAPK signaling pathway. Nutr Cancer, 2012,64:255-266.

[15] Shin YJ, Kim JH. The role of EZH2 in the regulation of the activity of matrix metalloproteinases in prostate cancer cells. PLoS One, 2012,7: e30393.

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(guó)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(zhǎng)辦公室(張瓊)

劉江偉，Email: ljw273@sohu.com