應(yīng)用RNA干擾技術(shù)下調(diào)ANXA3表達(dá)的MDA-MB-231乳腺癌細(xì)胞株的建立

周濤　楊麗　劉亮　巨英超

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·論著·

應(yīng)用RNA干擾技術(shù)下調(diào)ANXA3表達(dá)的MDA-MB-231乳腺癌細(xì)胞株的建立

周濤楊麗劉亮巨英超

目的建立針對(duì)膜聯(lián)蛋白A3(Annexin A3, ANXA3)的shRNA穩(wěn)定轉(zhuǎn)染的乳腺癌細(xì)胞株MDA-MB-231，為后續(xù)進(jìn)一步研究奠定基礎(chǔ)。方法熒光定量RT-PCR及western blot方法檢測(cè)兩種乳腺癌細(xì)胞株(MDA-MB-231及MCF-7)中ANXA3 mRNA及蛋白表達(dá)水平。構(gòu)建沉默ANXA3基因的shRNA質(zhì)粒3個(gè)(ANXA3-sh1-3)及陰性對(duì)照質(zhì)粒，脂質(zhì)體法轉(zhuǎn)染人乳腺癌細(xì)胞MDA-MB-231；選出ANXA3沉默效果最好的干擾質(zhì)粒做后續(xù)實(shí)驗(yàn)。嘌呤霉素篩選出穩(wěn)定轉(zhuǎn)染細(xì)胞；Western blot方法檢測(cè)轉(zhuǎn)染后細(xì)胞中ANXA3蛋白表達(dá)水平。結(jié)果MDA-MB-231細(xì)胞中ANXA3 mRNA 及蛋白表達(dá)水平顯著高于MCF-7中的表達(dá)(P<0.05)；成功構(gòu)建3個(gè)針對(duì)ANXA3基因的shRNA質(zhì)粒；轉(zhuǎn)染ANXA3-sh1～3及陰性對(duì)照質(zhì)粒后MDA-MB-231細(xì)胞中ANXA3 mRNA表達(dá)水平分別為(0.0196±0.0002)、(0.0085±0.0002)、(0.0220±0.0035)、(0.0661±0.0057)，未轉(zhuǎn)染的MDA-MB-231細(xì)胞中ANXA3 mRNA表達(dá)水平為(0.0692±0.0050)，脂質(zhì)體組MDA-MB-231細(xì)胞中ANXA3 mRNA表達(dá)水平為(0.0652±0.0118)，ANXA3-sh2對(duì)MDA-MB-231細(xì)胞中ANXA3 mRNA沉默效率最高，達(dá)87.72%，因此后續(xù)實(shí)驗(yàn)選擇ANXA3-sh2；嘌呤霉素篩選出穩(wěn)定轉(zhuǎn)染細(xì)胞分別命名為MDA-MB-231-Sh及MDA-MB-231-NC細(xì)胞。Western blot檢測(cè)結(jié)果顯示，MDA-MB-231-Sh細(xì)胞中ANXA3蛋白顯著低于MDA-MB-231及MDA-MB-231-NC細(xì)胞中的表達(dá)(P<0.01)。結(jié)論MDA-MB-231細(xì)胞較MCF-7細(xì)胞高表達(dá)ANXA3，成功的建立了穩(wěn)定下調(diào)ANXA3表達(dá)的乳腺癌細(xì)胞株MDA-MB-231，為后續(xù)進(jìn)一步研究ANXA3的表達(dá)及特性打下基礎(chǔ)。

乳腺癌；RNA干擾；膜聯(lián)蛋白A3；轉(zhuǎn)染

乳腺癌是女性最常見的惡性腫瘤之一，其發(fā)病率逐年上升，在歐美國(guó)家乳腺癌占女性惡性腫瘤的25%～30%，在我國(guó)乳腺癌發(fā)病率已上升為女性惡性腫瘤第一位。乳腺癌的侵襲轉(zhuǎn)移及復(fù)發(fā)是影響患者生存期及生活質(zhì)量的重要因素，探討乳腺癌發(fā)生發(fā)展、浸潤(rùn)轉(zhuǎn)移及復(fù)發(fā)機(jī)制，對(duì)治療乳腺癌和提高患者生存率具有決定性的意義。膜聯(lián)蛋白家族屬于Ca2+調(diào)節(jié)的磷脂和膜結(jié)合蛋白[1,2]。在細(xì)胞中參與膜轉(zhuǎn)運(yùn)及膜表面一系列依賴于鈣調(diào)蛋白的活動(dòng)，并調(diào)控炎癥反應(yīng)、細(xì)胞分化和細(xì)胞骨架蛋白間的相互作用，其表達(dá)失調(diào)與人類疾病相關(guān)，膜聯(lián)蛋白在腫瘤的發(fā)生發(fā)展中可介導(dǎo)細(xì)胞信號(hào)通路、細(xì)胞運(yùn)動(dòng)、腫瘤的侵襲轉(zhuǎn)移、細(xì)胞凋亡及抗藥性[3-6]。膜聯(lián)蛋白A3(Annexin A3, ANXA3)在腫瘤的形成、細(xì)胞增殖、凋亡、侵襲轉(zhuǎn)移及耐藥方面起著重要的作用[7,8]。但ANXA3在乳腺癌中的作用及功能仍未闡明，本研究主要采用RNAi技術(shù)篩選針對(duì)ANXA3的高效shRNA序列，并將其轉(zhuǎn)染乳腺癌細(xì)胞株MDA-MB-231，建立穩(wěn)定下調(diào)ANXA3表達(dá)的乳腺癌細(xì)胞株MDA-MB-231，為闡明ANXA3在乳腺癌發(fā)生發(fā)展及侵襲轉(zhuǎn)移機(jī)制提供實(shí)驗(yàn)基礎(chǔ)。

1　材料與方法

1.1材料

1.1.1細(xì)胞株:MDA-MB-231及MCF-7細(xì)胞株本實(shí)驗(yàn)室傳代培養(yǎng)。接種細(xì)胞于含10%胎牛血清的PRMI1640培養(yǎng)基中(補(bǔ)充青霉素、鏈霉素各100 U/L)，培養(yǎng)器皿置于37℃含5%CO2的細(xì)胞培養(yǎng)箱中。

1.1.2主要試劑及儀器:LipofectamineTM2000(Invintrogen,美國(guó))；PVDF膜、瓊脂糖(Bio-Rad,美國(guó))；RPMI-1640培養(yǎng)基、BSA (Sigma,美國(guó))；引物(上海生工，中國(guó))； 高純度質(zhì)粒小提中量試劑盒(天根生化科技有限公司,中國(guó))；RNA isolater total RNA extraction reagent、HiScript Ⅱ 1st Strand cDNA Synthesis Kit、qPCR SYBR Green Master mix試劑(Vazyme,中國(guó))；Annexin V-PE/7-AAD試劑盒、MatrigelTM Matrix Basement Membrane(BD公司,美國(guó))；Transwell 3422(Corning公司,美國(guó))；Epics-XL型流式細(xì)胞儀(Beckman Coulter公司,美國(guó))；PCR儀(Eppendorf，德國(guó))；Mx3000P熒光定量PCR儀(Agilent,美國(guó))。

1.2方法

1.2.1熒光定量RT-PCR檢測(cè)MDA-MB-231及MCF-7細(xì)胞中ANXA3 mRNA表達(dá)水平:取生長(zhǎng)狀態(tài)良好處于對(duì)數(shù)生長(zhǎng)期的MDA-MB-231及MCF-7細(xì)胞，冷PBS洗滌2次，加入1ml RNA isolater試劑，常規(guī)一步法提取總RNA。按照說(shuō)明書進(jìn)行反轉(zhuǎn)錄為cDNA，以cDNA為模板進(jìn)行PCR擴(kuò)增。Human GAPDH作為內(nèi)參，進(jìn)行標(biāo)準(zhǔn)化。按照標(biāo)準(zhǔn)的real-time PCR流程執(zhí)行，采用SYBR-Green Ⅰ作為熒光染料，每個(gè)樣品重復(fù)3次。ANXA3 上游引物5’-TCC GAA ACA TCT GGT GAC-3’， ANXA3 下游引物3’-TCA AGT TCT TCG TAA TAC CGA T-5’。內(nèi)參GAPDH上游引物3’-CACTACCGTACCTGACACCA-5’，GAPDH下游引物 3’-ATGTCGTTGTCCCACCACCT-5’。應(yīng)用2-ΔCt值法計(jì)算ANXA3 mRNA的相對(duì)表達(dá)量，ΔCt= ANXA3 CT值-GAPDH CT值。

1.2.2western blot方法檢測(cè)MDA-MB-231及MCF-7細(xì)胞中ANXA3 蛋白表達(dá)水平:取生長(zhǎng)狀態(tài)良好處于對(duì)數(shù)生長(zhǎng)期的MDA-MB-231及MCF-7細(xì)胞，冷PBS洗滌2次，用細(xì)胞裂解液提取細(xì)胞總蛋白，BCA方法檢測(cè)蛋白濃度。10% SDA-PAGE 分離，上樣量為 50 μg/孔，轉(zhuǎn)移蛋白質(zhì)至PVDF膜上，5%脫脂奶粉4℃封閉過(guò)夜，加入1∶500稀釋的鼠抗人ANXA3單抗或 1∶4 000稀釋的兔抗人β-actin單抗室溫孵育1 h，膜洗3次，加入1∶20 000稀釋的熒光標(biāo)記的二抗，室溫孵育2 h，用紅外成像顯影，以β-actin為內(nèi)參照分析ANXA3蛋白相對(duì)表達(dá)量。實(shí)驗(yàn)重復(fù)3次。

1.2.3慢病毒干擾載體構(gòu)建:RNAi慢病毒載體使用pGmlV-SC5，通過(guò)限制性內(nèi)切酶 BamH I(GGATCC)和EcoR I(GAATTC)使載體線性化，連接入目的RNAi sequence，構(gòu)建為帶有目的RNAi序列的慢病毒載體。①shRNA靶點(diǎn)的選擇:針對(duì)ANXA3基因序列，利用公用網(wǎng)站中提供的RNA干擾序列設(shè)計(jì)原則，設(shè)計(jì)多個(gè)RNA干擾靶點(diǎn)序列，根據(jù)設(shè)計(jì)經(jīng)驗(yàn)和設(shè)計(jì)軟件進(jìn)行評(píng)估測(cè)定，選擇最佳的動(dòng)力學(xué)參數(shù)靶點(diǎn)進(jìn)入后續(xù)實(shí)驗(yàn)流程。②shRNA引物的設(shè)計(jì):據(jù)基因序列分別設(shè)計(jì)并合成shRNA寡聚單鏈DNA，oligo序列見表2。③RNAi慢病毒重組質(zhì)粒的構(gòu)建:將寡聚單鏈DNA退火成雙鏈，將雙鏈的shRNA oligo分別插入到shRNA慢病毒載體中，構(gòu)建shRNA慢病毒重組質(zhì)粒，并轉(zhuǎn)化至感受態(tài)細(xì)胞DH5。挑取若干個(gè)單菌落，進(jìn)行小量搖菌培養(yǎng)。測(cè)序鑒定陽(yáng)性克隆。測(cè)序證實(shí)重組質(zhì)粒構(gòu)建成功后，搖菌抽提質(zhì)粒，測(cè)定質(zhì)粒濃度后，-20℃保存?zhèn)溆?。見?。

表1　ANXA3基因的shRNA靶點(diǎn)

1.2.4表達(dá)小發(fā)夾RNA(small hairpin RNA, shRNA)的質(zhì)粒轉(zhuǎn)染乳腺癌MDA-MB-231細(xì)胞沉默ANXA3基因:①細(xì)胞轉(zhuǎn)染液配制:A試劑1∶6 μl Lipofectamine TM 2000脂質(zhì)體與244 μl無(wú)牛清及抗生素的RPMI1640 培養(yǎng)液混合，室溫放置5 min。B試劑2∶10 μl shRNA質(zhì)粒(含4 μg質(zhì)粒;ANXA3-sh1～3及陰性對(duì)

表2　shRNA oligo序列

照質(zhì)粒)與240 μl無(wú)牛清及抗生素的RPMI1640 培養(yǎng)液混合，室溫放置5min。②細(xì)胞轉(zhuǎn)染步驟如下:取對(duì)數(shù)生長(zhǎng)期的MDA-MB-231細(xì)胞4×105個(gè)/孔接種于6孔板中，細(xì)胞貼壁后待融合度達(dá)到85%～90%時(shí)，進(jìn)行細(xì)胞轉(zhuǎn)染，加轉(zhuǎn)染試劑前用無(wú)血清及抗生素的RPMI-1640，培養(yǎng)液沖洗細(xì)胞2遍，每孔加2 ml無(wú)牛清及抗生素的RPMI-1640培養(yǎng)液，試劑1與試劑2混合(動(dòng)作輕柔)，室溫放置20 min后加入6孔板每孔中，6 h后換全培養(yǎng)基，轉(zhuǎn)染48 h后用熒光顯微鏡觀察。同時(shí)設(shè)置未轉(zhuǎn)染的空白組細(xì)胞及只加脂質(zhì)體的脂質(zhì)體組。

1.2.5qRT-PCR檢測(cè)轉(zhuǎn)染細(xì)胞中ANXA3 mRNA表達(dá):細(xì)胞轉(zhuǎn)染48 h后，收集細(xì)胞，PBS洗滌細(xì)胞2次，具體操作步驟同1.2.1，應(yīng)用2-ΔCt值法計(jì)算ANXA3 mRNA的相對(duì)表達(dá)量，ΔCt= ANXA3 CT值-GAPDH CT值，評(píng)估siRNA ANXA3表達(dá)質(zhì)粒轉(zhuǎn)染后對(duì)ANXA3基因的沉默效果，選出沉默效果最佳的干擾質(zhì)粒。實(shí)驗(yàn)重復(fù)3次。

1.2.6嘌呤霉素篩選穩(wěn)定轉(zhuǎn)染細(xì)胞:經(jīng)過(guò)實(shí)驗(yàn)驗(yàn)證，ANXA3-sh2質(zhì)粒沉默ANXA3表達(dá)效果最佳，因此后續(xù)的siRNA實(shí)驗(yàn)選擇ANXA3-sh2質(zhì)粒。MDA-MB-231細(xì)胞轉(zhuǎn)染ANXA3-sh2重組質(zhì)粒及陰性對(duì)照質(zhì)粒48 h后，將細(xì)胞用RPMI1640培養(yǎng)液按1∶10比例稀釋，以5×104/孔細(xì)胞加入6孔板中，向6孔板中加入篩選濃度的嘌呤霉素(600 ng/ml)。未轉(zhuǎn)染的MDA-MB-231細(xì)胞作為對(duì)照組。培養(yǎng)14 d，存活的細(xì)胞即為穩(wěn)定轉(zhuǎn)染的細(xì)胞。轉(zhuǎn)染ANXA3-sh2的細(xì)胞命名為MDA-MB-231-sh，轉(zhuǎn)染陰性對(duì)照(Negative control,NC)質(zhì)粒的細(xì)胞命名為MDA-MB-231-NC。MDA-MB-231-sh 及MDA-MB-231-NC細(xì)胞在含300 ng/ml嘌呤霉素的全培養(yǎng)基中培養(yǎng)。

1.2.7western blot 方法檢測(cè)轉(zhuǎn)染細(xì)胞中ANXA3蛋白表達(dá):取生長(zhǎng)狀態(tài)良好處于對(duì)數(shù)生長(zhǎng)期的MDA-MB-231、MDA-MB-231-NC及MDA-MB-231-sh 細(xì)胞，冷PBS洗滌2次后如方法1.2.2進(jìn)行western blot檢測(cè)。

2　結(jié)果

2.1熒光定量RT-PCR及Western blot檢測(cè)MDA-MB-231及MCF-7細(xì)胞中ANXA3 mRNA及蛋白表達(dá)水平MDA-MB-231細(xì)胞ANXA3 mRNA相對(duì)表達(dá)量為(0.0696±0.0248)與MCF-7細(xì)胞ANXA3 mRNA相對(duì)表達(dá)量(0.0236±0.0149)相比顯著增高(P<0.01)。MDA-MB-231細(xì)胞ANXA3 蛋白相對(duì)表達(dá)量顯著高于MCF-7細(xì)胞中ANXA3蛋白相對(duì)表達(dá)量(P<0.01)。基因及蛋白表達(dá)趨勢(shì)相一致。見圖1、2。

圖1熒光定量RT-PCR檢測(cè)MDA-MB-231及MCF-7細(xì)胞中ANXA3 mRNA表達(dá)水平

注：A：熒光定量RT-PCR檢測(cè)MDA-MB-231及MCF-7細(xì)胞ANXA3及GAPDH的擴(kuò)增曲線及溶解曲線。B：與MCF-7細(xì)胞中ANXA3 mRNA表達(dá)水平比較，*P<0.05

圖2Western blot 檢測(cè)MDA-MB-231及MCF-7細(xì)胞中ANXA3 蛋白表達(dá)水平

注：A：Western blot 檢測(cè)乳腺癌細(xì)胞中ANXA3 蛋白表達(dá)，顯示，MDA-MB-231細(xì)胞中ANXA3 蛋白表達(dá)高于MCF-7細(xì)胞中的表達(dá)；B：與MCF-7細(xì)胞中ANXA3 mRNA表達(dá)水平比較,*P<0.05

2.2shRNA慢病毒干擾載體構(gòu)建成功構(gòu)建了慢病毒干擾載體。shRNA干擾載體的測(cè)序結(jié)果經(jīng)過(guò)比對(duì)，重組克隆中插入片段序列與設(shè)計(jì)的oligo序列完全一致，因此慢病毒載體構(gòu)建成功。見表3，圖3。

2.3熒光定量RT-PCR檢測(cè)轉(zhuǎn)染細(xì)胞中ANXA3 mRNA表達(dá)水平轉(zhuǎn)染H_ANXA3-sh1、H_ANXA3-sh2、H_ANXA3-sh3及陰性對(duì)照質(zhì)粒的MDA-MB-231細(xì)胞ANXA3 mRNA表達(dá)水平分別為，(0.0196±0.0002)、(0.0085±0.0002)、(0.0220±0.0035)、(0.0661±0.0057)ANXA3 mRNA表達(dá)水平分別為(0.0692±0.0050)、(0.0652±0.0118)。轉(zhuǎn)染H_ANXA3-sh2的MDA-MB-231細(xì)胞ANXA3 mRNA表，空白組及脂質(zhì)體組MDA-MB-231細(xì)胞達(dá)水平顯著低于其他組中ANXA3 mRNA表達(dá)水平(P<0.05)提示，H_ANXA3-sh2對(duì)MDA-MB-231細(xì)胞ANXA3沉默效果最后，后續(xù)實(shí)驗(yàn)選用H_ANXA3-sh2進(jìn)行細(xì)胞轉(zhuǎn)染。見圖4。

2.4嘌呤霉素篩選穩(wěn)定轉(zhuǎn)染細(xì)胞600 ng/ml嘌呤霉素篩選細(xì)胞14 d后，空白組細(xì)胞全部死亡，轉(zhuǎn)染ANXA3-sh2及陰性對(duì)照(Negative control,NC)質(zhì)粒的細(xì)胞中有大量細(xì)胞存活，存活的細(xì)胞即為篩選出來(lái)的穩(wěn)定轉(zhuǎn)染細(xì)胞，轉(zhuǎn)染ANXA3-sh2的細(xì)胞命名為MDA-MB-231-Sh，轉(zhuǎn)染NC質(zhì)粒的細(xì)胞命名為MDA-MB-231-NC。見圖5。

2.5western blot 檢測(cè)轉(zhuǎn)染細(xì)胞中ANXA3 蛋白表達(dá)水平檢測(cè)結(jié)果顯示，MDA-MB-231-Sh細(xì)胞中高于ANXA3 蛋白表達(dá)量顯著低于MDA-MB-231-NC及MDA-MB-231細(xì)胞中的表達(dá)量(P<0.01)。見圖6。

3　討論

Annexin A3是Annexins家族成員之一。研究表明，Annexin A3基因定位于人染色體4q13～q22，由323個(gè)氨基酸殘基組成[9,10]。Annexin A3表達(dá)水平的改變，對(duì)腫瘤發(fā)生發(fā)展、耐藥性、轉(zhuǎn)移有重要影響。關(guān)于ANXA3與惡性腫瘤的相關(guān)研究目前開展并不廣泛，其深入程度也遠(yuǎn)不如ANXA家族ANXA1、ANXA2等的相關(guān)研究。Liu等[7]應(yīng)用熒光雙向差異凝膠電泳(2D-DIGE)與液相色譜質(zhì)譜(LC-MS)技術(shù)對(duì)胃癌分化相關(guān)蛋白質(zhì)進(jìn)行了篩選，結(jié)果發(fā)現(xiàn)ANXA3與胃癌分化關(guān)系密切。Xie等[11]研究60例直腸癌患者中ANXA3表達(dá)情況。采用了免疫組化的方法分析這些患者直腸癌組織中ANXA3蛋白表達(dá)情況發(fā)現(xiàn)，ANXA3在直腸癌組織中表達(dá)上調(diào)，且ANXA3過(guò)表達(dá)是預(yù)后較差的預(yù)后因素。國(guó)內(nèi)朱利芹等[12]報(bào)道ANXA3在耐藥細(xì)胞株中RNA基因水平和蛋白水平表達(dá)顯著高于親本細(xì)胞株HT-29的結(jié)論。但這些研究還比較零散，有待進(jìn)一步深入研究。Madoz-Gurpide等[13]研究發(fā)現(xiàn)在結(jié)直腸癌中ANXA3高表達(dá)率為63%，顯著高于正常組織。Liang等[14]報(bào)道Annexin A3在高淋巴道轉(zhuǎn)移肝癌細(xì)胞株Hca-F中表達(dá)明顯上調(diào)，其表達(dá)水平是低淋巴道轉(zhuǎn)移肝癌細(xì)胞株Hca-P的2.3倍，從而說(shuō)明ANXA3直接參與了肝癌的轉(zhuǎn)移過(guò)程。Zeng等[15,16]均報(bào)道ANXA3表達(dá)與腋淋巴結(jié)轉(zhuǎn)移呈正相關(guān)性。以上研究均提示ANXA3可能參與惡性腫瘤的發(fā)生、增值、遷移和轉(zhuǎn)移。這與我們研究的在乳腺癌中ANXA3表達(dá)結(jié)果相一致。有關(guān)ANXA3在乳腺癌方面的研究報(bào)道較少。我們的研究首先采用熒光定量RT-PCR及western blot方法檢測(cè)了兩株乳腺癌細(xì)胞(MDA-MB-231及MCF-7)中ANXA3 mRNA及蛋白表達(dá)水平。結(jié)果發(fā)現(xiàn)MDA-MB-231細(xì)胞中ANXA3 mRNA及蛋白表達(dá)水平顯著高于MCF-7中的表達(dá)，提示在高侵襲性乳腺癌細(xì)胞中ANXA3表達(dá)增高。我們成功構(gòu)建了3個(gè)針對(duì)ANXA3基因的shRNA質(zhì)粒；經(jīng)脂質(zhì)體法轉(zhuǎn)染人乳腺癌MDA-MB-231細(xì)胞48h觀察，ANXA3-sh2對(duì)MDA-MB-231細(xì)胞中ANXA3 mRNA沉默效率最高，達(dá)87.72%。經(jīng)嘌呤霉素篩選出穩(wěn)定轉(zhuǎn)染細(xì)胞；應(yīng)用了Western blot方法檢測(cè)轉(zhuǎn)染后細(xì)胞中ANXA3蛋白表達(dá)水平顯著低于MDA-MB-231細(xì)胞中的表達(dá)水平。本項(xiàng)研究，通過(guò)RNA干擾沉默乳腺癌MDA-MB-231細(xì)胞中ANXA3基因，從而建立穩(wěn)定下調(diào)ANXA3表達(dá)的乳腺癌細(xì)胞株MDA-MB-231，為進(jìn)一步研究ANXA3在乳腺癌發(fā)生發(fā)展及侵襲轉(zhuǎn)移機(jī)制提供實(shí)驗(yàn)基礎(chǔ)。

表3　shRNA干擾載體的測(cè)序結(jié)果

圖3　shRNA干擾載體的部分測(cè)序

圖4　熒光定量RT-PCR檢測(cè)轉(zhuǎn)染細(xì)胞中ANXA3 mRNA表達(dá)水平

注:A:熒光定量RT-PCR檢測(cè)細(xì)胞ANXA3及GAPDH的擴(kuò)增曲線及溶解曲線;B:與轉(zhuǎn)染H_ANXA3-sh2的MDA-MB-231細(xì)胞ANXA3 mRNA表達(dá)水平比較，*P<0.05

圖5　600ng/ml嘌呤霉素篩選細(xì)胞14日后細(xì)胞圖(×100)

圖6　western blot 檢測(cè)轉(zhuǎn)染細(xì)胞中ANXA3蛋白表達(dá)

注:A:western blot 檢測(cè)轉(zhuǎn)染細(xì)胞中ANXA3蛋白表達(dá)；B:與MDA-MB-231-Sh細(xì)胞比較,*P<0.05；MDA-MB-231-Sh細(xì)胞中ANXA3蛋白顯著降低，轉(zhuǎn)染針對(duì)ANXA3 的shRAN可以顯著下調(diào)ANXA3蛋白表達(dá)

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Establishment of breast cancer cell line-MDA-MB-231 through down-regulating the expressions of annexin A3 gene by RNA interference technique

ZHOUTao*,YANGLi,LIULiang,etal.

*CenterforBreastExamination,TheFourthHospitalofHebeiMedicalUniversity,Shijiazhuang050011,China

ObjectiveTo establish a breast cancer cell line-MDA-MB-231 transfected with shRNA (small hairpin RNA) targeting at annexin A3, in order to provide a basis for further study.MethodsThe expression levels of ANXA3 mRNA and protein in MDA-MB-231 cells and MCF-7 cells were detected by fluorescent quantitation RT-PCR and Western Blot,respectively. The three shRNAs (shRNA1, 2 and 3) plasmids of silencing ANXA3 gene and negative control plasmid were established. MDA-MB-231 human breast cancer cells were transfered with liposome method.The interference plasmid that could silence ANXA3 gene most effectively was selected for subsequent experiment.The stable transfection cells were selected out by using puromycin, and the expression levels of annexin A7 were detected by Western Blot.ResultsThe expression levels of ANXA3 mRNA and protein in MDA-MB-231 cells were significantly higher than those in MCF-7 cells (P<0.05). The three shRNAs plasmids targeting at annexin A3 gene were successfully established.The expression levels of ANXA3 mRNA in MDA-MB-231 cells after transfected with ANXA3-sh1～3 and negative control plasmid were 0.0196±0.0002, 0.0085±0.0002, 0.0220±0.0035, 0.0661±0.0057,respectively,however,which in MDA-MB-231 cells without transfection and in liposomes group were 0.0692±0.0050,0.0652±0.0118,respectively. The silencing efficiency of ANXA3-sh2 on ANXA3 mRNA in MDA-MB-231 cells was the highest (87.72%),thus, ANXA3-sh2 was selected for subsequent experiment. The stable transfection cells selected by puromycin were named as MDA-MB-231-Sh cells and MDA-MB-231-NC cells. Western Blot showed that the expression levels of ANXA3 protein in MDA-MB-231-Sh cells were significantly lower than those in MDA-MB-231 cells and in MDA-MB-231-NC cells (P<0.01).ConclusionThe expression levels of annexin A3 protein in MDA-MB-231 cells are higher than those in MCF-7 cells.The successful establishment of MDA-MB-231 cells of down-regulating the expressions of annexin A3 stably lays the foundation for furthere research about the expression and characteristics of ANXA3.

breast cancer; RNA interference; Annexin A3; transfection

10.3969/j.issn.1002-7386.2016.20.002

050011石家莊市，河北醫(yī)科大學(xué)第四醫(yī)院乳腺中心(周濤),CT室(楊麗)，腫瘤所(劉亮)，動(dòng)物中心(巨英超)

R 737.9

A

1002-7386(2016)20-3049-06

2016-05-19)