TLR4 shRNA質(zhì)粒的構(gòu)建及穩(wěn)定轉(zhuǎn)染人胰腺癌PANC1細(xì)胞株的篩選

張建軍 王博 田元 張景輝 吳河水

·論著·

TLR4 shRNA質(zhì)粒的構(gòu)建及穩(wěn)定轉(zhuǎn)染人胰腺癌PANC1細(xì)胞株的篩選

張建軍 王博 田元 張景輝 吳河水

目的構(gòu)建靶向人TLR4基因的短發(fā)夾RNA(shRNA)真核表達(dá)質(zhì)粒，轉(zhuǎn)染胰腺癌細(xì)胞，并篩選出穩(wěn)定轉(zhuǎn)染的克隆細(xì)胞株。方法采用小分子干擾RNA(siRNA)軟件設(shè)計(jì)3個(gè)靶向TLR4基因的shRNA，構(gòu)建3個(gè)質(zhì)粒表達(dá)載體，分別命名為TLR4-1、TLR4-2、TLR4-3；選取抑制效率最高的shRNA質(zhì)粒，采用脂質(zhì)體法轉(zhuǎn)染胰腺癌PANC1細(xì)胞；實(shí)時(shí)定量PCR和流式細(xì)胞技術(shù)檢測(cè)細(xì)胞TLR4 shRNA基因沉默效率和轉(zhuǎn)染效率；G418抗性篩選和有限稀釋單克隆形成法挑選培育穩(wěn)定轉(zhuǎn)染TLR4 shRNA的單克隆細(xì)胞系。結(jié)果轉(zhuǎn)染48 h后PANC1細(xì)胞瞬時(shí)轉(zhuǎn)染效率為(46.72±5.06)%。轉(zhuǎn)染TLR4-1、TLR4-2、TLR4-3的細(xì)胞的TLR4 mRNA表達(dá)水平分別為0.025±0.004、0.027±0.003、0.019±0.006，均顯著低于未轉(zhuǎn)染組的0.061±0.018和陰性對(duì)照轉(zhuǎn)染組的0.057±0.015(P值均<0.05)。以沉默效果最佳的TLR4-3轉(zhuǎn)染細(xì)胞所獲得的穩(wěn)轉(zhuǎn)細(xì)胞的轉(zhuǎn)染效率為(82.79±8.16)%，較瞬轉(zhuǎn)細(xì)胞顯著提高(P=0.001)；TLR4 mRNA表達(dá)水平為0.010±0.002,較瞬轉(zhuǎn)細(xì)胞顯著下調(diào)(P=0.001)；TLR4蛋白表達(dá)率為(0.54±0.32)%，顯著低于未轉(zhuǎn)染細(xì)胞的(87.42±5.00)%和轉(zhuǎn)染陰性對(duì)照細(xì)胞的(82.90±5.00)%(P=0.000)。結(jié)論成功篩選到TLR4基因沉默的穩(wěn)轉(zhuǎn)PANC1細(xì)胞。

胰腺腫瘤； Toll樣受體4； 轉(zhuǎn)染； RNA干擾

Toll樣受體4(Toll like receptor 4，TLR4)主要表達(dá)于上皮細(xì)胞和免疫細(xì)胞，在宿主防御反應(yīng)中起著重要作用[1]。近年來發(fā)現(xiàn)，TLR4在很多腫瘤細(xì)胞和腫瘤組織中表達(dá)上調(diào)[2]。我們?cè)鴪?bào)道TLR4在胰腺癌中高表達(dá)，且與腫瘤大小、淋巴結(jié)轉(zhuǎn)移、血管侵犯及臨床分期相關(guān)[3]。因此，TLR4可能成為治療胰腺癌的新靶點(diǎn)。本研究應(yīng)用RNA干擾(RNAi)技術(shù)構(gòu)建靶向TLR4的質(zhì)粒，轉(zhuǎn)染人胰腺癌PANC1細(xì)胞，篩選穩(wěn)轉(zhuǎn)細(xì)胞克隆。

材料和方法

一、靶向TLR4表達(dá)質(zhì)粒的構(gòu)建

用siRNA WizardTMInvivogen軟件設(shè)計(jì)3個(gè)靶向人TLR4基因cDNA序列的短發(fā)夾RNA(shRNA)，并應(yīng)用在線軟件BLAST檢索數(shù)據(jù)庫排除其他基因的同源性序列，其順序?yàn)?BamH酶切位點(diǎn)、19 nt正義序列、9 nt loop接頭序列、19 nt反義序列、 12 nt RNA聚合酶終止子(6個(gè)T)、Hind酶切位點(diǎn)，分別命名為TLR4-1、TLR4-2和TLR4-3(表1)。攜帶綠色熒光蛋白(GFP)的TLR4 shRNA 表達(dá)質(zhì)粒由上海吉?jiǎng)P基因公司構(gòu)建，并經(jīng)測(cè)序證實(shí)。陰性對(duì)照質(zhì)粒為該公司提供的攜帶 GFP的空質(zhì)粒載體。

表1 TLR4 shRNA構(gòu)建框架序列

二、TLR4 shRNA表達(dá)質(zhì)粒轉(zhuǎn)染細(xì)胞

胰腺癌細(xì)胞系PANC1由協(xié)和醫(yī)院普外科實(shí)驗(yàn)室提供,常規(guī)培養(yǎng)傳代。取對(duì)數(shù)生長(zhǎng)期細(xì)胞， 以2×105個(gè)/孔接種至6孔板中培養(yǎng)24 h，應(yīng)用LipofectamineTM2000(美國(guó)Invitrogen公司)將質(zhì)粒轉(zhuǎn)染細(xì)胞，按試劑盒說明書操作。在熒光顯微鏡下觀測(cè)轉(zhuǎn)染細(xì)胞GFP表達(dá)，并收集細(xì)胞上流式細(xì)胞儀檢測(cè)GFP表達(dá)，計(jì)算轉(zhuǎn)染效率。

三、實(shí)時(shí)定量PCR檢測(cè)TLR4 mRNA的表達(dá)

用Trizol提取轉(zhuǎn)染后細(xì)胞總RNA，隨機(jī)引物法逆轉(zhuǎn)錄合成cDNA。應(yīng)用熒光實(shí)時(shí)定量PCR盒(Promega公司)檢測(cè)TLR4 mRNA表達(dá)。 TLR4引物上游5′-ACCTGTCCCTGAACCCTATGAA-3′，下游5′-CTTCTAAACCAGCCAGACCTTG-3′ ，產(chǎn)物138 bp；內(nèi)參β-actin引物上游5′-AAGGCCAGGTAATTGTCACG-3′，下游5′-AGCAGCTCTGCAGTACGTC-3′，產(chǎn)物105 bp。PCR反應(yīng)程序：94℃ 15 s，58℃ 30 s，72℃ 45 s，共45個(gè)循環(huán)。 PCR結(jié)果采用2-△△Ct分析法[4]計(jì)算。

四、流式細(xì)胞儀檢測(cè)TLR4蛋白表達(dá)

胰酶消化轉(zhuǎn)染細(xì)胞，PBS洗滌后調(diào)整細(xì)胞濃度為106個(gè)/ml。取200 μl細(xì)胞懸液，加入藻紅蛋白(phycoerythrin)標(biāo)記的TLR4抗體(eBioscience公司)5 μl，4℃避光孵育30 min，上流式細(xì)胞儀檢測(cè)。

五、穩(wěn)定轉(zhuǎn)染TLR4 shRNA的細(xì)胞克隆篩選

選取沉默效率最佳的RNAi質(zhì)粒轉(zhuǎn)染細(xì)胞，按1∶10比例傳代于6孔板，細(xì)胞貼壁后用含G418(400～1000 mg/L)的培養(yǎng)基培養(yǎng)2周，挑取單個(gè)細(xì)胞集落，運(yùn)用有限稀釋單克隆形成法稀釋后，按每孔1個(gè)細(xì)胞接種于96孔板，繼續(xù)采用800 mg/L G418篩選。1個(gè)月后觀察單克隆形成情況，挑選綠色熒光強(qiáng)度最高的克隆進(jìn)行擴(kuò)大培養(yǎng)。

六、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、細(xì)胞瞬時(shí)轉(zhuǎn)染效率及TLR4 mRNA表達(dá)

重組質(zhì)粒轉(zhuǎn)染PANC1細(xì)胞48 h后,熒光顯微鏡下見細(xì)胞形態(tài)無明顯改變，但表達(dá)GFP(圖1a)，瞬轉(zhuǎn)效率為(46.72±5.06)%(圖1b、c)。

轉(zhuǎn)染TLR4-1、TLR4-2、TLR4-3的細(xì)胞的TLR4mRNA表達(dá)水平分別為0.025±0.004、0.027±0.003、0.019±0.006，均顯著低于未轉(zhuǎn)染組的0.061±0.018和陰性對(duì)照轉(zhuǎn)染組的0.057±0.015(P值均<0.05)。3個(gè)轉(zhuǎn)染組間比較差異無統(tǒng)計(jì)學(xué)意義，但以TLR4-3的沉默效果最佳。

圖1轉(zhuǎn)染48 h的PANC1細(xì)胞(a，熒光顯微鏡 ×100)及未轉(zhuǎn)染細(xì)胞(b)和轉(zhuǎn)染細(xì)胞(c)的熒光表達(dá)細(xì)胞比例(流式細(xì)胞儀)

二、 穩(wěn)轉(zhuǎn)細(xì)胞的GFP表達(dá)及TLR4 mRNA表達(dá)

穩(wěn)轉(zhuǎn)細(xì)胞廣泛表達(dá)GFP(圖2a)，GFP表達(dá)率(轉(zhuǎn)染效率)為(82.79±8.16)%(圖2b、c)，顯著高于瞬轉(zhuǎn)細(xì)胞的(46.72±5.06)%(P=0.001)。穩(wěn)轉(zhuǎn)細(xì)胞的TLR4 mRNA表達(dá)水平為0.010±0.002，顯著低于瞬轉(zhuǎn)細(xì)胞的0.019±0.006(P=0.001)。

圖2穩(wěn)轉(zhuǎn)細(xì)胞的GFP表達(dá)(a，熒光顯微鏡 ×100)及未轉(zhuǎn)染細(xì)胞(b)和穩(wěn)轉(zhuǎn)細(xì)胞(c)的熒光表達(dá)細(xì)胞比例(流式細(xì)胞儀)

三、穩(wěn)轉(zhuǎn)細(xì)胞的TLR4蛋白表達(dá)

穩(wěn)轉(zhuǎn)細(xì)胞TLR4蛋白表達(dá)率為(0.54±0.32)%，顯著低于未轉(zhuǎn)染組的(87.42±5.00)%(P=0.000)及陰性對(duì)照轉(zhuǎn)染組的(82.9±5.00)%(P=0.000, 圖3)。

圖3未轉(zhuǎn)染細(xì)胞(a)及穩(wěn)轉(zhuǎn)細(xì)胞(b)的TLR4蛋白表達(dá)(流式細(xì)胞儀)

討 論

RNAi具有高度的序列專一性和有效的干擾活力，可以應(yīng)用體外轉(zhuǎn)錄或化學(xué)合成siRNA,經(jīng)脂質(zhì)體或病毒載體轉(zhuǎn)染細(xì)胞，或應(yīng)用shRNA表達(dá)載體轉(zhuǎn)染細(xì)胞后特異地沉默特定基因?；瘜W(xué)合成siRNA抑制作用時(shí)間短,成本昂貴,并且易于污染 RNA酶；而shRNA轉(zhuǎn)染細(xì)胞后可以在RNA酶的作用下被切割成與體外合成siRNA相同的序列與結(jié)構(gòu)。因細(xì)胞可穩(wěn)定持續(xù)地表達(dá)shRNA,從而使RNAi作用持續(xù)時(shí)間更長(zhǎng)、更穩(wěn)[5]。

近年研究發(fā)現(xiàn)，TLR4在很多腫瘤細(xì)胞系和腫瘤組織中表達(dá)上調(diào)[6-7]。我們以往的研究也顯示TLR4在胰腺癌組織中高表達(dá)[3]。因此本研究設(shè)計(jì)合成了3個(gè)靶向TLR4的shRNA，將其插入U(xiǎn)6 RNA聚合酶 Ⅲ啟動(dòng)子、卡那耐藥基因、GFP基因的真核表達(dá)質(zhì)粒，具有利用相對(duì)單一的啟動(dòng)子和終止序列產(chǎn)生大量的小RNA，利于穩(wěn)定傳代細(xì)胞株的篩選及在熒光顯微鏡下觀察或流式細(xì)胞儀檢測(cè)等優(yōu)勢(shì)。本研究結(jié)果顯示，3個(gè)shRNA表達(dá)質(zhì)粒轉(zhuǎn)染細(xì)胞后均獲得滿意瞬間轉(zhuǎn)染效率及基因沉默效應(yīng)，選擇轉(zhuǎn)染率最高、基因沉默效應(yīng)最佳的TLR4-3表達(dá)質(zhì)粒轉(zhuǎn)染PANC1細(xì)胞所獲得的穩(wěn)轉(zhuǎn)細(xì)胞，其轉(zhuǎn)染效率顯著高于瞬轉(zhuǎn)細(xì)胞，目的基因的抑制效應(yīng)亦優(yōu)于瞬轉(zhuǎn)細(xì)胞，為以 TLR4為靶點(diǎn)的胰腺癌細(xì)胞生物學(xué)特性研究提供了良好基礎(chǔ)。

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ConstructionofTLR4shRNAplasmidandscreeningofhumanpancreaticcancerPANC1celllinewithstabletransfection

ZHANGJian-jun,WANGBo,TIANYuan,ZHANGJing-hui,WUHe-shui.

DepartmentofGastrointestinalSurgery,NanshanHospital,GuangdongMedicalCollege,Shenzhen518052,China

WUHe-shui,Email:heshuiwu@163.com

ObjectiveTo construct the eukaryotic plasmid expression vector mediated short hairpin RNA(shRNA) interference targeting TLR4 gene, and transfect it into pancreatic adenocarcinoma cell line PANC1, then screen stably transfected clonal cell line.MethodsThree shRNA interference expression plasmid vectors targeting the TLR4 gene were constructed, named TLR4-1, TLR4-2, TLR4-3.The shRNA plasmid with highest inhibitory efficiency was selected and transfected into PANC1 cells with liposome. The silencing efficiency and transfection efficiency of TLR4-shRNA was assayed with real-time quantitative PCR and flow cytometry analysis. Monoclonal cell with stable transfection of TLR4-shRNA were selected by geneticin 418(G418) and limiting dilution analysis.ResultsTransient transfection efficiency of PANC1 was (46.72±5.06)%. TLR4 mRNA expressions were 0.025±0.004,0.027±0.003,0.019±0.006in cells transfected with TLR4-1, TLR4-2, TLR4-3, respectively, which were significantly lower than that in untransfected group (0.061±0.018) and negative control group (0.057±0.015,P<0.05). The transfection efficiency of TLR4-3 vector in stably transfected clones [(82.79±8.16)%] was significantly higher than that of transient transfection (P=0.001). The expression of TLR4 mRNA was decreased to 0.010±0.002, which was significantly lower than that of transient transfection (P=0.001). The expression of TLR4 protein was (0.54±0.32)%, which was significantly lower than that of untransfected cells [(87.42±5.00)%] and that of negative control [(82.9±5.00)%,P=0.000].ConclusionsStable transfection PANC1 cell lines with TLR4 gene silencing are successfully identified.

Pancreatic neoplasms; Toll like receptor 4; Transfection; RNA interference

10.3760/cma.j.issn.1674-1935.2012.03.012

國(guó)家自然科學(xué)基金(30972898)

518052 深圳，廣東醫(yī)學(xué)院附屬南山醫(yī)院胃腸外科(張建平)；華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬協(xié)和醫(yī)院胰腺外科中心(張建軍、王博、田元、張景輝、吳河水)

吳河水，Email：heshuiwu@163.com

2011-11-25)

(本文編輯：呂芳萍)