江寅 侯寶華 簡(jiǎn)志祥 王慧玲 崔鵬 區(qū)金銳

·論著·

胰腺癌PANC1細(xì)胞microRNA-217靶基因ANLN的鑒定

江寅 侯寶華 簡(jiǎn)志祥 王慧玲 崔鵬 區(qū)金銳

目的通過實(shí)驗(yàn)驗(yàn)證ANLN為microRNA-217(miR-217)的靶基因。方法使用生物學(xué)軟件預(yù)測(cè)miR-217調(diào)控的靶基因可能為ANLN。設(shè)計(jì)并合成mR-217結(jié)合的ANLN及突變型ANLN(mutANLN)序列，將其PCR擴(kuò)增的片段插入表達(dá)質(zhì)粒psiCHECK-2，構(gòu)建重組質(zhì)粒psiCHECK-2-ANLN及psiCHECK-2-mutANLN。采用脂質(zhì)體法將2個(gè)重組質(zhì)粒單獨(dú)或分別與miR-217、miR-217 inhibitor及與人同源性遠(yuǎn)的miRNA序列(NC)、NC inhibitor共轉(zhuǎn)染胰腺癌PANC1細(xì)胞，采用雙熒光素酶報(bào)告系統(tǒng)檢測(cè)熒光素酶活性，采用蛋白質(zhì)印跡法檢測(cè)ANLN蛋白的表達(dá)。結(jié)果轉(zhuǎn)染psiCHECK-2-ANLN、psiCHECK-2-ANLN+miR-217、psiCHECK-2-ANLN+miR-217 inhibitor、psiCHECK-2-ANLN+NC、psiCHECK-2-ANLN+NC inhibitor各組間的熒光素酶活性分別為2.221±0.188、0.769±0.061、3.764±0.371、2.265±0.201、2.242±0.018，差異有統(tǒng)計(jì)學(xué)意義(F=77.405,P<0.01)，但psiCHECK-2-ANLN組、psiCHECK-2-ANLN+NC組及psiCHECK-2-ANLN+NC inhibitor組兩兩比較差異無統(tǒng)計(jì)學(xué)意義，而psiCHECK-2-ANLN+miR-217組的熒光素酶活性較這3組明顯下降，psiCHECK-2-ANLN+miR-217 inhibitor組活性較其他4組明顯升高(P值均<0.01)。轉(zhuǎn)染psiCHECK-2-mutANLN各組間熒光素酶活性差異無統(tǒng)計(jì)學(xué)意義(P=0.053)。psiCHECK-2-ANLN+miR-217共轉(zhuǎn)染組PANC1細(xì)胞的ANLN蛋白表達(dá)水平較單純轉(zhuǎn)染psiCHECK-2-ANLN組細(xì)胞的ANLN蛋白表達(dá)明顯下調(diào)。結(jié)論在胰腺癌中，ANLN可能是miR-217的直接靶基因。

胰腺腫瘤； 微RNAs； miR-217； 基因； ANLN

MicroRNA(miRNA)是一類由18～23個(gè)核苷酸構(gòu)成的單鏈非編碼RNA分子，通過靶向結(jié)合mRNA的3′非編碼區(qū)域(3′UTR)，降解mRNA或者抑制其翻譯，導(dǎo)致靶基因的轉(zhuǎn)錄后沉默，從而參與靶基因功能的調(diào)節(jié)。miRNA與胰腺癌的發(fā)生和發(fā)展存在密切關(guān)系，如miR-21、miR-34a、miR-10a、miR-155、miR-196a、miR-133a等在胰腺癌和正常胰腺中存在表達(dá)差異，可作為腫瘤標(biāo)志物用于胰腺癌與正常胰腺、慢性胰腺炎、胰腺內(nèi)分泌腫瘤等的鑒別診斷及預(yù)后預(yù)測(cè)[1-4]。我們前期的研究發(fā)現(xiàn)，miR-217在胰腺癌組織中的表達(dá)顯著下調(diào)。ANLN是一種肌動(dòng)蛋白結(jié)合蛋白，在包括胰腺癌在內(nèi)的多種惡性腫瘤中均有高表達(dá)。本研究進(jìn)一步驗(yàn)證ANLN為miR-217的靶基因。

材料與方法

一、雙熒光素酶報(bào)告載體構(gòu)建

通過TargetScan、picTar、miRanda和DIANA microT 4種生物學(xué)軟件分析并結(jié)合相關(guān)文獻(xiàn)資料，推測(cè)ANLN為miR-217調(diào)控的靶基因，且有2個(gè)結(jié)合位點(diǎn)，在132～138及660～666處。

從TargetScan網(wǎng)站上獲取人類ANLN基因3′UTR區(qū)與hsa-miR-217結(jié)合的序列，設(shè)計(jì)目標(biāo)序列和突變序列，兩端分別加XhoⅠ和NotⅠ酶切位點(diǎn)。野生型ANLN引物序列：正義 5′-CCGCTCGAGAC-CGGGAAATTTCCATGCTATC-3′，反義5′-AAG-GAAAAAAGCGGCCGCTCCTTTAGACATTTACAGGT-ATTTATTTGAG-3′；突變引物1序列：正義5′-CCA-ATATTCACTACGTATTGCTAGCTATTTATATCTTTTG-TATGT-3′，反義5′-ACATACAAAAGATATAAATAGCTAGCAATACGTAGTGAATATTGG-3′；突變引物2序列：正義5′-CATTTACTCAGCTACTATATGCTAGCT-GTGGTGCACATTTTCACAGAA-3′，反義5′-TTCTGTGAAAATGTGCACCACAGCTAGCATATAG-TAGCTGAGTAAATG-3′。以健康志愿者全血DNA為模板，將目標(biāo)序列和突變序列進(jìn)行PCR擴(kuò)增，PCR反應(yīng)條件：94℃ 5min，94℃ 30 s、 58℃ 30 s，72℃ 75 s，32個(gè)循環(huán)，72℃ 5 min。擴(kuò)增片段經(jīng)電泳分離、回收、純化、應(yīng)用限制酶(Xho Ⅰ和NotⅠ)切割后插入表達(dá)質(zhì)粒psiCHECK-2的多克隆位點(diǎn)。構(gòu)建含有miR-217結(jié)合位點(diǎn)的ANLN重組質(zhì)粒psiCHECK-2-ANLN。以psiCHECK-2-ANLN為模板，通過突變序列PCR擴(kuò)增，構(gòu)建缺失miR-217結(jié)合位點(diǎn)的突變型重組質(zhì)檢psiCHECK-2-mutANLN。

二、熒光素酶實(shí)驗(yàn)

胰腺癌PANC1細(xì)胞常規(guī)培養(yǎng)、傳代。取對(duì)數(shù)生長期細(xì)胞，按2×104個(gè)/孔密度接種于24孔板上，分為psiCHECK-2-ANLN和psiCHECK-2-mutANLN兩大組，每個(gè)大組又分為對(duì)照組、miR-217、miR-217 inhibitor、與人同源性遠(yuǎn)的miRNA序列(NC)、NC inhibitor 5組。對(duì)照組僅轉(zhuǎn)染各自的重組質(zhì)粒，其他4組則為重組質(zhì)粒分別與miR-217、miR-217 inhibitor、NC、NC inhibitor共轉(zhuǎn)染。細(xì)胞轉(zhuǎn)染采用LipofeetamineTM2000(美國Invitrogen公司)。轉(zhuǎn)染48 h后采用雙熒光報(bào)告系統(tǒng)檢測(cè)試劑盒(Promega公司，E1910)行熒光素酶活性檢測(cè)，以不轉(zhuǎn)染細(xì)胞為空白對(duì)照。實(shí)驗(yàn)重復(fù)3次，每次設(shè)3個(gè)復(fù)孔。

三、ANLN蛋白表達(dá)檢測(cè)

提取轉(zhuǎn)染psiCHECK-2-ANLN、psiCHECK-2-ANLN+miR-217、psiCHECK-2-ANLN+ miR-217 inhibitor組細(xì)胞總蛋白，牛血清蛋白(BSA)法測(cè)定蛋白含量。常規(guī)行蛋白質(zhì)印跡法檢測(cè)細(xì)胞ANLN蛋白的表達(dá)。兔抗人ANLN一抗購自Abcam公司(ab99352)，工作濃度1∶4000，羊抗兔辣根過氧化物酶(HRP)標(biāo)記的IgG 購自Southern Biotech公司，工作濃度1∶5000。采用數(shù)碼成像分析系統(tǒng)軟件IPWIN6.0對(duì)條帶進(jìn)行掃描。

四、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、雙熒光素酶報(bào)告載體

含有miR-217結(jié)合位點(diǎn)及缺失miR-217結(jié)合位點(diǎn)的熒光素酶報(bào)告載體psiCHECK-2-ANLN和psiCHECK-2-mutANLN測(cè)序結(jié)果如圖1、2。經(jīng)局部對(duì)比基本檢索工具(basic local alignment search tool, BLAST))結(jié)果分析，ANLN 3 ′UTR及mut ANLN 3 ′UTR片段成功地克隆入雙螢光報(bào)告載體psiCHECK-2中，可以用于后續(xù)螢光素酶的檢測(cè)。

圖1psiCHECK-2-ANLN測(cè)序圖(含有2個(gè)miR-217結(jié)合位點(diǎn))

圖2psiCHECK-2-mutANLN測(cè)序圖(2個(gè)miR-217結(jié)合位點(diǎn)已突變)

二、熒光素酶活性變化

轉(zhuǎn)染psiCHECK-2-ANLN各組間的熒光素酶活性差異有統(tǒng)計(jì)學(xué)意義(F=77.405,P<0.01)。而psiCHECK-2-ANLN組、psiCHECK-2-ANLN+NC組及psiCHECK-2-ANLN+NC inhibitor組兩兩比較差異無統(tǒng)計(jì)學(xué)意義。但psiCHECK-2-ANLN+miR-217組的熒光素酶活性較這3組明顯下降，差異有統(tǒng)計(jì)學(xué)意義(P值均<0.01)，psiCHECK-2-ANLN+miR-217 inhibitor組熒光素酶活性較其他4組明顯升高，差異有統(tǒng)計(jì)學(xué)意義(P值均<0.01，表1)。轉(zhuǎn)染psiCHECK-2-mutANLN各組間熒光素酶活性差異無統(tǒng)計(jì)學(xué)意義(P=0.053，表1)。

表1 各組細(xì)胞的熒光素酶活性

三、ANLN蛋白表達(dá)的變化

psiCHECK-2-ANLN+miR-217共轉(zhuǎn)染組PANC1細(xì)胞的ANLN蛋白表達(dá)水平較單純轉(zhuǎn)染psiCHECK-2-ANLN組細(xì)胞的ANLN蛋白表達(dá)明顯下調(diào)(63.23/10181.29對(duì)666.29/9581.17)，而psiCHECK-2-ANLN+miR-217 inhibitor組細(xì)胞的ANLN蛋白表達(dá)較單純轉(zhuǎn)染psiCHECK-2-ANLN組細(xì)胞上調(diào)(2141.22/7838.29對(duì)666.29/9581.17，圖3)。

1：psiCHECK-2-ANLN組；2：psiCHECK-2-ANLN+miR-217組；3：psiCHECK-2-ANLN+miR-217 inhibitor組

圖3各組細(xì)胞ANLN蛋白表達(dá)

討 論

我們前期的研究應(yīng)用高通量的miRNA芯片比較了20例胰腺癌和正常胰腺組織中miRNA表達(dá)譜，發(fā)現(xiàn)miR-217在胰腺癌組織中的表達(dá)較正常胰腺組織明顯下調(diào)，與Zhao等[5]、Szafranska等[6]和Greither等[7]的結(jié)果一致。

文獻(xiàn)報(bào)道，胰腺癌組織中miR-217通過靶向調(diào)節(jié)KRAS參與胰腺癌的發(fā)生和發(fā)展[7]。本研究通過4種生物學(xué)軟件分析并結(jié)合相關(guān)文獻(xiàn)資料，推測(cè)ANLN可能為miR-217調(diào)控的靶基因。

ANLN是一種肌動(dòng)蛋白結(jié)合蛋白，位于染色體7p14.2，編碼1125個(gè)氨基酸，包括一個(gè)保守的N端的肌動(dòng)蛋白(F-actin)和肌球蛋白(non muscle myosin II)結(jié)合區(qū)域、C端PH結(jié)構(gòu)域，在胞質(zhì)分裂中有重要作用[8-12]。研究發(fā)現(xiàn)[13-14]，ANLN在多種惡性腫瘤中均有高表達(dá)，而腫瘤組織中ANLN的基因位點(diǎn)并沒有顯著擴(kuò)增。Suzuki等[18]在非小細(xì)胞肺癌的研究中發(fā)現(xiàn)ANLN可能通過與RhoA相互作用提高癌細(xì)胞的遷徙能力；同時(shí)高表達(dá)ANLN的肺癌患者預(yù)后差。Olakowski等[15]報(bào)道，ANLN在約50%的胰腺癌中高表達(dá)可以作為診斷胰腺癌的一個(gè)新的分子標(biāo)記物。

本研究構(gòu)建雙熒光素酶報(bào)告載體檢測(cè)熒光素酶活性，結(jié)果顯示，ANLN 3′UTR能與miR-217相結(jié)合，而miR-217 inhibitor能夠阻斷miR-217與ANLN 3′UTR結(jié)合，突變的miR-217亦不能與ANLN 3′UTR結(jié)合，提示miR-217與ANLN 3′UTR的結(jié)合具有特異性。通過ANLN蛋白檢測(cè)，發(fā)現(xiàn)ANLN 3′UTR和miR-217共轉(zhuǎn)染的PANC1細(xì)胞的ANLN蛋白表達(dá)較單純轉(zhuǎn)染ANLN 3′UTR組細(xì)胞明顯下降，而ANLN 3′UTR和miR-217 inhibitor共轉(zhuǎn)染的PANC1細(xì)胞的ANLN蛋白表達(dá)有所提高，提示miR-217可以調(diào)控ANLN蛋白表達(dá)。因此，可以推測(cè)在胰腺癌細(xì)胞系PANC1中，ANLN是miR-217的靶基因。

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IdentificationofmicroRNA-217targetedgeneANLNinpancreaticcancerPANC1cells

JIANGYin,HOUBao-hua,JIANZhi-xiang,WANGHui-ling,CUIPeng,OUJin-rui.

DepartmentofGeneralSurgery,People′sHospitalofGuangdongProvince,Guangzhou510080,China

Correspondingauthor:OUJin-rui,Email:hbh1000@126.com

ObjectiveTo identify the miR-217 targeted gene ANLN by experiment.MethodsBioinformatic algorithms were used to predict the potential targets of miR-217. Then, ANLN binding with miR-217 and mutant ANLN (mutANLN) sequence were designed and synthesized, and their amplified fragments were inserted into plasmid psiCHECK-2, and recombinant plasmid psiCHECK-2-ANLN and psiCHECK-2-mutANLN were reconstructed. The two recombinant plasmids were co-transfected into pancreatic cancer cell line PANC1 with miR-217, miR-217 inhibitor, NC, NC inhibitor by liposome,respectively. Dual luciferase reporter system was used to determine the luciferase activity, and Western blot was used to measure the expression of ANLN protein.ResultsThe luciferase activities of psiCHECK-2-ANLN, psiCHECK-2-ANLN+miR-217, psiCHECK-2ANLN+miR-217 inhibitor, psiCHECK-2ANLN+NC, psiCHECK-2-ANLN+NC inhibitor were 2.221±0.188, 0.769±0.061, 3.764±0.371, 2.265±0.201, 2.242±0.018, and the difference among these groups was statistically significant (F=77.405,P<0.001), but the difference among psiCHECK-2ANLN group, psiCHECK-2-ANLN+NC group and psiCHECK-2-ANLN+NC inhibitor group was not statistically significant. However, luciferase activities of psiCHECK-2-ANLN+miR-217 group were significantly decreased when compared with other 3 groups, and luciferase activity of psiCHECK-2-ANLN+miR-217 inhibitor group were significantly increased when compared with other 4 groups (allP<0.001). Luciferase activities of groups transfected with psiCHECK-2-mutANLN was not significantly different (P=0.053). The expression of ANLN protein in PANC1 with psiCHECK-2-ANLN+miR-217 co-transfection was significantly down-regulated when compared with that with psiCHECK-2-ANLN transfection alone.ConclusionsANLN is one of the direct target genes of miR-217 in PANC1 cells.

Pancreatic neoplasms； MicroRNAs; miR-217； Gene； ANLN

2013-02-04)

(本文編輯：呂芳萍)

10.3760/cma.j.issn.1674-1935.2013.03.008

廣東省自然科學(xué)基金(2011010005036)；衛(wèi)生公益性行業(yè)科研專項(xiàng)經(jīng)費(fèi)項(xiàng)目(201202007)

510080 廣東廣州，廣東省人民醫(yī)院普通外科，廣東省醫(yī)學(xué)科學(xué)院

區(qū)金銳，Email:hbh1000@126.com