胰腺癌PANC1细胞microRNA-217靶基因ANLN的鉴定

江寅 侯宝华 简志祥 王慧玲 崔鹏 区金锐

·论著·

胰腺癌PANC1细胞microRNA-217靶基因ANLN的鉴定

江寅 侯宝华 简志祥 王慧玲 崔鹏 区金锐

目的通过实验验证ANLN为microRNA-217(miR-217)的靶基因。方法使用生物学软件预测miR-217调控的靶基因可能为ANLN。设计并合成mR-217结合的ANLN及突变型ANLN(mutANLN)序列，将其PCR扩增的片段插入表达质粒psiCHECK-2，构建重组质粒psiCHECK-2-ANLN及psiCHECK-2-mutANLN。采用脂质体法将2个重组质粒单独或分别与miR-217、miR-217 inhibitor及与人同源性远的miRNA序列(NC)、NC inhibitor共转染胰腺癌PANC1细胞，采用双荧光素酶报告系统检测荧光素酶活性，采用蛋白质印迹法检测ANLN蛋白的表达。结果转染psiCHECK-2-ANLN、psiCHECK-2-ANLN+miR-217、psiCHECK-2-ANLN+miR-217 inhibitor、psiCHECK-2-ANLN+NC、psiCHECK-2-ANLN+NC inhibitor各组间的荧光素酶活性分别为2.221±0.188、0.769±0.061、3.764±0.371、2.265±0.201、2.242±0.018，差异有统计学意义(F=77.405,P<0.01)，但psiCHECK-2-ANLN组、psiCHECK-2-ANLN+NC组及psiCHECK-2-ANLN+NC inhibitor组两两比较差异无统计学意义，而psiCHECK-2-ANLN+miR-217组的荧光素酶活性较这3组明显下降，psiCHECK-2-ANLN+miR-217 inhibitor组活性较其他4组明显升高(P值均<0.01)。转染psiCHECK-2-mutANLN各组间荧光素酶活性差异无统计学意义(P=0.053)。psiCHECK-2-ANLN+miR-217共转染组PANC1细胞的ANLN蛋白表达水平较单纯转染psiCHECK-2-ANLN组细胞的ANLN蛋白表达明显下调。结论在胰腺癌中，ANLN可能是miR-217的直接靶基因。

胰腺肿瘤； 微RNAs； miR-217； 基因； ANLN

MicroRNA(miRNA)是一类由18～23个核苷酸构成的单链非编码RNA分子，通过靶向结合mRNA的3′非编码区域(3′UTR)，降解mRNA或者抑制其翻译，导致靶基因的转录后沉默，从而参与靶基因功能的调节。miRNA与胰腺癌的发生和发展存在密切关系，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达差异，可作为肿瘤标志物用于胰腺癌与正常胰腺、慢性胰腺炎、胰腺内分泌肿瘤等的鉴别诊断及预后预测[1-4]。我们前期的研究发现，miR-217在胰腺癌组织中的表达显著下调。ANLN是一种肌动蛋白结合蛋白，在包括胰腺癌在内的多种恶性肿瘤中均有高表达。本研究进一步验证ANLN为miR-217的靶基因。

材料与方法

一、双荧光素酶报告载体构建

通过TargetScan、picTar、miRanda和DIANA microT 4种生物学软件分析并结合相关文献资料，推测ANLN为miR-217调控的靶基因，且有2个结合位点，在132～138及660～666处。

从TargetScan网站上获取人类ANLN基因3′UTR区与hsa-miR-217结合的序列，设计目标序列和突变序列，两端分别加XhoⅠ和NotⅠ酶切位点。野生型ANLN引物序列：正义 5′-CCGCTCGAGAC-CGGGAAATTTCCATGCTATC-3′，反义5′-AAG-GAAAAAAGCGGCCGCTCCTTTAGACATTTACAGGT-ATTTATTTGAG-3′；突变引物1序列：正义5′-CCA-ATATTCACTACGTATTGCTAGCTATTTATATCTTTTG-TATGT-3′，反义5′-ACATACAAAAGATATAAATAGCTAGCAATACGTAGTGAATATTGG-3′；突变引物2序列：正义5′-CATTTACTCAGCTACTATATGCTAGCT-GTGGTGCACATTTTCACAGAA-3′，反义5′-TTCTGTGAAAATGTGCACCACAGCTAGCATATAG-TAGCTGAGTAAATG-3′。以健康志愿者全血DNA为模板，将目标序列和突变序列进行PCR扩增，PCR反应条件：94℃ 5min，94℃ 30 s、 58℃ 30 s，72℃ 75 s，32个循环，72℃ 5 min。扩增片段经电泳分离、回收、纯化、应用限制酶(Xho Ⅰ和NotⅠ)切割后插入表达质粒psiCHECK-2的多克隆位点。构建含有miR-217结合位点的ANLN重组质粒psiCHECK-2-ANLN。以psiCHECK-2-ANLN为模板，通过突变序列PCR扩增，构建缺失miR-217结合位点的突变型重组质检psiCHECK-2-mutANLN。

二、荧光素酶实验

胰腺癌PANC1细胞常规培养、传代。取对数生长期细胞，按2×104个/孔密度接种于24孔板上，分为psiCHECK-2-ANLN和psiCHECK-2-mutANLN两大组，每个大组又分为对照组、miR-217、miR-217 inhibitor、与人同源性远的miRNA序列(NC)、NC inhibitor 5组。对照组仅转染各自的重组质粒，其他4组则为重组质粒分别与miR-217、miR-217 inhibitor、NC、NC inhibitor共转染。细胞转染采用LipofeetamineTM2000(美国Invitrogen公司)。转染48 h后采用双荧光报告系统检测试剂盒(Promega公司，E1910)行荧光素酶活性检测，以不转染细胞为空白对照。实验重复3次，每次设3个复孔。

三、ANLN蛋白表达检测

提取转染psiCHECK-2-ANLN、psiCHECK-2-ANLN+miR-217、psiCHECK-2-ANLN+ miR-217 inhibitor组细胞总蛋白，牛血清蛋白(BSA)法测定蛋白含量。常规行蛋白质印迹法检测细胞ANLN蛋白的表达。兔抗人ANLN一抗购自Abcam公司(ab99352)，工作浓度1∶4000，羊抗兔辣根过氧化物酶(HRP)标记的IgG 购自Southern Biotech公司，工作浓度1∶5000。采用数码成像分析系统软件IPWIN6.0对条带进行扫描。

四、统计学分析

结 果

一、双荧光素酶报告载体

含有miR-217结合位点及缺失miR-217结合位点的荧光素酶报告载体psiCHECK-2-ANLN和psiCHECK-2-mutANLN测序结果如图1、2。经局部对比基本检索工具(basic local alignment search tool, BLAST))结果分析，ANLN 3 ′UTR及mut ANLN 3 ′UTR片段成功地克隆入双萤光报告载体psiCHECK-2中，可以用于后续萤光素酶的检测。

图1psiCHECK-2-ANLN测序图(含有2个miR-217结合位点)

图2psiCHECK-2-mutANLN测序图(2个miR-217结合位点已突变)

二、荧光素酶活性变化

转染psiCHECK-2-ANLN各组间的荧光素酶活性差异有统计学意义(F=77.405,P<0.01)。而psiCHECK-2-ANLN组、psiCHECK-2-ANLN+NC组及psiCHECK-2-ANLN+NC inhibitor组两两比较差异无统计学意义。但psiCHECK-2-ANLN+miR-217组的荧光素酶活性较这3组明显下降，差异有统计学意义(P值均<0.01)，psiCHECK-2-ANLN+miR-217 inhibitor组荧光素酶活性较其他4组明显升高，差异有统计学意义(P值均<0.01，表1)。转染psiCHECK-2-mutANLN各组间荧光素酶活性差异无统计学意义(P=0.053，表1)。

表1 各组细胞的荧光素酶活性

三、ANLN蛋白表达的变化

psiCHECK-2-ANLN+miR-217共转染组PANC1细胞的ANLN蛋白表达水平较单纯转染psiCHECK-2-ANLN组细胞的ANLN蛋白表达明显下调(63.23/10181.29对666.29/9581.17)，而psiCHECK-2-ANLN+miR-217 inhibitor组细胞的ANLN蛋白表达较单纯转染psiCHECK-2-ANLN组细胞上调(2141.22/7838.29对666.29/9581.17，图3)。

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

图3各组细胞ANLN蛋白表达

讨 论

我们前期的研究应用高通量的miRNA芯片比较了20例胰腺癌和正常胰腺组织中miRNA表达谱，发现miR-217在胰腺癌组织中的表达较正常胰腺组织明显下调，与Zhao等[5]、Szafranska等[6]和Greither等[7]的结果一致。

文献报道，胰腺癌组织中miR-217通过靶向调节KRAS参与胰腺癌的发生和发展[7]。本研究通过4种生物学软件分析并结合相关文献资料，推测ANLN可能为miR-217调控的靶基因。

ANLN是一种肌动蛋白结合蛋白，位于染色体7p14.2，编码1125个氨基酸，包括一个保守的N端的肌动蛋白(F-actin)和肌球蛋白(non muscle myosin II)结合区域、C端PH结构域，在胞质分裂中有重要作用[8-12]。研究发现[13-14]，ANLN在多种恶性肿瘤中均有高表达，而肿瘤组织中ANLN的基因位点并没有显著扩增。Suzuki等[18]在非小细胞肺癌的研究中发现ANLN可能通过与RhoA相互作用提高癌细胞的迁徙能力；同时高表达ANLN的肺癌患者预后差。Olakowski等[15]报道，ANLN在约50%的胰腺癌中高表达可以作为诊断胰腺癌的一个新的分子标记物。

本研究构建双荧光素酶报告载体检测荧光素酶活性，结果显示，ANLN 3′UTR能与miR-217相结合，而miR-217 inhibitor能够阻断miR-217与ANLN 3′UTR结合，突变的miR-217亦不能与ANLN 3′UTR结合，提示miR-217与ANLN 3′UTR的结合具有特异性。通过ANLN蛋白检测，发现ANLN 3′UTR和miR-217共转染的PANC1细胞的ANLN蛋白表达较单纯转染ANLN 3′UTR组细胞明显下降，而ANLN 3′UTR和miR-217 inhibitor共转染的PANC1细胞的ANLN蛋白表达有所提高，提示miR-217可以调控ANLN蛋白表达。因此，可以推测在胰腺癌细胞系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)

(本文编辑：吕芳萍)

10.3760/cma.j.issn.1674-1935.2013.03.008

广东省自然科学基金(2011010005036)；卫生公益性行业科研专项经费项目(201202007)

510080 广东广州，广东省人民医院普通外科，广东省医学科学院

区金锐，Email:hbh1000@126.com