胰腺癌PANC1細胞microRNA-217靶基因ANLN的鑒定

江寅 侯寶華 簡志祥 王慧玲 崔鵬 區金銳

·論著·

胰腺癌PANC1細胞microRNA-217靶基因ANLN的鑒定

江寅 侯寶華 簡志祥 王慧玲 崔鵬 區金銳

目的通過實驗驗證ANLN為microRNA-217(miR-217)的靶基因。方法使用生物學軟件預測miR-217調控的靶基因可能為ANLN。設計并合成mR-217結合的ANLN及突變型ANLN(mutANLN)序列，將其PCR擴增的片段插入表達質粒psiCHECK-2，構建重組質粒psiCHECK-2-ANLN及psiCHECK-2-mutANLN。采用脂質體法將2個重組質粒單獨或分別與miR-217、miR-217 inhibitor及與人同源性遠的miRNA序列(NC)、NC inhibitor共轉染胰腺癌PANC1細胞，采用雙熒光素酶報告系統檢測熒光素酶活性，采用蛋白質印跡法檢測ANLN蛋白的表達。結果轉染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，差異有統計學意義(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計學意義，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)。轉染psiCHECK-2-mutANLN各組間熒光素酶活性差異無統計學意義(P=0.053)。psiCHECK-2-ANLN+miR-217共轉染組PANC1細胞的ANLN蛋白表達水平較單純轉染psiCHECK-2-ANLN組細胞的ANLN蛋白表達明顯下調。結論在胰腺癌中，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個核苷酸構成的單鏈非編碼RNA分子，通過靶向結合mRNA的3′非編碼區域(3′UTR)，降解mRNA或者抑制其翻譯，導致靶基因的轉錄后沉默，從而參與靶基因功能的調節。miRNA與胰腺癌的發生和發展存在密切關系，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差異，可作為腫瘤標志物用于胰腺癌與正常胰腺、慢性胰腺炎、胰腺內分泌腫瘤等的鑒別診斷及預后預測[1-4]。我們前期的研究發現，miR-217在胰腺癌組織中的表達顯著下調。ANLN是一種肌動蛋白結合蛋白，在包括胰腺癌在內的多種惡性腫瘤中均有高表達。本研究進一步驗證ANLN為miR-217的靶基因。

材料與方法

一、雙熒光素酶報告載體構建

通過TargetScan、picTar、miRanda和DIANA microT 4種生物學軟件分析并結合相關文獻資料，推測ANLN為miR-217調控的靶基因，且有2個結合位點，在132～138及660～666處。

從TargetScan網站上獲取人類ANLN基因3′UTR區與hsa-miR-217結合的序列，設計目標序列和突變序列，兩端分別加XhoⅠ和NotⅠ酶切位點。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，將目標序列和突變序列進行PCR擴增，PCR反應條件：94℃ 5min，94℃ 30 s、 58℃ 30 s，72℃ 75 s，32個循環，72℃ 5 min。擴增片段經電泳分離、回收、純化、應用限制酶(Xho Ⅰ和NotⅠ)切割后插入表達質粒psiCHECK-2的多克隆位點。構建含有miR-217結合位點的ANLN重組質粒psiCHECK-2-ANLN。以psiCHECK-2-ANLN為模板，通過突變序列PCR擴增，構建缺失miR-217結合位點的突變型重組質檢psiCHECK-2-mutANLN。

二、熒光素酶實驗

胰腺癌PANC1細胞常規培養、傳代。取對數生長期細胞，按2×104個/孔密度接種于24孔板上，分為psiCHECK-2-ANLN和psiCHECK-2-mutANLN兩大組，每個大組又分為對照組、miR-217、miR-217 inhibitor、與人同源性遠的miRNA序列(NC)、NC inhibitor 5組。對照組僅轉染各自的重組質粒，其他4組則為重組質粒分別與miR-217、miR-217 inhibitor、NC、NC inhibitor共轉染。細胞轉染采用LipofeetamineTM2000(美國Invitrogen公司)。轉染48 h后采用雙熒光報告系統檢測試劑盒(Promega公司，E1910)行熒光素酶活性檢測，以不轉染細胞為空白對照。實驗重復3次，每次設3個復孔。

三、ANLN蛋白表達檢測

提取轉染psiCHECK-2-ANLN、psiCHECK-2-ANLN+miR-217、psiCHECK-2-ANLN+ miR-217 inhibitor組細胞總蛋白，牛血清蛋白(BSA)法測定蛋白含量。常規行蛋白質印跡法檢測細胞ANLN蛋白的表達。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)標記的IgG 購自Southern Biotech公司，工作濃度1∶5000。采用數碼成像分析系統軟件IPWIN6.0對條帶進行掃描。

四、統計學分析

結 果

一、雙熒光素酶報告載體

含有miR-217結合位點及缺失miR-217結合位點的熒光素酶報告載體psiCHECK-2-ANLN和psiCHECK-2-mutANLN測序結果如圖1、2。經局部對比基本檢索工具(basic local alignment search tool, BLAST))結果分析，ANLN 3 ′UTR及mut ANLN 3 ′UTR片段成功地克隆入雙螢光報告載體psiCHECK-2中，可以用于后續螢光素酶的檢測。

圖1psiCHECK-2-ANLN測序圖(含有2個miR-217結合位點)

圖2psiCHECK-2-mutANLN測序圖(2個miR-217結合位點已突變)

二、熒光素酶活性變化

轉染psiCHECK-2-ANLN各組間的熒光素酶活性差異有統計學意義(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計學意義。但psiCHECK-2-ANLN+miR-217組的熒光素酶活性較這3組明顯下降，差異有統計學意義(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，差異有統計學意義(P值均<0.01，表1)。轉染psiCHECK-2-mutANLN各組間熒光素酶活性差異無統計學意義(P=0.053，表1)。

表1 各組細胞的熒光素酶活性

三、ANLN蛋白表達的變化

psiCHECK-2-ANLN+miR-217共轉染組PANC1細胞的ANLN蛋白表達水平較單純轉染psiCHECK-2-ANLN組細胞的ANLN蛋白表達明顯下調(63.23/10181.29對666.29/9581.17)，而psiCHECK-2-ANLN+miR-217 inhibitor組細胞的ANLN蛋白表達較單純轉染psiCHECK-2-ANLN組細胞上調(2141.22/7838.29對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各組細胞ANLN蛋白表達

討 論

我們前期的研究應用高通量的miRNA芯片比較了20例胰腺癌和正常胰腺組織中miRNA表達譜，發現miR-217在胰腺癌組織中的表達較正常胰腺組織明顯下調，與Zhao等[5]、Szafranska等[6]和Greither等[7]的結果一致。

文獻報道，胰腺癌組織中miR-217通過靶向調節KRAS參與胰腺癌的發生和發展[7]。本研究通過4種生物學軟件分析并結合相關文獻資料，推測ANLN可能為miR-217調控的靶基因。

ANLN是一種肌動蛋白結合蛋白，位于染色體7p14.2，編碼1125個氨基酸，包括一個保守的N端的肌動蛋白(F-actin)和肌球蛋白(non muscle myosin II)結合區域、C端PH結構域，在胞質分裂中有重要作用[8-12]。研究發現[13-14]，ANLN在多種惡性腫瘤中均有高表達，而腫瘤組織中ANLN的基因位點并沒有顯著擴增。Suzuki等[18]在非小細胞肺癌的研究中發現ANLN可能通過與RhoA相互作用提高癌細胞的遷徙能力；同時高表達ANLN的肺癌患者預后差。Olakowski等[15]報道，ANLN在約50%的胰腺癌中高表達可以作為診斷胰腺癌的一個新的分子標記物。

本研究構建雙熒光素酶報告載體檢測熒光素酶活性，結果顯示，ANLN 3′UTR能與miR-217相結合，而miR-217 inhibitor能夠阻斷miR-217與ANLN 3′UTR結合，突變的miR-217亦不能與ANLN 3′UTR結合，提示miR-217與ANLN 3′UTR的結合具有特異性。通過ANLN蛋白檢測，發現ANLN 3′UTR和miR-217共轉染的PANC1細胞的ANLN蛋白表達較單純轉染ANLN 3′UTR組細胞明顯下降，而ANLN 3′UTR和miR-217 inhibitor共轉染的PANC1細胞的ANLN蛋白表達有所提高，提示miR-217可以調控ANLN蛋白表達。因此，可以推測在胰腺癌細胞系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

廣東省自然科學基金(2011010005036)；衛生公益性行業科研專項經費項目(201202007)

510080 廣東廣州，廣東省人民醫院普通外科，廣東省醫學科學院

區金銳，Email:hbh1000@126.com