下調(diào)miR-21表達(dá)抑制鼻咽癌CNE2細(xì)胞增殖和侵襲

南華大學(xué)附屬南華醫(yī)院急診科，湖南 衡陽421002

下調(diào)miR-21表達(dá)抑制鼻咽癌CNE2細(xì)胞增殖和侵襲

周克兵 谷剛 曹昕

南華大學(xué)附屬南華醫(yī)院急診科，湖南 衡陽421002

背景與目的：miR-21在人類多種腫瘤中異常高表達(dá)。本研究探討干擾miR-21表達(dá)對(duì)鼻咽癌CNE2細(xì)胞增殖、遷移和侵襲的影響。方法：使用脂質(zhì)體將miR-21 inhibitor轉(zhuǎn)染CNE2細(xì)胞，以無關(guān)序列(NC inhibitor)作為陰性對(duì)照。采用qRT-PCR技術(shù)驗(yàn)證miR-21 inhibitor轉(zhuǎn)染的CNE2細(xì)胞中miR-21的表達(dá)水平；通過MTS法、細(xì)胞劃痕、Transwell侵襲實(shí)驗(yàn)觀察下調(diào)miR-21表達(dá)對(duì)CNE2細(xì)胞增殖、遷移和侵襲的影響。結(jié)果：轉(zhuǎn)染miR-21 inhibitor的CNE2細(xì)胞中miR-21的表達(dá)明顯下調(diào)，并且呈濃度依賴性。表明轉(zhuǎn)染miR-21 inhibitor能有效抑制CNE2細(xì)胞中miR-21的表達(dá)。轉(zhuǎn)染miR-21 inhibitor的CNE2細(xì)胞與對(duì)照細(xì)胞相比，增殖速度明顯減慢，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。細(xì)胞劃痕實(shí)驗(yàn)顯示，下調(diào)miR-21表達(dá)抑制CNE2細(xì)胞遷移(P＜0.05)。Transwell侵襲實(shí)驗(yàn)結(jié)果顯示，下調(diào)miR-21表達(dá)抑制CNE2細(xì)胞侵襲(P＜0.05)。結(jié)論：miR-21能促進(jìn)鼻咽癌細(xì)胞增殖、遷移和侵襲，其可能在鼻咽癌的發(fā)生、發(fā)展中發(fā)揮重要作用。

鼻咽癌；miR-21；細(xì)胞增殖；細(xì)胞侵襲

miRNA是由長(zhǎng)約19～24個(gè)核苷酸組成的非編碼小分子RNA，它通過降解靶mRNA或抑制靶mRNA的翻譯來調(diào)節(jié)靶基因表達(dá)。MiRNAs參與生命過程中一系列的重要進(jìn)程，包括生長(zhǎng)發(fā)育、細(xì)胞增殖、分化、細(xì)胞運(yùn)動(dòng)、新陳代謝等［1］。研究發(fā)現(xiàn)，人類多種腫瘤中存在miRNA基因異?；虮磉_(dá)異常［2］。miRNA通過癌基因和抑癌基因參與細(xì)胞增殖、凋亡和分化的調(diào)控，在腫瘤的發(fā)生、發(fā)展過程中發(fā)揮著重要的生物學(xué)功能［3-5］。研究表明，miR-21在乳腺癌、肺癌、胃癌、胰腺癌等組織和細(xì)胞中呈高表達(dá)［6-9］，其可能作為癌microRNA促進(jìn)腫瘤的發(fā)生發(fā)展［10-11］。相對(duì)于對(duì)照鼻咽黏膜組織，miR-21在鼻咽癌組織中表達(dá)顯著上調(diào)，并且與鼻咽癌臨床進(jìn)展及轉(zhuǎn)移呈正相關(guān)［12］。本研究通過在鼻咽癌CNE2細(xì)胞中轉(zhuǎn)染miR-21 inhibitor，探討miR-21下調(diào)對(duì)鼻咽癌細(xì)胞增殖、侵襲、遷移的影響，從而初步揭示miR-21在鼻咽癌中的生物學(xué)功能。

1 材料和方法

1.1 主要材料

LipofectamineTM2000轉(zhuǎn)染試劑購自美國Invitrogen公司。qRT-PCR miRNA 檢測(cè)試劑盒購自上海吉瑪生物公司。miRNA inhibitor為Ambion公司產(chǎn)品。miR-21 inhibitor序列為5’-UCAACAUCAGUCUGAUAAGCUA-3’，NC inhibitor序列為5’-CAGUACUUUUGUGUAGUA CAA-3’。RPMI-1640培養(yǎng)基購自Hyclone公司、胎牛血清購自杭州四季青公司。MTS細(xì)胞增殖和毒性檢測(cè)試劑盒購自美國Promega公司。鋪有基質(zhì)膠的Transwell侵襲小室(8.0 μm孔徑)購自BD Biosciences公司。

1.2 細(xì)胞培養(yǎng)

鼻咽癌細(xì)胞株CNE2為典型低分化鱗癌細(xì)胞，由本實(shí)驗(yàn)室保存，培養(yǎng)于含10%小牛血清的RPMI-1640，在95%濕度、CO2體積分?jǐn)?shù)為5%、37 ℃條件下培養(yǎng)。

1.3 細(xì)胞轉(zhuǎn)染

轉(zhuǎn)染前1d將細(xì)胞接種于6孔板中，待貼壁細(xì)胞融合度達(dá)40%～60%時(shí)，根據(jù)LipofectamineTM2000說明書用無血清培養(yǎng)基進(jìn)行轉(zhuǎn)染。轉(zhuǎn)染后6 h換新鮮完全培養(yǎng)基，繼續(xù)擴(kuò)大培養(yǎng)以用于后續(xù)實(shí)驗(yàn)。

1.4 qRT-PCR檢測(cè)

逆轉(zhuǎn)錄按上海吉瑪生物公司轉(zhuǎn)錄miRNA逆轉(zhuǎn)錄試劑盒進(jìn)行，反應(yīng)體系：總RNA 70 ℃預(yù)變性10 min，10×Buffer 2 μL，10 mmol/L MgCl24 μL，dNTP 2 μL，Ribonuclease Inhibitor 0.5 μL，AMV逆轉(zhuǎn)錄酶(20 U)0.6 μL，Oligo(dT)15 Primer 1 μL，總RNA 1 μg，無酶水至20 μL。反應(yīng)條件：42 ℃ 1 h，95 ℃ 5 min，4 ℃ 10 min。

PCR反應(yīng)體系(SYBR Premix Ex TaqTM 20 μL體系)：SYBR Premix Ex TaqTM 10 μL，特異性引物(10 μmol/L) 0.4 μL，連通Primer (10 μmol/L) 0.4 μL，cDNA模板2 μL，蒸餾水加至20 μL。采用Bio-Rad IQ5實(shí)時(shí)定量PCR儀進(jìn)行PCR反應(yīng)，反應(yīng)條件：95 ℃ 30 s；95 ℃延伸5 s，共40個(gè)循環(huán)，60 ℃退火30 s。miR-21特異性引物序列為5’-TAGCTTATCAGACTGATGTT-3’。

1.5 MTS法檢測(cè)細(xì)胞增殖活性

MTS是一種四唑鹽化合物，是一種常用的細(xì)胞存活和生長(zhǎng)的檢測(cè)方法［13］。其檢測(cè)原理是活細(xì)胞線粒體中琥珀酸脫氫酶能夠代謝還原MTS，生成紫色可溶于細(xì)胞培養(yǎng)液的甲瓚，可通過酶標(biāo)儀測(cè)定甲瓚的吸光度值來確定活細(xì)胞的相對(duì)數(shù)量。

miR-21 inhibitor轉(zhuǎn)染CNE2細(xì)胞24 h后，將細(xì)胞接種于96孔板中，每組設(shè)6個(gè)復(fù)孔，放置37 ℃、CO2體積分?jǐn)?shù)為5%的培養(yǎng)箱中培養(yǎng)。在未接種細(xì)胞的孔中加入RPMI-1640培養(yǎng)基中作為調(diào)零孔。接種后24、48、72和96 h各檢測(cè)1次。檢測(cè)時(shí)每孔加20 μL MTS檢測(cè)試劑，37 ℃溫育2 h，用酶標(biāo)儀測(cè)定570 nm波長(zhǎng)吸光度值(A570)，并繪制生長(zhǎng)曲線觀察miR-21下調(diào)對(duì)鼻咽癌細(xì)胞CNE2增殖的影響。實(shí)驗(yàn)重復(fù)3次。

1.6 細(xì)胞劃痕實(shí)驗(yàn)

miR-21 inhibitor轉(zhuǎn)染CNE2細(xì)胞24 h后，將細(xì)胞接種24孔板中，待細(xì)胞融合度達(dá)到90%時(shí)，用10 μL移液槍槍頭，以載玻片作依靠在每孔中心軸進(jìn)行劃痕并拍照(0 h)，37 ℃、CO2體積分?jǐn)?shù)為5%培養(yǎng)箱中繼續(xù)培養(yǎng)24 h并拍照，測(cè)量劃痕愈合程度。實(shí)驗(yàn)重復(fù)3次。

1.7 Transwell侵襲實(shí)驗(yàn)

鋪有基質(zhì)膠的Transwell侵襲實(shí)驗(yàn)可用于觀察細(xì)胞侵襲性［14］。miR-21 inhibitor轉(zhuǎn)染CNE2細(xì)胞24 h后，消化細(xì)胞，用無血清培養(yǎng)基洗滌2次，再用無血清培養(yǎng)基重懸細(xì)胞，細(xì)胞計(jì)數(shù)，調(diào)整細(xì)胞數(shù)為1×105/mL。在Transwell下室加入800 μL含15%FCS的培養(yǎng)基。在Transwell上室加入250 μL細(xì)胞懸液，37 ℃培養(yǎng)48 h。取出Transwell，擦掉靠上室側(cè)膜的細(xì)胞，PBS洗滌，將Transwell用4%甲醛溶液固定10 min，結(jié)晶紫染色，顯微鏡下觀察、照相，隨機(jī)選取5個(gè)高倍視野(×200)進(jìn)行細(xì)胞計(jì)數(shù)，并計(jì)算平均值。實(shí)驗(yàn)重復(fù)3次。

1.8 統(tǒng)計(jì)學(xué)處理

采用統(tǒng)計(jì)軟件SPSS 13.0進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果數(shù)據(jù)以x±s 表示，兩組間比較采用t檢驗(yàn)；3組或3組以上比較采用One-way ANOVA檢驗(yàn)。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 轉(zhuǎn)染miR-21 inhibitor有效抑制CNE2細(xì)胞中miR-21表達(dá)

首先，運(yùn)用qRT-PCR方法分析鼻咽癌CNE2細(xì)胞與正常鼻咽上皮細(xì)胞NP69細(xì)胞中miR-21表達(dá)情況，結(jié)果發(fā)現(xiàn)CNE2細(xì)胞中miR-21表達(dá)明顯高于NP69細(xì)胞，約為NP69的5.3倍(圖1)。然后分別用NC inhibitor及miR-21 inhibitor(終濃度為10、20、40 μmol/L)轉(zhuǎn)染CNE2細(xì)胞，48 h后抽提細(xì)胞總RNA，運(yùn)用qRTPCR驗(yàn)證miR-21 inhibitor轉(zhuǎn)染的細(xì)胞中miR-21的表達(dá)。結(jié)果表明，轉(zhuǎn)染不同濃度miR-21 inhibitor的CNE2細(xì)胞中，miR-21的表達(dá)明顯下調(diào)，并且呈濃度依賴性(圖2)。表明miR-21 inhibitor能有效抑制CNE2細(xì)胞中miR-21的表達(dá)。

圖 1 qRT-PCR檢測(cè)CNE2和NP69細(xì)胞中miR-21表達(dá)水平Fig. 1 Analysis of miR-21 expression in CNE2 and NP69 cells by qRT-PCR

圖 2 qRT-PCR驗(yàn)證miR-21 inhibitor轉(zhuǎn)染細(xì)胞后miR-21表達(dá)水平Fig. 2 Analysis of miR-21 expression in CNE2 cells transfected with miR-21 inhibitor by qRT-PCR

2.2 miR-21下調(diào)后CNE2細(xì)胞增殖速度減慢

MTS法檢測(cè)結(jié)果發(fā)現(xiàn)，轉(zhuǎn)染不同濃度miR-21 inhibitor的CNE2細(xì)胞從第48小時(shí)起增殖速度明顯減慢。統(tǒng)計(jì)分析各組在96 h時(shí)的細(xì)胞增殖情況發(fā)現(xiàn)，轉(zhuǎn)染10、20、40 μmol/L的miR-21 inhibitor組A570分別為1.18±0.09、0.98±0.09、0.71±0.06，與對(duì)照細(xì)胞組(1.51±0.12)相比，差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05)，并且呈現(xiàn)明顯的濃度依賴性(圖3)。這表明下調(diào)miR-21表達(dá)抑制鼻咽癌細(xì)胞CNE2增殖。

圖 3 下調(diào)miR-21對(duì)CNE2細(xì)胞增殖的影響Fig. 3 Effect of miR-21 downregulation in CNE2 cells on cell proliferation

2.3 miR-21表達(dá)下調(diào)抑制CNE2細(xì)胞遷移

細(xì)胞劃痕實(shí)驗(yàn)顯示劃痕后24 h，轉(zhuǎn)染miR-21 inhibitor(20 μmol/L)的CNE2細(xì)胞劃痕愈合率明顯低于對(duì)照組細(xì)胞(P＜0.05，圖4)，表明下調(diào)miR-21表達(dá)明顯抑制CNE2細(xì)胞的遷移。

2.4 下調(diào)miR-21表達(dá)抑制CNE2細(xì)胞侵襲

本研究利用鋪有基質(zhì)膠的Transwell小室觀察miR-21下調(diào)對(duì)細(xì)胞侵襲的影響。結(jié)果顯示，轉(zhuǎn)染miR-21 inhibitor(20 μmol/L)的CNE2細(xì)胞較對(duì)照細(xì)胞侵襲力明顯降低，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，圖5)。

圖 4 miR-21表達(dá)下調(diào)對(duì)CNE2細(xì)胞遷移的影響Fig. 4 Effect of miR-21 downregulation in CNE2 cells on cell migration

圖 5 miR-21表達(dá)下調(diào)對(duì)CNE2細(xì)胞侵襲的影響Fig. 5 Effect of miR-21 downregulation in CNE2 cells on cell invasion

3 討 論

miRNA是一類廣泛存在于動(dòng)植物體內(nèi)的非編碼小RNA，主要參與基因轉(zhuǎn)錄后水平調(diào)控，通過與其目標(biāo)mRNA分子的3'端非編碼區(qū)域(3'-untranslated region，3'UTR)互補(bǔ)匹配降解靶mRNA或抑制其翻譯。miRNA作為一類新型基因調(diào)控劑，與腫瘤的發(fā)生、發(fā)展密切相關(guān)。越來越多的研究顯示，在人類腫瘤中存在miRNA表達(dá)的失調(diào)，miRNA通過癌基因和抑癌基因參與細(xì)胞增殖、凋亡和分化的調(diào)控，在腫瘤的發(fā)生、發(fā)展過程中發(fā)揮著重要的生物學(xué)功能。

miR-21是最早發(fā)現(xiàn)的哺乳動(dòng)物microRNAs之一，成熟miR-21在不同物種之間高度保守。研究表明，miR-21在肺癌、前列腺癌、結(jié)腸癌、胃癌、肝癌等多種惡性腫瘤中過表達(dá)。Toiyama［15］等報(bào)道，外周血miR-21可能是結(jié)直腸癌的診斷和預(yù)后標(biāo)志物。鄧敏等［12］報(bào)道，miR-21在鼻咽癌組織中異常高表達(dá)，其表達(dá)與鼻咽癌臨床進(jìn)展及轉(zhuǎn)移呈正相關(guān)，Kaplan-Meier生存曲線分析發(fā)現(xiàn)，鼻咽癌患者miR-21表達(dá)越高，預(yù)后越差。這些結(jié)果提示，miR-21在鼻咽癌的發(fā)生、發(fā)展中起著重要作用。本研究將miR-21 inhibitor轉(zhuǎn)染至鼻咽癌CNE2細(xì)胞中，觀察miR-21下調(diào)對(duì)細(xì)胞增殖、遷移侵襲的影響。結(jié)果發(fā)現(xiàn)，轉(zhuǎn)染不同濃度miR-21 inhibitor的CNE2細(xì)胞從第48小時(shí)起增殖速度明顯減慢。細(xì)胞劃痕及Transwell侵襲實(shí)驗(yàn)結(jié)果顯示，miR-21表達(dá)下調(diào)抑制CNE2細(xì)胞遷移、侵襲性。這些結(jié)果表明，miR-21能促進(jìn)鼻咽癌細(xì)胞增殖和侵襲，很可能作為癌基因參與鼻咽癌的發(fā)生、發(fā)展。文獻(xiàn)報(bào)道m(xù)iR-21作為癌miRNA能夠通過調(diào)控靶基因如PTEN、CCL20、FAS，促進(jìn)肺癌、胃癌、結(jié)腸、腎癌等腫瘤細(xì)胞增殖、生長(zhǎng)、侵襲［7,9-11］。本研究結(jié)果與文獻(xiàn)報(bào)道的一致，miR-21將有可能成為治療鼻咽癌的潛在靶點(diǎn)。

［1］ HE L, HANNON G J. MicroRNAs: small RNAs with a big role in gene regulation ［J］.Nat Rev Genet, 2004, 5(7): 522-531.

［2］ CHEN P S, SU J L, HUNG M C. Dysregulation of MicroRNAs in cancer ［J］. J Biomed Sci, 2012, 19(1): 90.

［3］ MANIKANDAN J, AARTHI J J, KUMAR S D, et al. Oncomirs: the potential role of non-coding microRNAs in understanding cancer ［J］. Bioinformation, 2008, 2(8): 330-334.

［4］ CHIN L J, RATNER E, LENG S, et al. A SNP in a let-7 microRNA complementary site in the KRAS 3’ untranslated region increases non-small cell lung cancer risk ［J］. Cancer Res, 2008, 68(20): 8535-8540.

［5］ SENGUPTA S, DEN BOON J A, CHEN I H, et al. MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, upregulating mRNAs encoding extracellular matrix proteins［J］. Proc Natl Acad Sci U S A, 2008, 105(15): 5874-5878.

［6］ ZHU Q, WANG Z, HU Y, et al. miR-21 promotes migration and invasion by the miR-21-PDCD4-AP-1 feedback loop in human hepatocellular carcinoma ［J］. Oncol Rep, 2012, 27(5): 1660-1668.

［7］ ZHANG B G, LI J F, YU B Q, et al. microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN ［J］. Oncol Rep, 2012, 27(4): 1019-1026.

［8］ DARIDO C, GEORGY S R, WILANOWSKI T, et al. Targeting of the tumor suppressor GRHL3 by a miR-21-dependent proto-oncogenic network results in PTEN loss and tumorigenesis ［J］. Cancer Cell, 2011, 20(5): 635-648.

［9］ VICINUS B, RUBIE C, FAUST S K, et al. miR-21 functionally interacts with the 3’UTR of chemokine CCL20 and downregulates CCL20 expression in miR-21 transfected colorectal cancer cells ［J］. Cancer Lett, 2012, 316(1): 105-112.

［10］ MEDINA P P, NOLDE M, SLACK F J. OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma ［J］. Nature, 2010, 467(7311): 86-90.

［11］ HATLEY M E, PATRICK D M, GARCIA M R, et al. Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21 ［J］. Cancer Cell, 2010, 18(3): 282-293.

［12］ 鄧敏, 谷依學(xué), 鄭國沛, 等. 鼻咽癌組織中miR-21的表達(dá)變化及意義［J］. 山東醫(yī)藥, 2012, 52 (47): 10-12.

［13］ GAROFALO M, ROMANO G, DI LEVA G, et al. EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers［J］. Nat Med, 2011, 18(1): 74-82

［14］ DENG M, TANG H L, LU X H, et al. miR-26a suppresses tumor growth and metastasis by targeting FGF9 in gastric cancer ［J］. PLoS One, 2013, 8(8): e72662.

［15］ TOIYAMA Y, TAKAHASHI M, HUR K, et al. Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer［J］. J Natl Cancer Inst, 2013, 105(12): 849-859.

miR-21 downregulation attenuates cell proliferation, migration and invasion in nasopharyngeal carcinoma

ZHOU Ke-bing, GU Gang, CAO Xin (Emergency Department, Affiliated Nanhua Hospital, University of South China, Hengyang Hunan 421002, China)

ZHOU Ke-bing E-mail: 562136355@qq.com

Background and purpose: miR-21 is ovexpressed in various types of human cancers. This study was designed to investigate the effect of miR-21 knockdown on cell proliferation, migration and invasion of nasopharyngeal carcinoma (NPC) cell line CNE2. Methods: CNE2 was transfected with miR-21 inhibitor by LipofectamineTM2000. Meanwhile CNE2 was transfected with NC inhibitor as negative control. qRT-PCR was used to detect the miR-21 expression in these cells. The effects of miR-21 downregulation on cell proliferation, migration and invasion were evaluated by MTS, wound-healing Transwell and invasion assays. Results: miR-21 expression was remarkably downregulated in miR-21 inhibitor-transfected cells in concentration-dependent manner, indicating transfection with miR-21 inhibitor can effectively reduce expression level of miR-21 in CNE2 cells. Transfection of miR-21 inhibitor into CNE2 cells led to a significant decrease in cell proliferation rate compared with control cells (P＜0.05). miR-21 downregulation results in reduction of cell migration(P＜0.05). Moreover, the cell invasion by Transwell invasion assay was reduced in miR-21-downregulated cells relative to control cells. Conclusion: miR-21 can promote cell proliferation, migration and invasion of NPC cells. And it maybe plays an important role in tumorigenesis and development of NPC.

Nasopharyngeal carcinoma; MiR-21; Cell proliferation; Cell invasion

10.3969/j.issn.1007-3969.2013.11.002

R739.63

：A

：1007-3639(2013)11-0863-05

2013-06-11

2013-10-20）

國家自然科學(xué)基金(No：81101526)。

周克兵 E-mail: 562136355@qq.com