鄧敏劉季芳谷依學(xué)鄭國沛

1.廣州醫(yī)學(xué)院附屬腫瘤醫(yī)院，腫瘤研究所，廣東 廣州510095；

2.南華大學(xué)腫瘤研究所，湖南 衡陽 421001

miR-218對(duì)鼻咽癌細(xì)胞增殖和侵襲的影響

鄧敏1,2劉季芳1谷依學(xué)1鄭國沛1

1.廣州醫(yī)學(xué)院附屬腫瘤醫(yī)院，腫瘤研究所，廣東 廣州510095；

2.南華大學(xué)腫瘤研究所，湖南 衡陽 421001

背景與目的：miR-218在鼻咽癌組織中表達(dá)顯著下調(diào)。本研究探討過表達(dá)miR-218對(duì)人鼻咽癌細(xì)胞增殖和侵襲的影響。方法：使用脂質(zhì)體將成熟miR-218序列模擬物(miR-218 mimics)轉(zhuǎn)染人鼻咽癌細(xì)胞株5-8F，以無關(guān)序列(Scramble mimics)作為陰性對(duì)照。采用qRT-PCR技術(shù)驗(yàn)證miR-218 mimics轉(zhuǎn)染細(xì)胞中miR-218的表達(dá)水平；通過MTS法及Transwell侵襲實(shí)驗(yàn)觀察miR-218過表達(dá)對(duì)5-8F細(xì)胞增殖和侵襲力的影響。結(jié)果：在5-8F細(xì)胞中，轉(zhuǎn)染miR-218 mimics細(xì)胞中miR-218的表達(dá)量是Scramble mimics細(xì)胞的17倍。過表達(dá)miR-218的細(xì)胞與對(duì)照細(xì)胞相比，增殖速度明顯下降，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。Transwell侵襲實(shí)驗(yàn)結(jié)果顯示，轉(zhuǎn)染miR-218 mimics后，與Scramble mimics細(xì)胞相比，5-8F細(xì)胞的侵襲力明顯減慢(P＜0.05)。結(jié)論：miR-218能抑制鼻咽癌細(xì)胞增殖及侵襲，在鼻咽癌的發(fā)生和進(jìn)展中發(fā)揮重要作用。

鼻咽癌；miR-218；細(xì)胞增殖；細(xì)胞侵襲

miRNAs是由長約19～24個(gè)核苷酸組成的非編碼小分子RNA，能通過降解靶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，包括生長發(fā)育、細(xì)胞增殖、分化、細(xì)胞運(yùn)動(dòng)及新陳代謝等［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以發(fā)揮癌基因和抑癌基因的功能參與細(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［2-3］。例如，let-7通過靶向調(diào)控KRAS抑制肺癌的發(fā)生，且其表達(dá)水平與肺癌預(yù)后密切相關(guān)［4-5］。miR-182在黑素瘤中高表達(dá)，通過功能研究發(fā)現(xiàn)過表達(dá)miR-182促進(jìn)黑素瘤轉(zhuǎn)移［6］。近期，本研究通過qRT-PCR技術(shù)檢測了miR-218在鼻咽癌組織與對(duì)照鼻咽黏膜組織(包括正常及炎癥黏膜組織)中表達(dá)差異，發(fā)現(xiàn)鼻咽癌組織相對(duì)于對(duì)照鼻咽黏膜組織，miR-218表達(dá)顯著下調(diào)，并且miR-218表達(dá)與鼻咽癌臨床進(jìn)展及轉(zhuǎn)移呈負(fù)相關(guān)。本研究通過在鼻咽癌細(xì)胞中轉(zhuǎn)染成熟miR-218模擬物(miR-218 mimics)使miR-218高表達(dá)，探討miR-218對(duì)鼻咽癌細(xì)胞增殖、侵襲及遷移的影響，從而初步揭示miR-218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。miR-218 mimics為Ambion公司產(chǎn)品。RPMI-1640培養(yǎng)基購自Hyclone公司、胎牛血清購自杭州四季青。MTS細(xì)胞增殖/毒性檢測試劑盒購自美國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)和轉(zhuǎn)染

5-8F是高致瘤和高轉(zhuǎn)移的鼻咽癌細(xì)胞株，為典型低分化鱗癌細(xì)胞。由本實(shí)驗(yàn)室保存，均培養(yǎng)于含10%小牛血清的RPMI-1640液中，在95%濕度，CO2體積分?jǐn)?shù)為5%，37 ℃條件下。轉(zhuǎn)染前1天將細(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)。

1.3 MTS法檢測細(xì)胞增殖活性

使用脂質(zhì)體將miR-218 mimics轉(zhuǎn)染人鼻咽癌細(xì)胞株5-8F，以無關(guān)序列(Scramble mimics)作為陰性對(duì)照。取miR-218 mimics及Scramble mimics轉(zhuǎn)染24 h后細(xì)胞接種于96孔板中，每組設(shè)6個(gè)復(fù)孔，放入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1次。檢測時(shí)每孔加20 μL MTS檢測試劑，37 ℃溫育2 h，用酶標(biāo)儀測定570 nm波長的吸光度值。實(shí)驗(yàn)重復(fù)3次。

1.4 Transwell侵襲實(shí)驗(yàn)

先將24孔板預(yù)熱至37 ℃。消化miR-218 mimics 及control mimics轉(zhuǎn)染細(xì)胞，用無血清培基洗滌2次，再用無血清培基重懸細(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的培基。在Transwell上室加入250 μL細(xì)胞懸液，37 ℃培養(yǎng)36～48 h。取出Transwell，擦掉靠上室側(cè)膜的細(xì)胞，PBS洗滌，將Transwell用40%甲醛溶液中固定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。

1.5 qRT-PCR檢測

逆轉(zhuǎn)錄按吉瑪生物公司轉(zhuǎn)錄miRNA逆轉(zhuǎn)錄試劑盒進(jìn)行，反應(yīng)體系如下：總RNA 70 ℃預(yù)變性10 min，10×Buffer 2 μL，10 mmol/L MgCl24 μL，dNTP 2 μL，核糖核酸酶抑制劑 0.5 μL，AMV反轉(zhuǎn)錄酶(20 U) 0.6 μL，Oligo(dT)15 Primer 1 μL，總RNA 1 μg，加無酶水至20 μL。反應(yīng)條件42 ℃1 h，95 ℃ 5 min，4 ℃ 10 min。

PCR反應(yīng)體系如下：SYBR Premix Ex TaqTM 10 μL，F(xiàn)orward Primer (10 μmol/L) 0.4 μL，Reverse Primer (10 μmol/L) 0.4 μL，cDNA模板2.0 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)條件為：95.0 ℃變性30 s；60.0 ℃退火30 s，95.0 ℃延伸5 s，共40個(gè)循環(huán)。

1.6 統(tǒng)計(jì)學(xué)處理

2 結(jié) 果

2.1 轉(zhuǎn)染miR-218 mimics后細(xì)胞中miRNA-218表達(dá)變化

miR-218 mimics 及Scramble mimics(陰性對(duì)照)分別轉(zhuǎn)染5-8F細(xì)胞，48 h后抽提細(xì)胞總RNA，運(yùn)用qRT-PCR驗(yàn)證miR-218在細(xì)胞中的表達(dá)。結(jié)果表明，在5-8F細(xì)胞中，轉(zhuǎn)染miR-218 mimics細(xì)胞中miR-218的表達(dá)量是Scramble mimics細(xì)胞的17倍(圖1)。

圖 1 轉(zhuǎn)染miR-218 mimics后5-8F細(xì)胞中miRNA-218表達(dá)變化Fig. 1 Expression level of miRNA-218 in 5-8F cells transfected with miR-218 mimics

2.2 過表達(dá)miR-218對(duì)5-8F細(xì)胞增殖的影響

通過MTS法檢測，繪制生長曲線來觀察過表達(dá)miR-218對(duì)鼻咽癌細(xì)胞增殖的影響。結(jié)果發(fā)現(xiàn)，轉(zhuǎn)染miR-218 mimics的5-8F細(xì)胞從48 h起增殖速度明顯減慢，與Scramble mimics細(xì)胞相比，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，圖2)。

圖 2 過表達(dá)miR-218對(duì)5-8F細(xì)胞增殖的影響Fig. 2 Effect of miR-218 overexpression on cell proliferation in 5-8F cells

2.3 過表達(dá)miR-218對(duì)5-8F細(xì)胞侵襲的影響

本研究利用鋪有基質(zhì)膠的Transwell觀察miR-218對(duì)細(xì)胞侵襲的影響。在Transwell上室加入細(xì)胞懸液(無血清)，在下室加入含15% FCS培養(yǎng)基，顯微鏡下計(jì)數(shù)穿孔細(xì)胞數(shù)。結(jié)果顯示，轉(zhuǎn)染miR-218 mimics的5-8F細(xì)胞侵襲力均較Scramble mimics細(xì)胞明顯下降，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，圖3)。

圖 3 過表達(dá)miR-218對(duì)5-8F細(xì)胞侵襲力的影響Fig. 3 Effect of miR-218 overexpression on cell invasion in 5-8F cells

3 討 論

研究發(fā)現(xiàn)，miR-218在肺鱗癌、膀胱癌、胃癌、膠質(zhì)瘤中表達(dá)下調(diào)，且miR-218能抑制膠質(zhì)瘤細(xì)胞侵襲力［7-10］。本課題組前期通過qRTPCR技術(shù)檢測了miR-218在鼻咽癌組織與對(duì)照鼻咽黏膜組織(包括正常及炎癥黏膜組織)中差異表達(dá)。結(jié)果表明miR-218在鼻咽癌組織表達(dá)明顯下調(diào)；miR-218隨著鼻咽癌的臨床分期進(jìn)展而表達(dá)降低，且有淋巴結(jié)轉(zhuǎn)移的鼻咽癌表達(dá)明顯低于無淋巴結(jié)轉(zhuǎn)移的鼻咽癌(P＜0.05)，表明miR-218表達(dá)與鼻咽癌臨床進(jìn)展及轉(zhuǎn)移呈負(fù)相關(guān)。通過Kaplan-Meier生存曲線分析發(fā)現(xiàn)，miR-218表達(dá)越高的鼻咽癌患者預(yù)后越好。這些結(jié)果提示，miR-218在鼻咽癌的發(fā)生發(fā)展中起著重要作用。為了進(jìn)一步明確這一點(diǎn)，本研究將miR-218 mimics轉(zhuǎn)染至鼻咽癌細(xì)胞5-8F中，觀察miR-218高表達(dá)對(duì)人鼻咽癌細(xì)胞增殖、侵襲的影響。轉(zhuǎn)染miR-218 mimics的5-8F細(xì)胞從48 h起增殖速度明顯減慢，與scramble mimics細(xì)胞相比，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。Transwell侵襲實(shí)驗(yàn)結(jié)果顯示，轉(zhuǎn)染miR-218 mimics的5-8F細(xì)胞較Scramble mimics細(xì)胞侵襲力明顯下降。這些結(jié)果表明，miR-218能抑制鼻咽癌細(xì)胞增殖及侵襲，在鼻咽癌的發(fā)生、發(fā)展中發(fā)揮重要作用。

miRNA功能的發(fā)揮依賴于與靶基因的相互作用，探討miRNA的作用機(jī)制，關(guān)鍵是驗(yàn)證miRNA下游所調(diào)控的靶基因。本研究通過在線靶基因預(yù)測軟件預(yù)測miR-218的靶基因，通過Targestscan軟件得到931個(gè)靶基因，通過Pictar軟件得到551個(gè)靶基因，通過miRanda軟件得到7 337個(gè)靶基因。這些潛在的靶基因有許多與細(xì)胞增殖、粘附及遷移過程有關(guān)，如CDK6、CyclinD、E2F、CDH2、CHL1等。因而miR-218可能通過調(diào)控這些靶基因來發(fā)揮抑制鼻咽癌細(xì)胞增殖、侵襲的功能。當(dāng)然，這仍然有待進(jìn)一步的實(shí)驗(yàn)驗(yàn)證。

［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］ TAKAMIZAWA J, KONISHI H, YANAGISAWA K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival［J］. Cancer Res, 2004, 64(11): 3753-3756.

［5］ JOHNSON S M, GROSSHANS H, SHINGARA J, et al. RAS is regulated by the let-7 microRNA family ［J］. Cell, 2005, 120(5): 635-647.

［6］ SEGURA MF, HANNIFORD D, MENENDEZ S, et al. Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor ［J］. Proc Natl Acad Sci U S A, 2009, 106(6): 1814-1819.

［7］ DAVIDSON M R, LARSEN J E, YANG I A, et al. MicroRNA-218 is deleted and downregulated in lung squamous cell carcinoma ［J］. PLoS One, 2010, 5: e12560.

［8］ MARTINEZ I, GARDINER A S, BOARD K F, et al. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells ［J］. Oncogene, 2008, 27(18): 2575-2582.

［9］ TIE J, PAN Y, ZHAO L, et al. miR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor［J］. PLoS Genet, 2010, 6: e1000879.

［10］ SONG L, HUANG Q, CHEN K, et al. miR-218 inhibits the invasive ability of glioma cells by direct downregulation of IKK-β ［J］. Biochem Biophys Res Commun, 2010, 402(1): 135-140.

Effect of miR-218 on tumor cell proliferation and invasion in nasopharyngeal carcinoma

DENG Min1,2, LIU Ji-fang1, GU Yi-xue1, ZHENG Guo-pei1(1. Cancer Research Institute and Cancer Hospital, Guangzhou Medical University, Guangzhou Guangdong 510183, China; 2. Cancer Research Institute, University of South China, Hengyang Hunan 421001, China)

DENG Min E-mail: dengmin2006@yahoo.com.cn

Background and purpose：miR-218 is downregulated in nasopharyngeal carcinoma (NPC). This study was designed to investigate the effect of miR-218 on tumor cell proliferation and invasion in NPC. Methods：Human NPC cell line 5-8F was transfected with miR-218 mimics by LipofectamineTM2000. Meanwhile 5-8F cells were transfected with control mimics (Scramble mimics) as negative control. qRT-PCR was used to detect the miR-218 expression in these cells. The effects of miR-218 overexpression on cell proliferation and invasion were evaluated by MTS and Transwell invasion assay. Results：miR-218 expression was remarkably upregulated in miR-218-transfected cells, and the fold difference between miR-218-transfected cells and control cells was 17-fold. Transfection of miR-218 mimics into 5-8F cells led to a significant decrease in cell proliferation rate compared with control cells (P＜0.05). The cell invasion by Transwell invasion assay was reduced in miR-218-overpressing cells relative to control cells (P＜0.05). Conclusion：miR-218 can suppress cell proliferation and invasion in NPC. And it plays an important role in tumorigenesis and development of NPC.

Nasopharyngeal carcinoma; miR-218; Cell proliferation; Cell invasion

10.3969/j.issn.1007-3969.2013.03.005

R769.63

：A

：1007-3639(2013)03-0184-04

2012-10-23

2012-12-25）

國家自然科學(xué)基金(No: 81101526)。

鄧敏 E-mail:dengmin2006@yahoo.com.cn