miR-106b在鼻咽癌組織中表達(dá)及對(duì)鼻咽癌CNE-2細(xì)胞生物學(xué)特性的影響

周士霞，王海莉，王豪勛

(鄭州大學(xué)第二附屬醫(yī)院，鄭州 450014)

miR-106b在鼻咽癌組織中表達(dá)及對(duì)鼻咽癌CNE-2細(xì)胞生物學(xué)特性的影響

周士霞，王海莉，王豪勛

(鄭州大學(xué)第二附屬醫(yī)院，鄭州 450014)

目的 探討miR-106b在鼻咽癌組織中的表達(dá)及其對(duì)鼻咽癌CNE-2細(xì)胞生物學(xué)特性的影響。方法 選取68例份初治鼻咽癌患者的手術(shù)切除標(biāo)本和45例份鼻咽炎患者的鼻咽部活檢組織標(biāo)本，采用實(shí)時(shí)熒光定量PCR法檢測miR-106b表達(dá)，分析miR-106b表達(dá)與鼻咽癌患者臨床病理參數(shù)的關(guān)系。取體外培養(yǎng)的對(duì)數(shù)生長期人鼻咽癌細(xì)胞株CNE-2分為四組(1×106個(gè)/組)。miR-106b模擬物組、miR-106b抑制物組分別轉(zhuǎn)染miR-106b模擬物及miR-106b抑制物序列，陰性對(duì)照組轉(zhuǎn)染陰性對(duì)照序列，空白對(duì)照組不處理；采用實(shí)時(shí)熒光定量PCR技術(shù)檢測各組miR-106b表達(dá)，MTT法檢測各組細(xì)胞增殖情況(以吸光度值表示)，流式細(xì)胞儀檢測各組細(xì)胞周期，Transwell試驗(yàn)檢測各組細(xì)胞遷移和侵襲能力。結(jié)果 鼻咽癌及鼻咽炎組織miR-106b相對(duì)表達(dá)量分別為1.57±0.15、1.14±0.12，二者比較P<0.05； miR-106b相對(duì)表達(dá)量與鼻咽癌患者淋巴結(jié)轉(zhuǎn)移、侵犯頸動(dòng)脈鞘和侵犯顱底有關(guān)(P均<0.05)。各組miR-106b相對(duì)表達(dá)量：miR-106b模擬物組高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，miR-106b抑制物組低于陰性對(duì)照組和空白對(duì)照組，P均<0.05；各組細(xì)胞增殖情況：miR-106b模擬物組細(xì)胞接種后24、48、72和96 h時(shí)吸光度值均高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，而miR-106b抑制物組低于陰性對(duì)照組和空白對(duì)照組，P均<0.05；各組細(xì)胞周期：miR-106b模擬物組S期細(xì)胞比例高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，而miR-106b抑制物組細(xì)胞S期比例低于陰性對(duì)照組和空白對(duì)照組，P均<0.05；各組細(xì)胞遷移和侵襲情況：miR-106b模擬物組遷移和侵襲細(xì)胞數(shù)均高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，且miR-106b抑制物組均低于陰性對(duì)照組和空白對(duì)照組，P均<0.05。結(jié)論 miR-106b在鼻咽癌組織中呈高表達(dá)； miR-106b過表達(dá)可促進(jìn)鼻咽癌CNE-2細(xì)胞增殖、遷移和侵襲能力。

鼻咽癌；微小RNA-106b；CNE-2細(xì)胞；細(xì)胞增殖；細(xì)胞遷移

微小RNA(miRNA)是廣泛存在于生物體內(nèi)的高度保守的短小RNA，在細(xì)胞增殖、分化、遷移等多種生物學(xué)過程中發(fā)展重要作用，且參與了多種惡性腫瘤的發(fā)生、侵襲、轉(zhuǎn)移過程[1～3]。miR-106b作為miRNA的重要成員，與原癌基因簇成員miR-17、miR-20有高度同源性，高表達(dá)于多種惡性腫瘤[4]，且與腫瘤細(xì)胞增殖關(guān)系密切[5]。但有關(guān)miR-106b與鼻咽癌發(fā)病的相關(guān)性鮮有報(bào)道。本研究對(duì)鼻咽癌組織中miR-106b表達(dá)變化進(jìn)行分析，并通過轉(zhuǎn)染miR-106b模擬物和抑制物，探討其對(duì)人鼻咽癌細(xì)胞株CNE-2增殖、遷移及侵襲能力的影響，以期為鼻咽癌發(fā)病機(jī)制的研究提供依據(jù)。

1 材料與方法

1.1 材料 選取68例份2014年2月有～2016年2月我院初治鼻咽癌患者[男43例、女25例，年齡27～81(47.6±11.5)歲；排除心肝腎等重要臟器嚴(yán)重功能障礙者以及糖尿病、惡性腫瘤患者]手術(shù)切除的癌組織標(biāo)本，經(jīng)病理學(xué)檢查確診。臨床分期：Ⅰ～Ⅱ期31例，Ⅲ～Ⅳ期37例；發(fā)生頸淋巴結(jié)轉(zhuǎn)移51例。選取同期行鼻咽部活檢的45例慢性鼻咽炎患者[男29例、女16例，年齡(48.0±12.1)歲]手術(shù)切除的正常胃咽部組織標(biāo)本，經(jīng)病理學(xué)檢查確診。鼻咽癌患者與鼻咽炎患者性別、年齡具有可比性(P均>0.05)。人鼻咽癌細(xì)胞株CNE-2購自中科院上海生科院細(xì)胞資源中心，F(xiàn)BS、DMEM培養(yǎng)基、Lipofectamine 2000轉(zhuǎn)染試劑盒均購自美國Invitrogen公司，MTT細(xì)胞增殖及細(xì)胞毒性檢測試劑盒購自上海威奧生物公司，Transwell小室購自美國Corning公司，實(shí)時(shí)熒光定量PCR儀購自美國Bio-rad公司，F(xiàn)ACSCalibur流式細(xì)胞儀購自美國BD公司。TRIzol總RNA提取試劑盒購自加拿大BBI公司，逆轉(zhuǎn)錄試劑盒和PCR試劑盒均購自大連寶生物公司，miR-106b及內(nèi)參U6引物、miR-106b模擬物、miR-抑制物和陰性對(duì)照均由上海生工公司設(shè)計(jì)合成。

1.2 鼻咽組織miR-106b表達(dá)檢測 采用實(shí)時(shí)熒光定量PCR法。取鼻咽癌及慢性鼻咽炎患者鼻咽部活檢組織，研磨后加入細(xì)胞裂解液進(jìn)行裂解，用TRIzol總RNA提取試劑盒對(duì)總RNA進(jìn)行提取，并檢測其純度，取A260/A280≥1.80的標(biāo)本作為合格樣品。將總RNA逆轉(zhuǎn)錄為模板單鏈cDNA，以cDNA為模板，用PCR儀進(jìn)行PCR擴(kuò)增。PCR反應(yīng)條件：94 ℃、60 s，92 ℃、30 s，56 ℃、30 s，74 ℃、30 s，連續(xù)進(jìn)行38個(gè)循環(huán)。每個(gè)樣品均設(shè)置3個(gè)平行反應(yīng)復(fù)孔。采用2-ΔΔCt法獲得不同組織中miR-106b相對(duì)表達(dá)量。分析miR-106b表達(dá)與鼻咽癌患者臨床病理參數(shù)的關(guān)系。

1.3 miR-106b對(duì)CNE-2細(xì)胞生物學(xué)特性影響的觀察

1.3.1 CNE-2細(xì)胞培養(yǎng)及分組處理 將CNE-2細(xì)胞培養(yǎng)于含100 μg/mL鏈霉素、100 U/mL青霉素和10% FBS中的DMEM完全培養(yǎng)基中，于5%CO2、37 ℃恒溫培養(yǎng)箱中培養(yǎng)，待細(xì)胞生長豐度達(dá)到80%以上時(shí)，胰酶消化后傳代培養(yǎng)。取對(duì)數(shù)生長期的CNE-2細(xì)胞，用無血清DMEM培養(yǎng)基重懸后，接種于6孔板，細(xì)胞密度5×104/mL，待細(xì)胞貼壁生長融合度達(dá)到50%以上后，用Lipofectamine 2000轉(zhuǎn)染試劑盒按照操作說明對(duì)細(xì)胞進(jìn)行轉(zhuǎn)染。將細(xì)胞分成四組(1×106個(gè)/組)：miR-106b模擬物組：轉(zhuǎn)染miR-106b模擬物序列：5′-TAAAGTGCTGACAGTGCAGAT-3′；miR-106b抑制物組：轉(zhuǎn)染miR-106b抑制物序列：5′-AUCUGCACUGUCAGCACUUUA-3′；陰性對(duì)照組：轉(zhuǎn)染陰性對(duì)照序列：5′-CAGUACUUUUGUGUAGUACAA-3′；空白對(duì)照組：不做任何處理。

1.3.2 miR-106b表達(dá)檢測 各轉(zhuǎn)染組細(xì)胞培養(yǎng)48 h后加入細(xì)胞裂解液進(jìn)行裂解，采用實(shí)時(shí)熒光定量PCR法檢測miR-106b相對(duì)表達(dá)量。

1.3.3 細(xì)胞增殖率的測算 采用MTT法。取轉(zhuǎn)染24 h細(xì)胞，胰酶消化后，接種于96孔板內(nèi)，細(xì)胞數(shù)2.0×103/孔，分別于接種后12、24、48、72和96 h時(shí)，將孔板中液體吸出，加入新鮮培養(yǎng)基和MTT，置于5% CO2、37 ℃恒溫箱中培養(yǎng)4 h，加入二甲基亞砜(DMSO)，振蕩12 min后，用酶標(biāo)儀進(jìn)行檢測，取490 nm處吸光度(A)值。重復(fù)實(shí)驗(yàn)3次，取平均值。

1.3.4 細(xì)胞周期檢測 采用流式細(xì)胞儀檢測。取各轉(zhuǎn)染組對(duì)數(shù)生長的細(xì)胞，接種于6孔板，細(xì)胞數(shù)1×105/孔，培養(yǎng)24 h后，胰酶消化后收集細(xì)胞，用預(yù)冷PBS洗滌3次，用乙醇固定，4 ℃下過夜孵育。加入碘化丙啶避光染色25 min，利用流式細(xì)胞儀檢測各轉(zhuǎn)染組細(xì)胞周期。重復(fù)實(shí)驗(yàn)3次，取平均值。

1.3.5 細(xì)胞遷移能力及侵襲能力檢測 采用Transwell試驗(yàn)。細(xì)胞遷移能力：取各組轉(zhuǎn)染后培養(yǎng)48 h細(xì)胞，胰酶消化后收集細(xì)胞，用無血清DMEM培養(yǎng)基重懸，調(diào)整細(xì)胞濃度為5×105個(gè)/mL，取200 μL細(xì)胞加入Transwell小室的上室，將含10%胎牛血清的DMEM培養(yǎng)基加入下室，于37 ℃培養(yǎng)箱中培養(yǎng)24 h，用甲醛進(jìn)行固定，結(jié)晶紫染色后，用顯微鏡進(jìn)行觀察，隨機(jī)選取5個(gè)高倍視野對(duì)穿膜細(xì)胞數(shù)進(jìn)行計(jì)數(shù)，取平均數(shù)。重復(fù)實(shí)驗(yàn)3次。細(xì)胞侵襲能力：將50 μg的Matrigel膠將Transwell小室底部膜包被，37 ℃成膠30 min，其余步驟同上。重復(fù)實(shí)驗(yàn)3次，取平均值。

2 結(jié)果

2.1 鼻咽癌及鼻咽炎組織miR-106b表達(dá)比較 鼻咽癌組織、慢性鼻咽炎鼻咽組織中miR-106b相對(duì)表達(dá)量分別為1.57±0.15、1.14±0.12，二者比較差異有統(tǒng)計(jì)學(xué)意義(t=15.433，P<0.05)。miR-106b表達(dá)與鼻咽癌患者年齡、性別、臨床分期無關(guān)(P均>0.05)，而與淋巴結(jié)轉(zhuǎn)移、侵犯頸動(dòng)脈鞘和侵犯顱底有關(guān)(P均<0.05)，詳見表1。

表1 miR-106b表達(dá)與鼻咽癌患者臨床病理參數(shù)的關(guān)系

2.2 miR-106b對(duì)鼻咽癌細(xì)胞生物學(xué)特性的影響

2.2.1 各組細(xì)胞中miR-106b表達(dá)比較 miR-106b模擬物組miR-106b相對(duì)表達(dá)量為1.82±0.16，顯著高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，分別為1.07±0.09、1.31±0.13和1.33±0.14，且miR-106b抑制物組低于陰性對(duì)照組和空白對(duì)照組(P均<0.05)。

2.2.2 各組細(xì)胞增殖情況比較 見表2。

2.2.3 各組細(xì)胞周期比較 見表3。

表2 各組細(xì)胞增殖情況比較

注：與空白對(duì)照組相比，*P<0.05；與陰性對(duì)照組相比，△P<0.05；與miR-106b抑制物組相比，#P<0.05。

2.2.4 各組細(xì)胞遷移和侵襲能力比較 見表4。

3 討論

miR-106b為miRNA的重要類型，參與了細(xì)胞增殖、分化、凋亡過程。研究發(fā)現(xiàn)， miR-106b過表達(dá)可促進(jìn)肝細(xì)胞肝癌細(xì)胞增殖和轉(zhuǎn)移[7]；miR-106b高表達(dá)于喉鱗狀細(xì)胞癌，通過負(fù)性調(diào)控APC基因而促進(jìn)喉鱗狀細(xì)胞癌Hep-2細(xì)胞增殖[8]。本研究結(jié)果顯示，鼻咽癌組織中miR-106b相對(duì)表達(dá)量顯著高于慢性鼻咽炎組織，提示miR-106b可能參與了鼻咽癌的發(fā)生過程； miR-106b表達(dá)量與鼻咽癌患者年齡、 性別、原發(fā)灶大小和臨床分期無關(guān)，而與淋巴結(jié)轉(zhuǎn)移、侵犯頸動(dòng)脈鞘和侵犯顱底有關(guān)，提示鼻咽癌患者發(fā)生頸淋巴結(jié)轉(zhuǎn)移、出現(xiàn)侵犯頸動(dòng)脈鞘和侵犯顱底時(shí)，腫瘤組織中miR-106表達(dá)水平顯著升高，miR-106b可能與鼻咽癌局部侵襲和淋巴結(jié)及遠(yuǎn)處轉(zhuǎn)移有關(guān)。

表3 各組細(xì)胞周期情況比較

注：與空白對(duì)照組相比，*P<0.05；與陰性對(duì)照組相比，△P<0.05；與miR-106b抑制物組相比，#P<0.05。

表4 各組細(xì)胞遷移和侵襲能力比較(個(gè)

注：與空白對(duì)照組相比，*P<0.05；與陰性對(duì)照組相比，△P<0.05；與miR-106b抑制物組相比，#P<0.05。

本研究為進(jìn)一步研究miR-106b對(duì)鼻咽癌細(xì)胞株CNE-2增殖、遷移及侵襲能力的影響，利用細(xì)胞轉(zhuǎn)染技術(shù)，特異性上調(diào)或抑制細(xì)胞中miR-106b表達(dá)，結(jié)果顯示，miR-106b模擬物組細(xì)胞中miR-106b相對(duì)表達(dá)量顯著高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，且miR-106b抑制物組明顯低于陰性對(duì)照組和空白對(duì)照組，說明轉(zhuǎn)染miR-106b模擬物或抑制物可特異性使細(xì)胞中miR-106b表達(dá)上調(diào)或抑制。miR-106b模擬物組細(xì)胞接種后24、48、72和96 h時(shí)A值均明顯高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，而miR-106b抑制物組明顯低于陰性對(duì)照組和空白對(duì)照組，說明上調(diào)miR-106b可加速CNE-2細(xì)胞增殖，而抑制miR-106b表達(dá)則可抑制CNE-2細(xì)胞增殖，提示miR-106b參與了CNE-2細(xì)胞增殖過程。miR-106b模擬物組細(xì)胞S期比例明顯高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，miR-106b抑制物組細(xì)胞S期比例明顯低于陰性對(duì)照組和空白對(duì)照組，說明miR-106b可能通過調(diào)控細(xì)胞周期而參與細(xì)胞增殖過程，與葉敏華等[9]研究結(jié)論相同。近年來文獻(xiàn)報(bào)道，miR-106b調(diào)控細(xì)胞周期的機(jī)制可能與調(diào)控了細(xì)胞周期中的一些相關(guān)因子有關(guān)[10]。有研究通過對(duì)調(diào)控細(xì)胞周期的相關(guān)基因進(jìn)行篩選發(fā)現(xiàn)，p21/CDKN1A是miR-106b的直接作用靶位[11]；miR-106b可通過激活轉(zhuǎn)錄因子E2F1而促使G1/S期的轉(zhuǎn)化[12]。本研究結(jié)果顯示，miR-106b模擬物組遷移細(xì)胞數(shù)和侵襲細(xì)胞數(shù)均明顯高于miR-106b抑制物組、陰性對(duì)照組和空白對(duì)照組，且miR-106b抑制物組均低于陰性對(duì)照組和空白對(duì)照組，說明miR-106b可能參與了CNE-2細(xì)胞遷移和侵襲過程，但具體機(jī)制尚待進(jìn)一步研究明確。

綜上所述，miR-106b在鼻咽癌組織中呈高表達(dá)，過表達(dá)miR-106b可促進(jìn)鼻咽癌CNE-2細(xì)胞增殖、遷移和侵襲能力，其機(jī)制可能與促進(jìn)細(xì)胞進(jìn)入S期有關(guān)；提示miR-106b有望成為鼻咽癌基因治療的潛在靶點(diǎn)。

[1] 王盼盼,季明芳,吳標(biāo)華.鼻咽癌高發(fā)區(qū)人群患鼻咽癌風(fēng)險(xiǎn)率的動(dòng)態(tài)觀察[J].中華腫瘤防治雜志,2014,21(15):1144-1147.

[2] Kim W, Lee WB, Lee J, et al. Traditional herbal medicine as adjunctive therapy for nasopharyngeal cancer: a systematic review and meta-analysis[J]. Integr Cancer Ther, 2015,14(3):212-220.

[3] Mori M, Triboulet R, Mohseni M, et al. Hippo signaling regulates microprocessor and links cell-density-dependent miRNA biogenesis to cancer[J]. Cell, 2014,156(5):893-906.

[4] 董曦,孫桂波,邢小燕,等.Micro RNA-106b-25簇與腫瘤[J].中國藥理學(xué)通報(bào),2014,30(12):1639-1641.

[5] Tan W, Li Y, Lim SG, et al. miR-106b-25/miR-17-92 clusters: polycistrons with oncogenic roles in hepatocellular carcinoma[J]. World J Gastroenterol, 2014,20(20):5962-5972.

[6] Tian Y, Tian Y, Zhang W, et al. Junctional adhesion molecule-A, an epithelial-mesenchymal transition inducer, correlates with metastasis and poor prognosis in human nasopharyngeal cancer[J]. Carcinogenesis, 2015,36(1):41-48.

[7] 申剛,嘉紅云,陳德基,等.miR-106b表達(dá)對(duì)人肝細(xì)胞肝癌細(xì)胞增殖能力的影響[J].中華腫瘤雜志,2014,36(7):489-495.

[8] 段繼惠,楊磊,王巍,等.微小RNA-106b靶向下調(diào)腺瘤性息肉病基因表達(dá)促進(jìn)Hep-2細(xì)胞的增殖[J].中華實(shí)驗(yàn)外科雜志,2015,32(7):1650-1652.

[9] 葉敏華,張安玲,吳雷,等.反義抑制miR-106b表達(dá)對(duì)人腦膠質(zhì)瘤細(xì)胞增殖和侵襲的影響[J].中華神經(jīng)外科雜志,2015,31(1):60-65.

[10] Xiang W, He J, Huang C, et al. miR-106b-5p targets tumor suppressor gene SETD2 to inactive its function in clear cell renal cell carcinoma[J]. Oncotarget, 2015,6(6):4066-4079.

[11] Prasad R, Katiyar SK. Down-regulation of miRNA-106b inhibits growth of melanoma cells by promoting G1-phase cell cycle arrest and reactivation of p21/WAF1/Cip1 protein[J]. Oncotarget, 2014,5(21):10636-10649.

[12] Zhang H, Yan X. Cantharidin modulates the E2F1/MCM7-miR-106b-93/p21- PTEN signaling axis in MCF-7 breast cancer cells[J]. Oncol Lett, 2015,10(5):2849-2855.

Expression of miR-106b in nasopharyngeal carcinoma tissues and its effects on the biological characteristics of nasopharyngeal carcinoma CNE-2 cells

ZHOUShixia,WANGHaili,WANGHaoxun

(TheSecondAffiliatedHospitalofZhengzhouUniversity,Zhengzhou450014,China)

ObjectiveTo investigate the expression of miR-106b in nasopharyngeal carcinoma tissues and its effects on the biological characteristics of nasopharyngeal carcinoma CNE-2 cells.MethodsSixty-eight cases of surgical resection specimens from first treatment patients with nasopharyngeal carcinoma and 45 cases of nasopharyngeal biopsy specimens from patients with chronic nasopharyngitis were selected. The expression of miR-106b was detected by using real-time PCR technology. The relationship between the expression of miR-106b and clinicopathological parameters in patients with nasopharyngeal carcinoma was analyzed. The nasopharyngeal carcinoma CNE-2 cells in the logarithmic growth phase were divided into four groups (1×106cells/group). Cells in the miR-106b mimics group and miR-106b inhibitor group were transected with miR-106b mimics and miR-106 inhibitor, respectively. Cells in the negative control group were transfected with negative control sequence, while cells in the blank control group were not treated. The cell proliferation of each transfected group was tested by using MTT assay. The cell cycle of each transfected group was detected by using flow cytometry. The cell migration and invasion of each transfected group were detected by using Transwell experiment.ResultsThe relative expression levels of miR-106b in the nasopharyngeal carcinoma tissues and chronic nasopharyngitis nasopharyngeal tissues were 1.57±0.15 and 1.14±0.12, respectively,P<0.05. The expression of miR-106b in the nasopharyngeal carcinoma tissues was related with lymph node metastasis, carotid sheath violations, and skull base violations (allP<0.05). The relative expression level in each transfection group: the miR-106b mimic group was significantly higher than the miR-106b inhibitor group, the negative control group and blank control group, and the miR-106b inhibitor group was lower than the negative control group and blank control group, allP<0.05. The proliferation of each transfection group: A values after cell inoculation 24, 48, 72 and 96 h in the miR-106b mimic group were higher than those of the miR-106b inhibitor group, negative control group and blank control group, while the miR-106b inhibitor group was lower than the negative control group and blank control group, allP<0.05. The cell cycle of each transfection group: the proportion of S phase in the miR-106b mimics group was higher than that of the miR-106b inhibitor group, the negative control group and blank control group, while the miR-106b inhibitor group was lower than the negative control group and blank control group, allP<0.05. The cell migration and cell invasion in each transfection group: the number of migration cells and invasion cells in the miR-106b mimics group was higher than that of the miR-106b inhibitor group, negative control group and blank control group, and the miR-106b inhibitor group was lower than the negative control group and blank control group, allP<0.05.ConclusionmiR-106b is highly expressed in nasopharyngeal carcinoma tissue, and the miR-106b overexpression may promote proliferation, migration, and invasion of nasopharyngeal carcinoma CNE-2 cells.

nasopharyngeal carcinoma; miR-106b; CNE-2 cells; cell proliferation; cell migration

河南省科技發(fā)展計(jì)劃(142102310087)。

周士霞(1979-)，女，主治醫(yī)師，主要研究方向?yàn)閻盒阅[瘤的臨床及基礎(chǔ)研究。E-mail: 1541421042@qq.com

10.3969/j.issn.1002-266X.2017.20.003

R739.6

A

1002-266X(2017)20-0009-04

2016-07-05)