轉(zhuǎn)染miR-214抑制物的口腔鱗癌細(xì)胞SCC15增殖、遷移、侵襲能力變化及其機(jī)制

許馳強(qiáng)，肖金剛

(西南醫(yī)科大學(xué)附屬口腔醫(yī)院，四川瀘州646000)

轉(zhuǎn)染miR-214抑制物的口腔鱗癌細(xì)胞SCC15增殖、遷移、侵襲能力變化及其機(jī)制

許馳強(qiáng)，肖金剛

(西南醫(yī)科大學(xué)附屬口腔醫(yī)院，四川瀘州646000)

目的 觀察轉(zhuǎn)染miR-214抑制劑的口腔鱗癌SCC15細(xì)胞增殖、遷移、侵襲能力的變化，并探討其機(jī)制。方法 取體外培養(yǎng)的口腔鱗癌SCC15細(xì)胞，分為觀察組、陰性對(duì)照組和空白對(duì)照組,觀察組和陰性對(duì)照組分別用Lipofectamine2000轉(zhuǎn)染miR-214 抑制物、無關(guān)序列，空白對(duì)照組不轉(zhuǎn)染。采用實(shí)時(shí)熒光定量PCR法檢測(cè)轉(zhuǎn)染24 h時(shí)各組細(xì)胞中miR-214的表達(dá)，用CCK-8法觀察轉(zhuǎn)染24、48、72 h各組細(xì)胞增殖情況，用劃痕實(shí)驗(yàn)觀察轉(zhuǎn)染24 h時(shí)各組細(xì)胞遷移能力，用Transwell實(shí)驗(yàn)觀察轉(zhuǎn)染24 h時(shí)各組侵襲能力，用Western blotting法檢測(cè)轉(zhuǎn)染24 h時(shí)各組細(xì)胞中的Wnt通路拮抗因子Dickkopf 相關(guān)蛋白-3(DKK-3)及Wnt通路關(guān)鍵基因β-catenin蛋白。結(jié)果 轉(zhuǎn)染24 h時(shí)，與陰性對(duì)照組和空白對(duì)照組相比，觀察組細(xì)胞miR-214相對(duì)表達(dá)量低，劃痕愈合百分比低，穿膜細(xì)胞數(shù)少，β-catenin蛋白相對(duì)表達(dá)量低，DKK-3蛋白相對(duì)表達(dá)量高(P均<0.05)。轉(zhuǎn)染48、72 h觀察組OD值低于陰性對(duì)照組和空白對(duì)照組(P均<0.05)。結(jié)論 轉(zhuǎn)染miR-214抑制物的口腔鱗癌SCC15細(xì)胞增殖、遷移及侵襲能力下降。其機(jī)制可能是因?yàn)檗D(zhuǎn)染miR-214抑制物能抑制Wnt/β-catenin信號(hào)通路相關(guān)蛋白DKK-3、β-catenin蛋白表達(dá)。

微小RNA；miR-214；口腔鱗狀細(xì)胞癌；細(xì)胞增殖；細(xì)胞遷移；細(xì)胞侵襲；Wnt信號(hào)通路；Dickkopf 相關(guān)蛋白-3；β-catenin蛋白

口腔癌是頭頸部較常見的惡性腫瘤之一，以鱗狀細(xì)胞癌最為常見，約占全身惡性腫瘤的3%[1]，雖然近年來醫(yī)療水平不斷提高，新型藥物不斷開發(fā)應(yīng)用，但口腔癌患者總生存率卻沒有得到明顯的提高[2、3],腫瘤侵襲及轉(zhuǎn)移是口腔癌患者死亡的重要原因。微小RNA(miRNAs)是一種單鏈非編碼RNA，與惡性腫瘤關(guān)系密切[4]。miR-214在口腔鱗癌中表達(dá)增高[5]。近期有研究報(bào)道m(xù)iR-214通過Wnt信號(hào)通路影響食管癌[6]和肺癌[7]細(xì)胞的增殖、侵襲、轉(zhuǎn)移，但miR-214在口腔鱗癌增殖、侵襲、轉(zhuǎn)移過程中發(fā)揮的作用及機(jī)制尚不明確。本研究觀察了轉(zhuǎn)染miR-214 抑制物的口腔鱗癌SCC15細(xì)胞增殖、遷移、侵襲能力及Wnt通路拮抗因子Dickkopf相關(guān)蛋白-3(DKK-3)和Wnt通路關(guān)鍵蛋白β-catenin蛋白的表達(dá)變化，旨在明確轉(zhuǎn)染miR-214抑制物的口腔鱗癌SCC15細(xì)胞增殖、侵襲、轉(zhuǎn)移能力的變化及其可能機(jī)制。

1 材料與方法

1.1 材料 miR-214抑制物及陰性對(duì)照無關(guān)序列均購自廣州銳博生物科技公司，TRIzol試劑和脂質(zhì)體2000(Lipofectamine2000)購自美國Invitrogen公司，實(shí)時(shí)熒光定量PCR相關(guān)試劑購自北京Promega公司，引物由上海生工公司設(shè)計(jì)合成，Transwell購自美國Corning公司，基質(zhì)膠購自美國BD公司，CCK-8試劑盒購自上海東仁化學(xué)科技公司，β-catenin、DKK-3單克隆抗體和HRP標(biāo)記羊抗鼠二抗購自美國Abcam公司。人口腔鱗狀細(xì)胞癌細(xì)胞系SCC15購自中國科學(xué)院典型培養(yǎng)物保藏細(xì)胞庫，人正?？谇火つそ腔?xì)胞系HOK購自美國Sciencell公司。

1.2 細(xì)胞分組及轉(zhuǎn)染方法 口腔鱗癌SCC15細(xì)胞用含10%胎牛血清的RPMI1640培養(yǎng)基在37 ℃、5%CO2培養(yǎng)箱中常規(guī)傳代，取對(duì)數(shù)生長(zhǎng)期細(xì)胞進(jìn)行實(shí)驗(yàn)。設(shè)觀察組、陰性對(duì)照組和空白對(duì)照組。觀察組和陰性對(duì)照組分別轉(zhuǎn)染轉(zhuǎn)染miR-214抑制物、無關(guān)序列，操作按Lipofectamine2000試劑盒說明書進(jìn)行。空白對(duì)照組不轉(zhuǎn)染。

1.3 各組miR-214檢測(cè) 轉(zhuǎn)染24 h時(shí)采用實(shí)時(shí)熒光定量PCR法檢測(cè)各組miR-214相對(duì)表達(dá)量，TRIzol法提取細(xì)胞總RNA, 測(cè)RNA濃度，按逆轉(zhuǎn)錄試劑盒說明書逆轉(zhuǎn)錄合成cDNA。以2 μL cDNA配置PCR體系，PCR反應(yīng)條件: 94 ℃ 2 min，94 ℃ 20 s，60 ℃ 30s，40個(gè)循環(huán)。以U6作為內(nèi)參基因。每次實(shí)驗(yàn)至少重復(fù)3次，miR-214表達(dá)量采用2-ΔΔCt法計(jì)算。

1.4 各組細(xì)胞增殖情況觀察 采用CCK-8法。 取轉(zhuǎn)染24、48、72 h各組細(xì)胞，將細(xì)胞接種于96孔板，向每孔加入10 μL CCK-8溶液，培養(yǎng)箱孵育2 h，用酶標(biāo)儀測(cè)定450 nm處的光密度(OD)值。每組細(xì)胞設(shè)6個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次。

1.5 各組細(xì)胞遷移能力觀察 采用細(xì)胞劃痕實(shí)驗(yàn)。取轉(zhuǎn)染24 h各組細(xì)胞，將細(xì)胞接種于6孔板中，細(xì)胞生長(zhǎng)完全融合后，用10 μL槍頭在孔板中心軸處沿直線輕輕劃痕，PBS沖洗去除漂浮細(xì)胞后繼續(xù)培養(yǎng)，培養(yǎng)24 h后在顯微鏡下觀察拍照，以細(xì)胞劃痕愈合百分比表示細(xì)胞遷移能力，細(xì)胞愈合百分比=(實(shí)驗(yàn)前劃痕寬度-培養(yǎng)24 h后劃痕寬度)/實(shí)驗(yàn)前劃痕寬度×100%。

1.6 各組細(xì)胞侵襲能力觀察 采用Transwell實(shí)驗(yàn)。用Matrigel基質(zhì)膠包被Transwell小室基底膜。收集轉(zhuǎn)染24 h各組細(xì)胞，重懸于無血清培液中饑餓培養(yǎng)16 h，向上室加入含1×105個(gè)細(xì)胞的細(xì)胞懸液，下層加入含10%FBS的培養(yǎng)基，37 ℃和5%CO2下孵箱培養(yǎng)24 h后取出，擦凈上層小室內(nèi)未遷移的細(xì)胞，4%多聚甲醛固定，0.5%結(jié)晶紫染色，隨機(jī)選取顯微鏡下選5個(gè)視野，計(jì)數(shù)穿膜細(xì)胞數(shù)，取平均值。

1.7 各組SCC15細(xì)胞中DKK-3、β-catenin檢測(cè) 采用Western blotting法。收集轉(zhuǎn)染24 h各組細(xì)胞，加入配置預(yù)冷的RIPA裂解液，充分勻漿裂解提取總蛋白，BCA法測(cè)定蛋白樣品濃度。每組取30 μg蛋白上樣，SDS-聚丙烯酰胺凝膠上電泳30 min，5%脫脂牛奶室溫封閉1 h，加入適當(dāng)濃度一抗，4 ℃過夜，次日洗膜后，加入HRP標(biāo)記的二抗，室溫孵育1 h，洗膜后，將膜置于ECL中，于凝膠成像系統(tǒng)中曝光并采集圖像。結(jié)果以目的基因條帶灰度值與GAPDH條帶灰度值的比值表示。

2 結(jié)果

2.1 各組細(xì)胞轉(zhuǎn)染24 h miR-214相對(duì)表達(dá)量比較 觀察組、陰性對(duì)照組、空白對(duì)照組miR-214相對(duì)表達(dá)量分別為0.41±0.07、1.03±0.02、1.04±0.05。觀察組細(xì)胞miR-214相對(duì)表達(dá)量低于陰性對(duì)照組和空白對(duì)照組(P均<0.05)；陰性對(duì)照組和空白對(duì)照組miR-214相對(duì)表達(dá)量差異無統(tǒng)計(jì)學(xué)意義。

2.2 各組轉(zhuǎn)染24、48、72 h細(xì)胞增殖情況比較 各組轉(zhuǎn)染24、48、72 h時(shí)OD值見表1。轉(zhuǎn)染48、72 h觀察組OD值低于陰性對(duì)照組和空白對(duì)照組(P均<0.05)；陰性對(duì)照組和空白對(duì)照組OD值差異無統(tǒng)計(jì)學(xué)意義。轉(zhuǎn)染24 h各組OD值差異無統(tǒng)計(jì)學(xué)意義。

表1 不同時(shí)點(diǎn)各組OD值比較

2.3 各組轉(zhuǎn)染24 h細(xì)胞遷移能力比較 觀察組、陰性對(duì)照組、空白對(duì)照組劃痕愈合百分比分別為41.5%±3.1%、80.1%±5.0%、83.1%±6.1%。觀察組劃痕愈合百分比低于陰性對(duì)照組和空白對(duì)照組(P均<0.05)，陰性對(duì)照組和空白對(duì)照組劃痕愈合百分比差異無統(tǒng)計(jì)學(xué)意義。

2.4 各組轉(zhuǎn)染 24 h細(xì)胞侵襲能力比較 觀察組、陰性對(duì)照組、空白對(duì)照組穿膜細(xì)胞數(shù)分別為(54±10)、(185±24)、(191±26)個(gè)/HP。觀察組穿膜細(xì)胞數(shù)少于陰性對(duì)照組和空白對(duì)照組(P均<0.05)，陰性對(duì)照組和空白對(duì)照組穿膜細(xì)胞數(shù)差異無統(tǒng)計(jì)學(xué)意義。

2.5 各組轉(zhuǎn)染24 h細(xì)胞中DKK-3、β-catenin 蛋白表達(dá)比較 觀察組、陰性對(duì)照組、空白對(duì)照組DKK-3蛋白相對(duì)表達(dá)量分別為0.61±0.11、0.12±0.03、0.11±0.04，β-catenin 蛋白相對(duì)表達(dá)量分別為0.54±0.12、0.95±0.14、0.93±0.16。觀察組DKK-3蛋白相對(duì)表達(dá)量高于陰性對(duì)照組和空白對(duì)照組(P均<0.05)，β-catenin蛋白相對(duì)表達(dá)量低于陰性對(duì)照組和空白對(duì)照組(P均<0.05)；陰性對(duì)照組和空白對(duì)照組DKK-3、β-catenin 蛋白相對(duì)表達(dá)量差異無統(tǒng)計(jì)學(xué)意義。

3 討論

miR-214位于人1號(hào)染色體長(zhǎng)臂(1q24.3)非編碼基因Dnm3os內(nèi)[8]，參與機(jī)體的細(xì)胞發(fā)育、細(xì)胞衰老及血管形成等生理過程[9、10]。近年研究發(fā)現(xiàn)miR-214在在多種惡性腫瘤中表達(dá)異常，與腫瘤發(fā)生發(fā)展關(guān)系密切[6]。Zhang等[11]發(fā)現(xiàn)miR-214在鼻咽癌組織和細(xì)胞中高表達(dá)，抑制鼻咽癌細(xì)胞miR-214的表達(dá)能夠促進(jìn)癌細(xì)胞的凋亡并抑制其增殖。Penna等[12]報(bào)道m(xù)iR-214在惡性黑色素瘤中表達(dá)增高，并且通過對(duì)抑癌基因TFAP2C的抑制促進(jìn)腫瘤的進(jìn)展。Zhang等[13]發(fā)現(xiàn)胰腺癌中miR-214的表達(dá)較癌旁組織明顯增高，上調(diào)胰腺癌細(xì)胞miR-214的表達(dá)能夠降低癌細(xì)胞對(duì)吉西他濱的敏感性，并且可能與miR-214對(duì)Wnt通路相關(guān)基因Wnt3A的調(diào)控有關(guān)。目前研究證實(shí)在多數(shù)惡性腫瘤中miR-214高表達(dá)，但Derfoul等[14]發(fā)現(xiàn)，乳腺癌組織中miR-214的表達(dá)較癌旁組織顯著下降。Wang等[15]發(fā)現(xiàn)miR-214在胃癌組織和胃癌細(xì)胞中表達(dá)下調(diào)，miR-214通過靶向CSF1影響癌增殖、侵襲和轉(zhuǎn)移。以上結(jié)果說明miR-214在不同的惡性腫瘤中存在異質(zhì)性，可能與miR-214在不同惡性腫瘤中通過不同的靶基因參與惡性腫瘤的進(jìn)展有關(guān)。Scapoli等[16]發(fā)現(xiàn)口腔鱗癌組織miR-214表達(dá)顯著增高，但未進(jìn)行機(jī)制方面的研究。腫瘤的侵襲及轉(zhuǎn)移是腫瘤患者死亡的主要原因之一，研究證實(shí)miR-214通過不同的機(jī)制影響癌細(xì)胞的增殖、侵襲及轉(zhuǎn)移[6,7，11～15]等生物學(xué)行為。本研究結(jié)果顯示，觀察組口腔鱗癌SCC15細(xì)胞轉(zhuǎn)染miR-214 抑制物后，miR-214表達(dá)下調(diào)，細(xì)胞增殖、遷移、侵襲能力下降，說明miR-214在口腔鱗癌的進(jìn)展中起著促癌基因的作用。

Wnt信號(hào)通路是多細(xì)胞真核生物中高度保守的信號(hào)通路[17]，調(diào)控細(xì)胞的增殖、凋亡、分化等生理過程，Wnt/β-catenin通路是經(jīng)典Wnt通路，也是目前研究最多、最深入的Wnt通路，Wnt/β-catenin通路主要通過激活β-catenin在核內(nèi)的功能來調(diào)節(jié)靶基因，Wnt信號(hào)通路的異常激活是以細(xì)胞內(nèi)β-catenin的積聚為特征，激活許多下游靶基因[18]參與多種惡性腫瘤的發(fā)生發(fā)展[19～22]。DKK-3是Wnt信號(hào)通路拮抗因子DDK家族成員之一，能夠阻止LRP-5、LRP-6與Wnt、Fz配體或受體復(fù)合物的結(jié)合，從而影響β-catenin在細(xì)胞質(zhì)內(nèi)的積聚，抑制經(jīng)典Wnt信號(hào)通路[23，24]。已有研究表明miR-214在食管癌[6]、肺癌[7]、胰腺癌[13]等腫瘤中發(fā)揮的作用均與Wnt通路有關(guān)。本研究發(fā)現(xiàn)觀察組轉(zhuǎn)染miR-214抑制物下調(diào)SCC15細(xì)胞miR-214表達(dá)后，細(xì)胞內(nèi)DDK-3表達(dá)上升，β-catenin的表達(dá)下降，說明miR-214對(duì)口腔鱗癌細(xì)胞增殖、遷移及侵襲行為的影響與Wnt/β-catenin通路的調(diào)控有關(guān)，miR-214可能通過對(duì)DDK-3及β-catenin表達(dá)的調(diào)控影響口腔鱗癌細(xì)胞的生物學(xué)行為，miR-214調(diào)控Wnt/β-catenin通路具體的機(jī)制尚需在后面的實(shí)驗(yàn)中進(jìn)一步研究證實(shí)。

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Changes in proliferation, migration and invasion abilities of oral squamous-cell carcinoma SCC15 cells transfected by miR-214 inhibitor and its mechanism

XUChiqiang,XIAOJingang

(TheAffiliatedOralHospitalofSouthwestMedicalUniversity,Luzhou646000,China)

Objective To investigate the changes in proliferation, migration and invasion abilities of oral squamous-cell carcinoma SCC15 cells transfected by miR-214 inhibitor, and to explore the mechanism. Methods The oral squamous-cell carcinoma SCC15 cells cultured in vitro were randomly divided into the observation group, negative control group and blank control group. SCC15 cells in the observation group and negative control group were transfected with miR-214 inhibitor and inhibitor-NC in vitro by using Lipofectamine2000, respectively, and SCC15 cells in the blank control group were not given any intervention. The real-time PCR was used to detect the expression of miR-214 at 24 h after transfection. CCK-8 assay was used to observe the proliferation of the cells at 24, 48 and 72 h after transfection. Scratch test and Transwell assay were used to observe the migration and invasion abilities of the cells at 24 h after transfection. Western blotting was used to explore the expression of Dikkopf-related protein-3 DKK-3 and β-catenin protein at 24 h after transfection. Results At 24 h after transfection, compared with the negative control group and blank control group, the expression of miR-214 and β-catenin protein and wound healing rate was significantly lower, the number of penetrating cells was significantly less and the expression of DKK-3 protein was significantly higher in the observation group (allP<0.05). The A450 in the observation group was lower than that in the negative control group and blank control group at 48 and 72 h after transfection (allP<0.05).Conclusion The proliferation, migration and invasion abilities of oral squamous-cell carcinoma SCC15 cells transfected with miR-214 inhibitor decrease, and the mechanism may be that the transfection of miR-214 inhibitor can inhibit the expression of DKK-3 andβ-catenin protein in Wnt/β-catenin signaling pathway.

micro RNA; miR-214; oral squamous-cell carcinoma; cell proliferation; cell migration; cell invasion; Wnt signal; Dickkopf-related protein-3; β-catenin protein

國家自然科學(xué)基金資助項(xiàng)目(81371125)；四川省科技廳科研基金資助項(xiàng)目(2014JY0044)；四川省教育廳科研基金資助項(xiàng)目(10ZB030)；四川省衛(wèi)計(jì)委科研基金資助項(xiàng)目(80170)；西南醫(yī)科大學(xué)重點(diǎn)項(xiàng)目(201207)；西南醫(yī)科大學(xué)科研基金資助項(xiàng)目(2014070)。

許馳強(qiáng)(1983-)，男，醫(yī)學(xué)碩士，主治醫(yī)師，主要研究方向?yàn)榭谇荒[瘤的分子生物學(xué)機(jī)制。E-mail： dr_xcq@yeah.net

簡(jiǎn)介：肖金剛(1975-)，男，醫(yī)學(xué)博士，主任醫(yī)師，教授，碩士研究導(dǎo)師,主要研究方向?yàn)榭谇活M面修復(fù)重建。E-mail: xjgkqk@yeah.net

10.3969/j.issn.1002-266X.2016.47.003

R739.8

A

1002-266X(2016)47-0009-04

2016-08-11)