李磊，倪正義，湯中文，周密

(武漢市醫(yī)療救治中心，武漢430000)

miR-106b在非小細(xì)胞肺癌細(xì)胞A549中的表達變化及意義

李磊，倪正義，湯中文，周密

(武漢市醫(yī)療救治中心，武漢430000)

目的觀察miR-106b在非小細(xì)胞肺癌細(xì)胞A549中的表達變化，并探討其對細(xì)胞遷移、侵襲的影響及可能機制。方法采用熒光定量PCR法檢測非小細(xì)胞肺癌細(xì)胞A549及正常肺表皮細(xì)胞系HPL-1中miR-106b相對表達量。將非小細(xì)胞肺癌細(xì)胞A549分為miR-106b模擬物組、miR-106b抑制物組和陰性對照組，采用Lipofectamine 2000轉(zhuǎn)染法分別轉(zhuǎn)染miR-106b mimics、miR-106b inhibit及scramble。培養(yǎng)24 h，采用熒光定量PCR法檢測miR-106b相對表達量，進行細(xì)胞劃痕試驗、Transwell細(xì)胞侵襲試驗檢測細(xì)胞遷移和侵襲能力(分別以劃痕愈合率、穿膜細(xì)胞數(shù)表示)，采用熒光定量PCR法和Western blotting法檢測墨角藻糖基轉(zhuǎn)移酶6(FUT6)mRNA及蛋白表達。結(jié)果非小細(xì)胞肺癌細(xì)胞A549及正常肺表皮細(xì)胞HPL-1中miR-106b相對表達量分別為5.37±0.42、1.00，二者比較P<0.01。miR-106b模擬物組、miR-106b抑制物組及陰性對照組miR-106b相對表達量分別為8.94±0.73、0.21±0.03、1.00，組間兩兩比較P均<0.05。miR-106b模擬物組、陰性對照組劃痕愈合率、穿膜細(xì)胞數(shù)均高于miR-106b抑制物組，F(xiàn)UT6 mRNA 及蛋白相對表達量均低于miR-106b抑制物組，但miR-106b模擬物組變化更明顯(P均<0.05)。結(jié)論非小細(xì)胞肺癌細(xì)胞A549中miR-106b表達升高；過表達miR-106b可能通過下調(diào)FUT6表達而促進非小細(xì)胞肺癌細(xì)胞的遷移與侵襲。

非小細(xì)胞肺癌；微小RNA-106b；細(xì)胞遷移；細(xì)胞侵襲；墨角藻糖基轉(zhuǎn)移酶6

據(jù)國際腫瘤研究機構(gòu)統(tǒng)計，2012年約180萬人被診斷為肺癌，占所有惡性腫瘤的13%[1,2]。肺癌可分為小細(xì)胞肺癌和非小細(xì)胞肺癌，小細(xì)胞肺癌主要采用化療等全身治療，非小細(xì)胞肺癌可采用手術(shù)、化療等綜合治療，目前關(guān)于非小細(xì)胞肺癌的發(fā)病機制仍不十分清楚。微小RNA(miRNA)是一類非編碼長鏈RNA，長度為19～22 nt，可在轉(zhuǎn)錄后水平調(diào)控靶基因表達[3]，廣泛參與腫瘤細(xì)胞的增殖、凋亡、侵襲及轉(zhuǎn)移等生物學(xué)過程[4,5]。miR-106b定位于染色體7q22.1上[6]，其在非小細(xì)胞肺癌中的作用鮮見報道。2015年5月～2016年5月，本研究觀察了miR-106b在非小細(xì)胞肺癌細(xì)胞中的表達變化及其對細(xì)胞遷移、侵襲的影響，并探討其可能的機制。

1 材料與方法

1.1 材料 細(xì)胞：非小細(xì)胞肺癌細(xì)胞A549及正常肺表皮細(xì)胞HPL-1均購自協(xié)和醫(yī)科大學(xué)實驗中心。主要試劑：DMEM培養(yǎng)基、TRIzol試劑均購自美國Invitrogen公司，實驗所需一抗均購自美國BD公司，二抗購自武漢博士德生物科技有限公司。主要儀器：NanoDrop1000紫外分光光度計購自美國Thermo Scientific公司，7900HT熒光定量PCR系統(tǒng)購自美國Applied Biosystems公司，WimScratch在線圖像分析系統(tǒng)購于德國Wimasis GmbH公司。miR-106b mimics及scramble質(zhì)檢均由廣州銳博生物科技有限公司合成。

1.2 A549細(xì)胞及HPL-1細(xì)胞miR-106b表達檢測 采用熒光定量PCR法。取A549細(xì)胞及HPL-1細(xì)胞，以1×103個接種于DMEM培養(yǎng)基(添加10% FBS)，37 ℃、5% CO2條件下，培養(yǎng)48 h，待細(xì)胞長至鋪滿培養(yǎng)皿后，加入細(xì)胞裂解液，TRIzol試劑提取總RNA，采用NanoDrop1000紫外分光光度計檢測RNA濃度合格。采用7900HT熒光定量PCR儀進行PCR反應(yīng)。引物序列：miR-106b正向引物：5′-CCTGCCGGGGCTAAAGTGCT-3′，反向引物：5′-TGCTGGAGCAGCAAGTACCC-3′，引物長度20 bp。內(nèi)參β-actin正向引物: 5′-TGGCACCCAGCACAATGAA-3′， 反向引物：5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′，引物長度19 bp。PCR反應(yīng)體系：1×Taq Buffer 2 μL，MgCl21.2 μL，dNTP 0.2 μL，上下游引物各0.4 μL， cDNA模板 1 μL，DEPC水14.7 μL，Taq酶0.1 μL。反應(yīng)條件：95 ℃預(yù)變性10 min，95 ℃變性15 s，60 ℃退火1 min，72 ℃延伸45 s，4 ℃條件下共40個循環(huán)。以2-ΔΔCt法計算miR-106b相對表達量。

1.3 A549細(xì)胞分組處理 將A549細(xì)胞以2×103/孔的密度接種于24孔板(DMEM培養(yǎng)基)，置于37 ℃、5% CO2培養(yǎng)箱中，培養(yǎng)48 h后進行傳代。將對數(shù)生長期的A549細(xì)胞隨機分為miR-106b模擬物組、miR-106b抑制物組及陰性對照組，采用Lipofectamine 2000脂質(zhì)體轉(zhuǎn)染試劑盒分別轉(zhuǎn)染miR-106b mimics、miR-106b inhibit及scramble，轉(zhuǎn)染RNA濃度為300 nmol/孔。

1.4 miR-106b表達檢測 取處理24 h的三組細(xì)胞，采用熒光定量PCR法檢測miR-106b相對表達量。具體步驟同1.2。

1.5 細(xì)胞遷移能力檢測 采用細(xì)胞劃痕試驗。將三組細(xì)胞以2×104個/孔接種于6孔板中，培養(yǎng)24 h。待細(xì)胞融合至100%后，用滅菌槍頭沿直線用力劃直線。劃痕后即刻及48 h后在顯微鏡下觀察細(xì)胞劃痕修復(fù)情況，使用WimScratch在線圖像分析系統(tǒng)檢測細(xì)胞劃痕面積，計算劃痕愈合率。劃痕愈合率=(劃痕后即刻劃痕面積-劃痕后48 h劃痕面積)/劃痕后即刻劃痕面積×100%。實驗重復(fù)3次，取平均值。劃痕愈合率越高，表示細(xì)胞遷移能力越強。

1.6 細(xì)胞侵襲能力檢測 采用Transwell細(xì)胞侵襲試驗。將三組細(xì)胞以3×104個/孔接種于Transwell小室的碳酸磷脂表面，上室BioCoat TM包被Matrigel基質(zhì)膠，37 ℃培養(yǎng)24 h。取出上層小室，將膜下面的細(xì)胞與1%多聚甲醛混合，用0.2%結(jié)晶紫溶液染色15 min。光學(xué)顯微鏡下隨機取10個視野，計數(shù)進入膜下的細(xì)胞數(shù)量(下稱穿膜細(xì)胞數(shù))。實驗重復(fù)3次，取平均值。

1.7 細(xì)胞墨角藻糖基轉(zhuǎn)移酶6(FUT6)mRNA及蛋白表達檢測 ① FUT6 mRNA表達：采用熒光定量PCR法。取三組對數(shù)生長期細(xì)胞，參照1.2的步驟采用熒光定量PCR法檢測三組細(xì)胞FUT6 mRNA相對表達量。FUT6上游引物：5′-AAGATGAAACAGA-TCTGGAC-3′，下游引物：5′-CACGATGACGAAACTGCAGT-3′；內(nèi)參GAPDH上游引物：5′-TATGCTCTCCTCATGCATTG-3′，下游引物： 5′-GGGACGACCTTC-GATCTACC-3′。②FUT6蛋白表達：采用Western blotting法。取三組對數(shù)生長期細(xì)胞，待細(xì)胞融合至100%后，加入裂解液提取細(xì)胞總蛋白，以每孔30 μg總蛋白上樣，常規(guī)濕法轉(zhuǎn)膜，5%脫脂奶粉封閉2 h。分別加入FUT6、GAPDH一抗過夜，抗體稀釋度均為1∶200；加入二抗山羊抗鼠(1∶1 000)孵育2 h。ECL化學(xué)發(fā)光，凝膠成像系統(tǒng)顯影。采用Quantity One 1-D分析軟件對蛋白質(zhì)印跡條帶進行定量。以目的蛋白與GAPDH條帶灰度值的比值作為FUT6蛋白相對表達量。

2 結(jié)果

2.1 A549細(xì)胞及HPL-1細(xì)胞miR-106b表達比較 A549細(xì)胞及HPL-1細(xì)胞miR-106b相對表達量分別為5.37±0.42、1.00，二者比較P<0.01。

2.2 三組miR-106b表達比較 miR-106b模擬物組、miR-106b抑制物組及陰性對照組miR-106b相對表達量分別為8.94±0.73、0.21±0.03、1.00，組間兩兩比較P均<0.05。

2.3 三組細(xì)胞遷移、侵襲能力比較 miR-106b模擬物組、陰性對照組劃痕愈合率、穿膜細(xì)胞數(shù)均高于miR-106b抑制物組，但miR-106b模擬物組升高更明顯(P均<0.05)。見表1。

表1 三組劃痕愈合率、穿膜細(xì)胞數(shù)比較

注：與陰性對照組比較，*P<0.05；與miR-106b抑制物組比較，#P<0.05。

2.4 三組FUT6 mRNA及蛋白表達比較 miR-106b模擬物組、陰性對照組FUT6 mRNA 及蛋白相對表達量均低于miR-106b抑制物組，但miR-106b模擬物組降低更明顯(P均<0.05)。見表2。

表2 三組FUT6 mRNA及蛋白相對表達量比較

注：與陰性對照組比較，*P<0.05；與miR-106b抑制物組比較，#P<0.05。

3 討論

腫瘤的發(fā)生、發(fā)展是多種癌基因與抑制基因突變的結(jié)果，包括p53[7]、p21[8]、miRNA[5]等。miRNA屬于非編碼長鏈RNA，長度為19～22 nt，可在轉(zhuǎn)錄后水平通過與靶基因結(jié)合而下調(diào)靶基因表達。miRNA廣泛參與腫瘤細(xì)胞的增殖、凋亡、侵襲及轉(zhuǎn)移等生物學(xué)過程[4,5]。miR-106b是miR-106b-25家族中的一員[6]，在膀胱癌[9]、腎細(xì)胞癌[10]、喉癌[11]、肝細(xì)胞癌[12]細(xì)胞中表達升高，而在子宮內(nèi)膜癌[13]細(xì)胞中表達降低。腎細(xì)胞癌組織miR-106b表達較癌旁正常組織升高，應(yīng)用miR-106b抑制劑轉(zhuǎn)染腎細(xì)胞癌細(xì)胞系后，可抑制其增殖、遷移，誘導(dǎo)細(xì)胞凋亡[14]。Yang等[15]報道，miR-106b在胃癌細(xì)胞中高表達，并可促進胃癌細(xì)胞侵襲和遷移，與患者預(yù)后密切相關(guān)。miR-106b在乳腺癌細(xì)胞中呈高表達，與淋巴結(jié)轉(zhuǎn)移相關(guān)；miR-106b可激活Wnt信號通路，并介導(dǎo)β-鏈蛋白(β-catenin)入核，進而增強乳腺癌細(xì)胞的侵襲和遷移能力[16]。miR-106b還可抑制Smad7蛋白表達，激活轉(zhuǎn)化生長因子α(TGF-α)TGF-α/Smad信號通路，進而誘導(dǎo)乳腺癌細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化[17]。在卵巢癌細(xì)胞中，miR-106b呈高表達，并通過靶向結(jié)合DAB2基因、促進TGF-α表達而參與卵巢癌細(xì)胞遷移[18]。結(jié)腸癌細(xì)胞miR-106b表達高于正常結(jié)腸細(xì)胞，miR-106b高表達與結(jié)腸癌患者的臨床分期及淋巴結(jié)轉(zhuǎn)移相關(guān)，可能通過靶向結(jié)合DLC1基因而促進結(jié)腸癌細(xì)胞遷移和侵襲[19]。miR-106b在垂體瘤細(xì)胞中高表達，雙熒光素酶實驗顯示miR-106b與PTEN 基因的3′-UTR區(qū)靶向結(jié)合，通過PI3K/AKT信號通路而調(diào)控腫瘤細(xì)胞的增殖與侵襲[20]。與之相反，miR-106b在甲狀腺癌細(xì)胞中呈低表達，雙熒光素酶實驗顯示miR-106b可與C1orf24基因靶向結(jié)合，并誘導(dǎo)甲狀腺癌細(xì)胞凋亡、抑制細(xì)胞侵襲[21]。以上研究結(jié)果提示miR-106b在不同的腫瘤中可能具有不同的作用。

本研究結(jié)果顯示，A549細(xì)胞miR-106b相對表達量明顯高于HPL-1細(xì)胞，提示miR-106b在非小細(xì)胞肺癌細(xì)胞中呈高表達，與其在胃癌、乳腺癌、腎細(xì)胞癌、卵巢癌、結(jié)腸癌及垂體瘤組織中的表達變化一致[14～20]。本研究結(jié)果顯示，miR-106b模擬物組、陰性對照組劃痕愈合率、穿膜細(xì)胞數(shù)均高于miR-106b抑制物組，但miR-106b模擬物組升高更明顯；說明升高miR-106b表達可促進A549細(xì)胞的遷移和侵襲，miR-106b在非小細(xì)胞肺癌的發(fā)生過程中具有促癌基因的作用。FUT6是墨角藻糖基轉(zhuǎn)移酶家族中的一員，在乳腺癌細(xì)胞中呈低表達，上調(diào)FUT6表達可降低乳腺癌細(xì)胞系MDA-MB-231增殖、侵襲和遷移能力[22]。在肝癌細(xì)胞系中，siRNA 靶向沉默F(xiàn)UT6可降低腫瘤相關(guān)抗原sLeX表達及人肝癌細(xì)胞株HepG2的遷移和侵襲能力[23]。本研究結(jié)果顯示，miR-106b模擬物組、陰性對照組FUT6 mRNA 及蛋白相對表達量均低于miR-106b抑制物組，但miR-106b模擬物組降低更明顯；說明升高miR-106b表達可降低A549細(xì)胞FUT6表達，F(xiàn)UT6可能是miR-106b的負(fù)向靶控基因。

綜上所述，A549細(xì)胞中miR-106b升高；過表達miR-106b可能通過下調(diào)FUT6表達而促進A549細(xì)胞的遷移與侵襲。miR-106b可能成為非小細(xì)胞肺癌的一個新的治療靶點，但是其促進A549細(xì)胞遷移、侵襲的相關(guān)機制以及FUT6是否為miR-106b的靶基因仍需進一步探討。

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ExpressionchangesofmiR-106binnon-small-celllungcancercelllineA549

LILei,NIZhengyi,TANGZhongwen,ZHOUMi

(WuhanMedicalTreatmentCenter,Wuhan430000,China)

ObjectiveTo observe the expression changes of miR-106b in non-small-cell lung cancer (NSCLC) cell line A549, and to investigate its effects on cell migration and invasion, as well as the regulatory mechanism.MethodsThe expression level of miR-106b was measured in A549 and HPL-1 cells by the fluorescent quantitative PCR. A549 cells were divided into three groups. The miR-106b mimics group was transfected with miR-106b mimics, the miR-106b inhibition group with miR-106b inhibit, and the control group with scramble. The relative expression level of miR-106b was measured by real-time fluorescent quantitative PCR (qRT-PCR) at 24 h after culture. The migration and invasion abilities in the three groups were measured by Scratch test and Transwell invasion assay. The expression levels of FUT6 mRNA and protein were measured in these three groups by fluorescent quantitative PCR and Western blotting, respectively.ResultsThe expression level of miR-106b in the A549 cells was 5.37±0.42, which was higher than that of HPL-1 cells (1.0),P<0.01. The expression level of miR-106b was 8.94±0.73 in the miR-106b mimics group, 0.21±0.03 in the miR-106b inhibit group and 1.0 in the control group, respectively, and the difference was significant, allP<0.05. The wound healing rate and the number of invasive cells in the miR-106b mimics group and control group were significantly higher than those of the miR-106 inhibition group, while the expression levels of FUT6 mRNA and protein were significantly lower than those in the miR-106b inhibit group, and the change in the miR-106b mimics group was more significance (allP<0.05).ConclusionThe miR-106b is up-regulated in the NSCLC A549 cells and the over-expression of miR-106b promotes the migration and invasion of NSCLC by down-regulating FUT6 expression.

non-small-cell lung cancer; microRNA-106b; cell migration; cell invasion; fucosyltransferase VI

武漢市應(yīng)用基礎(chǔ)研究計劃項目。

李磊(1979-)，男，主治醫(yī)師，研究方向為肺癌的基礎(chǔ)與臨床。E-mail： lileiwuhan899@126.com

周密(1974-)，男，副主任醫(yī)師，研究方向為肺癌的基礎(chǔ)與臨床。E-mail： mizhou768@126.com

10.3969/j.issn.1002-266X.2017.44.006

R734.2

A

1002-266X(2017)44-0022-04

2017-03-27)