劉行仁 白義鳳 梁 良 馮 靜 鄧 菲

(四川省醫(yī)學(xué)科學(xué)院·四川省人民醫(yī)院呼吸科,成都610072)

·基礎(chǔ)免疫學(xué)·

miR-34a通過Snail誘導(dǎo)肺癌EMT及促進(jìn)其轉(zhuǎn)移的分子機(jī)制①

劉行仁 白義鳳②梁 良②馮 靜③鄧 菲④

(四川省醫(yī)學(xué)科學(xué)院·四川省人民醫(yī)院呼吸科,成都610072)

目的：探討miR-34a在肺癌組織中的表達(dá)情況以及miR-34a在肺癌細(xì)胞侵襲和遷移過程中的作用及其機(jī)制。方法：qPCR檢測肺癌和正常肺組織中miR-34a的表達(dá)情況；使用miR-34a-mimic和miR-34a-inhibitor過表達(dá)和沉默miR-34a，qPCR檢測沉默和過表達(dá)效果；Western blot檢測沉默和過表達(dá)miR-34a后Snail蛋白的表達(dá)情況；熒光素酶報(bào)告基因檢測miR-34a與Snail的相互作用；Transwell侵襲實(shí)驗(yàn)檢測miR-34a的表達(dá)對肺癌細(xì)胞侵襲能力的影響；劃痕實(shí)驗(yàn)檢測miR-34a的表達(dá)對肺癌細(xì)胞遷移能力的影響；Western blot檢測E-Cadherin、Vimentin和Twist蛋白的表達(dá)情況。結(jié)果：與正常肺組織相比，肺癌組織中miR-34a表達(dá)明顯降低；且晚期、低分化和有淋巴結(jié)轉(zhuǎn)移的肺癌組織miR-34a表達(dá)明顯較早期、高分化和無淋巴結(jié)轉(zhuǎn)移的肺癌組織低；miR-34a-mimic和miR-34a-inhibitor可以有效抑制和過表達(dá)miR-34a的表達(dá)；miR-34a能與Snail的3′ UTR特異性結(jié)合；miR-34a可以調(diào)控肺癌H1650細(xì)胞的侵襲遷移能力；過表達(dá)miR-34a上調(diào)E-Cadherin，同時(shí)下調(diào)Vimentin和Twist蛋白的表達(dá)，沉默miR-34a則相反。結(jié)論：miR-34a在肺癌中表達(dá)明顯降低，且跟肺癌分期分級以及淋巴結(jié)轉(zhuǎn)移與否密切相關(guān)，同時(shí)miR-34a可以通過上皮間質(zhì)轉(zhuǎn)化調(diào)節(jié)肺癌細(xì)胞侵襲和遷移能力。

miR-34a；肺癌；上皮間質(zhì)轉(zhuǎn)化；E-Cadherin；Transwell；Snail

肺癌是世界上發(fā)病率最高的惡性腫瘤。肺癌也是我國發(fā)病率和死亡率最高的惡性腫瘤[1]。全球每年有超過160萬新發(fā)病例，占所有新發(fā)腫瘤的12.7%。同時(shí)肺癌也是死亡率最高的惡性腫瘤，每年有超過140萬肺癌患者死亡，占所有腫瘤死亡人數(shù)的19%[2]。雖然近年來肺癌治療手段不斷進(jìn)步，但是肺癌患者尤其是晚期肺癌患者的五年生存率仍然很低。局部復(fù)發(fā)、淋巴結(jié)轉(zhuǎn)移以及遠(yuǎn)處轉(zhuǎn)移是導(dǎo)致患者死亡的主要原因[3]。

腫瘤轉(zhuǎn)移是個(gè)多步驟，多階段的復(fù)雜過程，主要包括局部浸潤、浸入血管、隨血液循環(huán)系統(tǒng)轉(zhuǎn)移并在其中存活、移出血管和在新的部位定居并增殖，而腫瘤轉(zhuǎn)移的第一步是細(xì)胞與細(xì)胞間黏附的破裂[4，5]。Snail是近年發(fā)現(xiàn)的鋅指轉(zhuǎn)錄因子，是腫瘤進(jìn)展過程中一個(gè)重要的調(diào)節(jié)子，可以促進(jìn)腫瘤浸潤和轉(zhuǎn)移的因子[6]。Burton等[7]研究表明，Snail與乳腺癌組織分期和分級、淋巴結(jié)轉(zhuǎn)移的情況密切相關(guān)。Palma等[8]發(fā)現(xiàn)，Snail的高表達(dá)可以促進(jìn)上皮間質(zhì)轉(zhuǎn)化的發(fā)生，從而促進(jìn)腫瘤細(xì)胞的侵襲和遷移。

miRNA是高度保守的非編碼RNA，通過調(diào)控相應(yīng)基因的表達(dá)，參與細(xì)胞的增殖、凋亡、細(xì)胞分化等生物學(xué)行為，同時(shí)也可參與惡性腫瘤的侵襲、遷移、腫瘤微環(huán)境的調(diào)節(jié)以及腫瘤干細(xì)胞的調(diào)控等[9，10]。許多研究表明，miR-34a的缺失可能與肝癌、乳腺癌和結(jié)腸癌等腫瘤發(fā)生和發(fā)展密切相關(guān)[11-13]。但是miR-34a在肺癌發(fā)生發(fā)展過程中機(jī)制不甚明確。故本研究擬探討miR-34a在肺癌中的表達(dá)情況，以及在肺癌轉(zhuǎn)移過程中的作用及機(jī)制。

1 材料與方法

1.1 材料

1.1.1 臨床標(biāo)本采集及處理 收集2015年1月-2016年3月在我院收治的100例肺癌患者，其中男性65例，女性35例；年齡(61.24±3.61)歲。所有患者術(shù)前無化療或放療，全部患者術(shù)后病理分期均經(jīng)兩名副高以上病理科醫(yī)師共同閱片確定，根據(jù)肺癌TNM分期標(biāo)準(zhǔn)，Ⅰ級39例、Ⅱ級18例、Ⅲ級37例、Ⅳ級6例。低分化28例，中分化32例，高分化40例。45例發(fā)生淋巴結(jié)轉(zhuǎn)移，55例未發(fā)生淋巴結(jié)轉(zhuǎn)移。同時(shí)取癌旁正常組織100例。腫瘤組織離體后分兩塊，一塊迅速投入RNA保存液中，另一塊用經(jīng)焦碳酸二乙酯處理的冷磷酸緩沖液沖洗，去除血跡，迅速投入液氮凍存。

1.1.2 細(xì)胞株與主要試劑 人肺癌細(xì)胞株H1650購自ATCC。細(xì)胞培養(yǎng)條件：含10%胎牛血清的RPMI DMEM，37℃，5%CO2條件下培養(yǎng)。胎牛血清，RPMI DMEM培養(yǎng)基均購自Gibco公司。Snail、E-cadherin、Vimentin、Twist單克隆抗體購自CST。Transwell小室購自美國Millipore公司。Matrigel購自美國BD公司。脂質(zhì)體LipofectamineTM2000、miR-34a mimic、miR-34a-inhibitor購自上海吉?jiǎng)P基因。Trizol購自美國Ambion公司。逆轉(zhuǎn)錄試劑盒(FSQ-101)購自日本TOYOBO公司。PCR試劑盒購自美國Kapa公司。熒光素酶活性檢測試劑盒購自Promega公司。熒光素酶報(bào)告載體由Promega公司合成。

1.2 方法

1.2.1 qPCR檢測miR-34a的表達(dá) 按照Trizol說明書提取組織中總RNA，超微量分光光度計(jì)測定RNA濃度，復(fù)能基因有限公司設(shè)計(jì)miR-34a引物，上游引物：5′-GTGCAGGGTCCGAGGT-3′；下游引物：5′-GCCGCTGGCAGTGTCTTAGCTG-3′。以100 ng總RNA為模板，逆轉(zhuǎn)錄cDNA，反應(yīng)條件為：37℃ 15 min，98℃ 5 min。后根據(jù)Kapa PCR試劑盒說明書進(jìn)行PCR反應(yīng)。獲得數(shù)據(jù)以RQ=2-ΔΔCt計(jì)算mRNA表達(dá)量。實(shí)驗(yàn)重復(fù)3次。

1.2.2 細(xì)胞轉(zhuǎn)染 取對數(shù)生長期的肺癌細(xì)胞H1650細(xì)胞，按照LipofectamineTM2000轉(zhuǎn)染試劑盒說明書將miR-34a-mimic、miR-34a-inhibitor和陰性對照轉(zhuǎn)染細(xì)胞。轉(zhuǎn)染后qPCR檢測miR-34a的表達(dá)情況。

1.2.3 Western blot檢測細(xì)胞轉(zhuǎn)染后中Snail、E-cadherin、Vimentin、Twist的表達(dá) 取轉(zhuǎn)染miR-34a-mimic和miR-34a-inhibitor 48 h的細(xì)胞蛋白，BCA法測定蛋白濃度，加入Loading buffer后變性蛋白。配制10%SDS-PAGE，每孔加入20 μg蛋白樣品。使用濕轉(zhuǎn)法電轉(zhuǎn)至PVDF膜，5%脫脂奶粉封閉2 h，1∶1 000 TBST稀釋一抗，4℃過夜；加入羊抗兔二抗1∶5 000 稀釋，室溫孵育2 h，ECL發(fā)光。重復(fù)3次。

1.2.4 Transwell侵襲實(shí)驗(yàn)檢測miR-34a對肺癌細(xì)胞侵襲能力的影響 所有試劑及器材均于冰上預(yù)冷，將Transwell小室置于24孔板內(nèi)，將Transwell小室內(nèi)膜均勻涂抹Matrigel膠 50 μl (0.2 μg/μl)，37℃孵育15 min，使膠凝固；消化、離心、計(jì)數(shù)細(xì)胞后，按照2.5×104個(gè)/ml用無血清培養(yǎng)基稀釋細(xì)胞，制成細(xì)胞懸液；按照每孔200 μl，將細(xì)胞懸液加入Transwell上室，同時(shí)在Transwell下室加入10%FBS+培養(yǎng)基500 μl，放入37℃孵箱培養(yǎng)；甲醛固定，結(jié)晶紫染色15 min，然后用棉簽輕輕擦拭內(nèi)膜上的細(xì)胞。顯微鏡下技術(shù)，計(jì)數(shù)4個(gè)高倍視野(×40)下穿過濾膜的細(xì)胞數(shù)。實(shí)驗(yàn)重復(fù)3次。

1.2.5 劃痕實(shí)驗(yàn)檢測miR-34a對肺癌細(xì)胞遷移能力的影響 劃痕實(shí)驗(yàn)：將H1650細(xì)胞接種于6孔板，待細(xì)胞融合度生長在90%時(shí)，用200 μl消毒槍頭從上而下劃線，并在顯微鏡下觀察，測量劃痕的初始距離(0 time)；在24、48、72 h后，分別測量劃痕的距離，并拍照，計(jì)算細(xì)胞的遷移率。實(shí)驗(yàn)重復(fù)3次。

1.2.6 熒光素酶活性檢測 將熒光素酶報(bào)告載體與miR-34a-mimic共轉(zhuǎn)染H1650細(xì)胞。以轉(zhuǎn)染pRL-TK作為標(biāo)準(zhǔn)內(nèi)質(zhì)控。轉(zhuǎn)染36 h后，收獲細(xì)胞。按Promega公司熒光素酶活性檢測試劑盒說明書檢測H1650細(xì)胞熒光素酶活性。相對熒光素酶活性=螢火蟲熒光素酶活性值/海腎熒光素酶活性值。實(shí)驗(yàn)重復(fù)3次。

2 結(jié)果

2.1 肺癌組織和正常肺組織中miR-34a mRNA的表達(dá) qPCR結(jié)果表明：與癌旁正常組織相比，肺癌組織中miR-34a mRNA表達(dá)水平明顯降低[(2.51±0.31) vs (0.58±0.17),P<0.05]，差異有統(tǒng)計(jì)學(xué)意義(圖1)。同時(shí)檢測miR-34a在肺癌H1650和H889細(xì)胞中的表達(dá)情況。qRCR結(jié)果表明：H1650肺癌細(xì)胞中miR-34a表達(dá)水平低，H889肺癌細(xì)胞中miR-34a表達(dá)水平高。

2.2 miR-34a表達(dá)與臨床病理資料的關(guān)系 統(tǒng)計(jì)分析表明：miR-34a的表達(dá)水平隨肺癌病理分期的增加而降低；隨分化程度的降低，miR-34a的表達(dá)水平逐漸降低；有淋巴結(jié)轉(zhuǎn)移的肺癌組織中，miR-34a的表達(dá)明顯降低；miR-34a的表達(dá)水平與年齡和性別無關(guān)。結(jié)果表明：miR-34a與肺癌病理分期分級以及淋巴結(jié)轉(zhuǎn)移與否有關(guān)，而與性別、年齡等無關(guān)(表1)。

圖1 qPCR 檢測肺癌組織和細(xì)胞株中miR-34a 的表達(dá)情況Fig.1 qPCR was used to detect expression of miR-34a in lung cancer tissues and cell linesNote: *.P<0.05.

2.3 miR-34a-mimic和miR-34a-inhibitor轉(zhuǎn)染后顯著提高和降低miR-34a mRNA的表達(dá) miR-34a-mimic轉(zhuǎn)染肺癌細(xì)胞H1650 48 h后，qPCR結(jié)果表明：與NC組相比，miR-34a-mimic組miR-34a mRNA水平明顯提高[ (0.22±0.03)vs(0.89±0.13),P<0.01]，差異有統(tǒng)計(jì)學(xué)意義(圖2A)；與NC組相比，miR-34a-inhibitor組miR-34a mRNA水平明顯提高[(0.22±0.03)vs(0.89±0.13),P<0.01]，差異有統(tǒng)計(jì)學(xué)意義(圖2B)。結(jié)果表明，miR-34a-mimic可以顯著提高miR-34a表達(dá)水平；miR-34a-inhibitor可以顯著降低miR-34a表達(dá)水平。

2.4 miR-34a可以調(diào)節(jié)Snail的表達(dá) Western blot結(jié)果顯示：與NC組相比，miR-34a-mimic組中Snail蛋白表達(dá)水平明顯下降(51.4±2.8) vs (13.7±0.8),P<0.05，而miR-34a-inhibitor組中Snail蛋白表達(dá)水平明顯提高[(72.4±3.1) vs (13.7±0.8),P<0.05]，表明miR-34a可以調(diào)節(jié)Snail的表達(dá)(圖3)。

2.5 熒光素酶報(bào)告基因檢測Snail和miR-34a的作用關(guān)系 為明確miR-34a能否與Snail 3′UTR結(jié)合，將miR-34a-inhibitor與Snail-Wt、Snail-Mut共轉(zhuǎn)染到肺癌H1650細(xì)胞中。熒光素酶報(bào)告基因結(jié)果顯示：miR-34a-inhibitor可以明顯抑制Snail-Wt的熒光素酶活性；miR-34a-mimic對Snail-Mut的熒光素酶無明顯抑制作用。結(jié)果表明，miR-34a能與Snail的3′ UTR特異性結(jié)合(圖4)。

表1 miR-34a表達(dá)與肺癌臨床病理特征的關(guān)系

Tab.1 Relationship between miR-34a expression and clinicopathological features of lung cancer

Clinicopatho-logicaldataQuantityLowlevelofmiR-34aHighlevelofmiR-34aPGender0.612Male653233Female351817Age(year)0.821≤60532627>60472324Differentiation0.004High402416Medium321616Low28622Pathologicalstages0.003Ⅰ392019Ⅱ18513Ⅲ37532Ⅳ606Lymphnodemetastasis0.01No553718Yes451233

2.6 miR-34a可以抑制人肺癌細(xì)胞H1650的侵襲能力 細(xì)胞侵襲穿過Matrigel的能力可以反映細(xì)胞的侵襲能力。Transwell結(jié)果顯示：NC組通過Matrigel基質(zhì)膠的細(xì)胞數(shù)量為(346.57±28.70)，明顯多于miR-34a-mimic組(131.92±13.80)，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)(圖5A)；NC組通過Matrigel基質(zhì)膠的細(xì)胞數(shù)量為(72.57±3.20)，明顯少于miR-34a-mimic組(406.57±31.90)，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)(圖5B)，表明miR-34a可以抑制人肺癌細(xì)胞H1650的侵襲能力。

2.7 miR-34a可以抑制人肺癌細(xì)胞H1650的遷移能力 在顯微鏡下分別測量0、24、48、72 h時(shí)，各組細(xì)胞任意三個(gè)部位的劃痕的寬度，遷移率=[D(t=24，48，72 h)-D(t=0 h)]/D(t=0 h)。劃痕實(shí)驗(yàn)結(jié)果表明：與NC組相比，在24、48、72 h時(shí)，miR-34a-mimic組遷移率明顯降低[24 h (54.51±3.15)% vs (13.07±1.12)%,P<0.05;48 h (77.51±5.35)% vs (33.24±2.23)%,P<0.05;72 h (84.22±7.12)% vs (48.21±4.27)%,P<0.01]，差異有統(tǒng)計(jì)學(xué)意義(圖6A)；與NC組相比，在24、48、72 h時(shí)，miR-34a-inhibitor組遷移率明顯增高[24 h (55.83±6.25)% vs (14.02±1.29)%,P<0.05;48 h (78.52±5.18)% vs (32.55±2.18)%,P<0.05;72 h (86.52±8.49)% vs (38.23±4.42)%,P<0.01]，差異有統(tǒng)計(jì)學(xué)意義(圖6B)，表明miR-34a可以抑制人肺癌細(xì)胞H1650的遷移能力。

圖2 qPCR 檢測過表達(dá)和沉默miR-34a后的mRNA表達(dá)水平Fig.2 qPCR was used to detect mRNA expression levels after overexpression and silencing miR-34aNote: *.P<0.05.

圖3 Western blot檢測細(xì)胞轉(zhuǎn)染后Snail蛋白的表達(dá)Fig.3 Expression of Snail were detected by Western blot after transfected with miR-34a-mimic and miR-34a-inhibitorNote: Error bars represent standard error.*.P<0.05.

2.8 miR-34a可以調(diào)節(jié)E-cadherin、Vimentin和Twist的表達(dá) 許多研究表明，上皮間質(zhì)轉(zhuǎn)化在上皮性腫瘤的侵襲和遷移中激活，是上皮性腫瘤細(xì)胞獲得侵襲性的關(guān)鍵分子事件，在惡性腫瘤的侵襲和遷移中發(fā)揮重要的作用[14]。而E-Cadherin蛋白是上皮間質(zhì)轉(zhuǎn)化過程中上皮性標(biāo)志物，而Vimentin和Twist蛋白為上皮間質(zhì)轉(zhuǎn)化過程中間質(zhì)性標(biāo)志物。

圖4 熒光素酶報(bào)告基因檢測Snail是miR-34a的直接靶點(diǎn)Fig.4 Luciferase report gene detects Snail direct target of miR-34aNote: *.P<0.05.

圖5 Transwell侵襲實(shí)驗(yàn)檢測miR-34a對人肺癌細(xì)胞H1650侵襲能力的影響Fig.5 Effect of miR-34a on invasion ability of H1650 cells were detected by Transwell matrigel invasion assaysNote: Error bars represent standard error.*.P<0.05.

圖6 劃痕實(shí)驗(yàn)檢測miR-34a-mimic和miR-34a-inhibitor對人肺癌細(xì)胞H1650遷移能力的影響Fig.6 Effeet of miR-34a on H1650 cell migration ability by wound healing a assaysNote: A.Effect of miR-34a-mimic on H1650 cells migration ability were detected by wound healing assays;B.Effect of miR-34a-inhibitor on H1650 cells migration ability were detected by wound healing assays.Error bars represent standard error.*.P<0.05.

圖7 Western blot檢測過表達(dá)和沉默miR-34a后Ecadherin、Vimentin 和Twist 蛋白的表達(dá)水平Fig.7 Western blot was used to detect Ecadherin,Vimentin and Twist protein expression levels after overexpression and silencing miR-34a Note: Error bars represent standard error.*.P<0.05.

Western blot結(jié)果顯示：與NC組相比，miR-34a-mimic組中Vimentin、Twist蛋白表達(dá)水平明顯下降[Vimentin (0.12±0.01)% vs (0.82±0.06)%，Twist (0.11±0.02)% vs (0.72±0.06)%,P<0.05]，但E-Cadherin表達(dá)水平明顯增高[(0.76±0.05)% vs (0.22±0.02)% ,P<0.05]。與NC組相比，miR-34a-inhibitor組中Vimentin、Twist蛋白表達(dá)水平明顯增高[Vimentin (0.13±0.01)% vs (0.92±0.08)%;Twist (0.12±0.02)% vs (0.68±0.06)%,P<0.05]，但E-Cadherin表達(dá)水平明顯降低[(0.83±0.06)% vs (0.21±0.02)% ,P<0.05]。表明miR-34a可以調(diào)節(jié)E-cadherin、Vimentin和Twist的表達(dá)，表明miR-34a可以促進(jìn)肺癌細(xì)胞的上皮間質(zhì)轉(zhuǎn)化，見圖7。

3 討論

miRNA是一類內(nèi)源性的、高度保守的非編碼單鏈RNA，廣泛存在于植物和多細(xì)胞動(dòng)物的基因組中[15]。成熟的miRNA經(jīng)過Drosha和Dicer酶剪切，可以與靶基因的3-UTR區(qū)結(jié)合降解或者抑制靶基因的翻譯[16]。miR-34a的編碼基因位于染色體1p36，研究表明，miR-34a受p53的直接調(diào)控，p54的低表達(dá)或者功能異常可以降低對miR-34a轉(zhuǎn)錄的刺激信號，從而降低miR-34a的表達(dá)，從而影響下游靶基因發(fā)揮生物學(xué)功能[17,18]。Garofalo等[19]使用miR-34a-mimic轉(zhuǎn)染非小細(xì)胞肺癌細(xì)胞系進(jìn)行轉(zhuǎn)染，發(fā)現(xiàn)肺癌細(xì)胞的增殖能力明顯減弱。Gougelet等[20]對肝癌組織中miR-34a的表達(dá)分析發(fā)現(xiàn)，肝癌組織中miR-34a的表達(dá)水平明顯降低，且跟肝癌分期和患者不良預(yù)后密切相關(guān)。

本研究通過檢測肺癌和正常肺組織中miR-34a的表達(dá)發(fā)現(xiàn)，肺癌組織中miR-34a的表達(dá)明顯降低，miR-34a的表達(dá)水平隨肺癌病理分期的增加而降低；隨分化程度的降低，miR-34a的表達(dá)水平逐漸降低；有淋巴結(jié)轉(zhuǎn)移的肺癌組織中，miR-34a的表達(dá)明顯降低，表明miR-34a在肺癌組織中起抑癌作用。

上皮間質(zhì)轉(zhuǎn)化發(fā)生于多種生理和病理過程中，表現(xiàn)為上皮標(biāo)記基因的表達(dá)與細(xì)胞間黏附力的喪失，間質(zhì)標(biāo)記基因、細(xì)胞遷移與運(yùn)動(dòng)力的獲得和細(xì)胞形態(tài)的改變[21]。E-cadherin是維持上皮細(xì)胞結(jié)構(gòu)極性和完整性的糖蛋白，對于維持細(xì)胞與細(xì)胞間的黏附能力非常重要。許多研究表明，E-cadherin在許多惡性腫瘤中表達(dá)低下，且E-cadherin的低表達(dá)與腫瘤侵襲、遷移和不良預(yù)后密切相關(guān)。而Snail是E-cadherin的抑制子，可以負(fù)性調(diào)節(jié)E-cadherin的表達(dá)[22]。Chen等[23]在肝癌中的研究發(fā)現(xiàn)，Snail高表達(dá)的腫瘤組織中，E-cadherin表達(dá)常常下降，同時(shí)與腫瘤轉(zhuǎn)移密切相關(guān)。

本研究通過使用miR-34a-mimic和miR-34a-inhibitor后，Snail表達(dá)明顯降低和提高，使用熒光素酶報(bào)告基因檢測miR-34a和Snail相互作用發(fā)現(xiàn)，miR-34a能與Snail的3′ UTR特異性結(jié)合。Transwell和劃痕實(shí)驗(yàn)表明，miR-34a可以調(diào)控肺癌細(xì)胞的侵襲和遷移能力。Western blot檢測E-cadherin、Vimentin和Twist蛋白的表達(dá)水平發(fā)現(xiàn)，E-cadherin表達(dá)水平與Snail相反，但Vimentin和Twist蛋白的表達(dá)水平與Snail表達(dá)趨勢一致。

本研究表明miR-34a在肺癌組織中表達(dá)明顯降低，而且與腫瘤分期分級以及淋巴結(jié)轉(zhuǎn)移密切相關(guān)，同時(shí)發(fā)現(xiàn)miR-34a可以通過誘導(dǎo)上皮間質(zhì)轉(zhuǎn)化來促進(jìn)肺癌細(xì)胞的侵襲和遷移。提示miR-34a可能參與肺癌的發(fā)展過程，有可能成為預(yù)測肺癌進(jìn)展和預(yù)后，以至可能成為肺癌治療的靶標(biāo)。

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[收稿2016-09-28 修回2017-04-20]

(編輯 許四平)

Mechanism of miR-34a on invasion and migration ability of human lung carcinoma by Snail induced EMT

LIUXing-Ren,BAIYI-Feng,LIANGLiang,FENGJing,DENGFei.

DepartmentofRespiration,SichuanProvincialPeople′sHospital,SichuanAcademyofMedicalSciences，Chengdu610072，China

Objective:To investigate the exression of miR-34a on lung cancer and normal lung tissues,and the effect and mechanism of miR-34a in lung cancer cell invasion and migration.Methods: qPCR was used to detect the expression of miR-34a on lung cancer.miR-34a-mimic and miR-34a-inhibitor were used to overexpress and knockdown miR-34a.qPCR was used to detect the effectiveness.Western blot was used to detect the expression of Snail after induced with miR-34a-mimic and miR-34a-inhibitor.Luciferase reporter gene was used to detect interaction between miR-34a and Snail.Transwell invasion assay was used to detect invasion ability after induced with miR-34a-mimic and miR-34a-inhibitor.Scratch assay was used to detect migration ability after induced with miR-34a-mimic and miR-34a-inhibitor.The expression of E-cadherin，Vimentin and Twist were detected by Western blot.Results: miR-34a expression was significantly reduced in lung cancer.With the stage of lung cancer progression,the expression of miR-34a reduced.With the differentiation of lung cancer progression,the expression of miR-34a decreased.Decreasing of miR-34a was associated with lung cancer lymph node metastasis.miR-34a-mimic and miR-34a-inhibitor could overexpress and knockdown miR-34a.miR-34a could regulate expression of Snail.Snail was the direct target of miR-34a;miR-34a could regulate the invasion ability of human lung carcinoma H1650 cells;miR-34a could regulate the migration of human lung carcinoma H1650 cells;miR-34a could regulate the expression of E-cadherin，Vimentin and Twist.Conclusion: miR-34a plays the role of tumor suppressor factor in lung cancer.miR-34a can regulate the invasion and migration ability of lung carcinoma H1650 cells by Snail induced EMT.

miR-34a;Lung cancer;EMT；E-Cadherin；Transwell；Snail

10.3969/j.issn.1000-484X.2017.05.002

①本文受國家自然科學(xué)基金(No.81301910)和四川省衛(wèi)計(jì)委科研課題(No.110137)資助。

劉行仁(1980年-)，男，主治醫(yī)師，主要從事肺部感染及肺部腫瘤治療方面的研究，E-mail：syyliudoc@sina.com。

R541.6 R332

A

1000-484X(2017)05-0646-06

②四川省醫(yī)學(xué)科學(xué)院·四川省人民醫(yī)院腫瘤科,成都610072。

③四川省醫(yī)學(xué)科學(xué)院·四川省人民醫(yī)院中醫(yī)科,成都610072。

④通訊作者，四川省醫(yī)學(xué)科學(xué)院·四川省人民醫(yī)院腎內(nèi)科,成都610072，E-mail：dengfei_here@163.com。