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        MicroRNA-219-5p靶向E-鈣黏蛋白調(diào)控上皮間質(zhì)轉(zhuǎn)化抑制肝癌細(xì)胞侵襲轉(zhuǎn)移

        2016-10-22 07:46:45朱理輝羅勇廖文秋張琍李國慶
        關(guān)鍵詞:肝癌水平

        朱理輝,羅勇,廖文秋,張琍,李國慶

        (南華大學(xué)附屬第二醫(yī)院1.消化內(nèi)科,2.重癥醫(yī)學(xué)科,湖南衡陽421001)

        MicroRNA-219-5p靶向E-鈣黏蛋白調(diào)控上皮間質(zhì)轉(zhuǎn)化抑制肝癌細(xì)胞侵襲轉(zhuǎn)移

        朱理輝1,羅勇2,廖文秋1,張琍1,李國慶1

        (南華大學(xué)附屬第二醫(yī)院1.消化內(nèi)科,2.重癥醫(yī)學(xué)科,湖南衡陽421001)

        目的研究MicroR NA-219-5p(miR-219-5p)靶向E-鈣黏蛋白(E-Cadherin)調(diào)控上皮間質(zhì)轉(zhuǎn)化(EMT)抑制肝癌細(xì)胞侵襲轉(zhuǎn)移的分子機(jī)制。方法首先通過生物信息學(xué)方法,尋找與E-Cadherin結(jié)合特異性最好、穩(wěn)定性強(qiáng)的miR NAs。在30例肝癌組織和20例癌旁肝組織中,通過蛋白免疫印跡法(Western blot)檢測E-Cadherin的表達(dá)水平;實時熒光定量PCR檢測(qR T-PCR)miR-219-5p表達(dá),分析兩者之間的相關(guān)性。在高轉(zhuǎn)移和低轉(zhuǎn)移的肝癌細(xì)胞株中,qR T-PCR檢測miR-219-5p表達(dá),Western blot檢測E-Cadherin、N-cadherin表達(dá)。采用Lipofectamine 2000將miR-219-5p模擬子(mimic)、抑制子(inhibitor)、陰性對照組(negative contral)轉(zhuǎn)染到肝癌HepG2細(xì)胞系,qR T-PCR檢測miR-219-5p表達(dá),Western blot檢測E-Cadherin、N-cadherin的表達(dá);Transwell方法檢測miR-219-5p表達(dá)改變對肝癌細(xì)胞侵襲轉(zhuǎn)移能力的影響。結(jié)果生物信息學(xué)發(fā)現(xiàn)miR-219-5p與E-Cadherin結(jié)合最穩(wěn)定,且特異性最好。肝癌組織中miR-219-5p低表達(dá)、E-Cadherin高表達(dá),而癌旁組織中miR-219-5p高表達(dá)、E-Cadherin低表達(dá)。高轉(zhuǎn)移細(xì)胞株中miR-219-5p高表達(dá)、E-Cadherin低表達(dá)而N-cadherin高表達(dá),在低轉(zhuǎn)移細(xì)胞株中miR-219-5p低表達(dá)、E-Cadherin高表達(dá)而N-cadherin低表達(dá)(P<0.05)。miR-219-5p水平表達(dá)增高,引起E-Cadherin表達(dá)下調(diào),N-cadherin高表達(dá);miR-219-5p表達(dá)下調(diào),可引起E-Cadherin表達(dá)上調(diào),N-cadherin低表達(dá)(P<0.05);HepG2-miR-219-5p模擬子細(xì)胞株中侵襲細(xì)胞計數(shù)為(24±3)個/高倍鏡視野,明顯低于對照組細(xì)胞株(P<0.05)。結(jié)論miR NA-219-5p可通過靶向結(jié)合E-Cadherin,調(diào)控EMT信號通路,抑制肝癌細(xì)胞的侵襲轉(zhuǎn)移。

        miR NA;肝癌;侵襲轉(zhuǎn)移;E-Cadherin;上皮間質(zhì)轉(zhuǎn)化

        原發(fā)性肝細(xì)胞癌(hepatocellular carcinoma,HCC)是原發(fā)于肝細(xì)胞或肝內(nèi)膽管細(xì)胞的惡性腫瘤,惡性程度高,容易發(fā)生血道轉(zhuǎn)移,早期診斷困難。因此,臨床上大多數(shù)肝癌患者就診時,已處于臨床中晚期,預(yù)后較差[1]。目前,臨床上治療肝癌總的療效仍不理想,大多數(shù)患者治療失敗的原因是由于腫瘤發(fā)生轉(zhuǎn)移[2-3]。目前針對肝癌轉(zhuǎn)移復(fù)發(fā)的分子機(jī)制的研究,對于探索肝癌的臨床治療新方法和提高療效,具有重要的臨床和現(xiàn)實意義。

        上皮間質(zhì)轉(zhuǎn)化(epithelial mesenchymal transition,EMT)指上皮細(xì)胞轉(zhuǎn)變?yōu)榫哂虚g質(zhì)細(xì)胞表型的生物學(xué)過程。在胚胎的發(fā)育、炎癥、組織的重建、癌癥轉(zhuǎn)移過程中具有重要作用[4-5],其主要的特征有:如E-鈣黏蛋白(E-cadherin)、β-catenin、角蛋白等表達(dá)下調(diào),而間質(zhì)細(xì)胞表型的N-鈣黏蛋白(N-cadherin)、波形蛋白等表達(dá)上調(diào),并且誘導(dǎo)上皮-間質(zhì)轉(zhuǎn)化的細(xì)胞因子和轉(zhuǎn)錄因子表達(dá)上調(diào)等[6-7]。腫瘤細(xì)胞發(fā)生EMT,導(dǎo)致上皮細(xì)胞失去極性,黏附能力下降,從而失去與基底膜的連接,獲得較高的遷移與侵襲能力,以及降解胞外基質(zhì)等能力[8]。EMT與腫瘤侵襲轉(zhuǎn)移關(guān)系密切,但有關(guān)EMT的調(diào)控分子機(jī)制目前尚不清楚。研究發(fā)現(xiàn),在HCC臨床研究和動物實驗中,發(fā)現(xiàn)EMT的發(fā)生與肝癌細(xì)胞的轉(zhuǎn)移以及化療藥物耐藥有關(guān),在此過程中,許多microRNAs(miR)分子參與了EMT的調(diào)節(jié)[9]。本研究擬探討肝癌細(xì)胞中,miR調(diào)控E-Cadherin影響EMT與肝癌細(xì)胞侵襲轉(zhuǎn)移的分子機(jī)制。

        1 資料與方法

        1.1臨床標(biāo)本及資料

        30例原發(fā)性肝癌組織標(biāo)本及20例癌旁正常肝組織標(biāo)本均來自南華大學(xué)附屬第二醫(yī)院消化內(nèi)科住院患者活檢或手術(shù)切除的新鮮標(biāo)本。原發(fā)性肝癌患者,男性19例,女性11例;年齡32~75歲,中位平均(55.5±7.2)歲。20例癌旁正常肝組織來源于肝組織活檢。所有病例均經(jīng)兩位高年資副主任以上職稱的病理醫(yī)生診斷,結(jié)果均證實準(zhǔn)確無誤。置入-80℃冰箱冷凍保存?zhèn)溆谩?/p>

        1.2細(xì)胞株

        肝癌細(xì)胞系HepG2、人肝癌細(xì)胞低轉(zhuǎn)移細(xì)胞系MHCC97-L、人肝癌細(xì)胞高轉(zhuǎn)移細(xì)胞系MHCC97-H均購自中南大學(xué)細(xì)胞中心。

        1.3主要試劑

        免疫組織化學(xué)SP法試劑盒為福州邁新生物公司產(chǎn)品,鼠抗人單克隆E-Cadherin、N-cadherin抗體、β-actin抗體、過氧化物酶標(biāo)記的二抗、脂質(zhì)體Lipofectamine 2000均購自美國Santa Cruz公司,qRT-PCR試劑為日本TaKaRa公司產(chǎn)品,Transwell侵襲試劑盒為美國Corning公司產(chǎn)品,Matrigel膠為美國B&D公司產(chǎn)品。

        1.4生物信息學(xué)分析

        E-Cadherin(CDH1)的靶miRNAs預(yù)測:通過miRwalk在線程序進(jìn)行,利用10個常用的生物信息學(xué)軟件對miRNA-CDH1之間的結(jié)合進(jìn)行預(yù)測和分析。預(yù)測與CDH1結(jié)合的最佳靶miRNA:通過mi-Randa在線程序進(jìn)行熱力學(xué)穩(wěn)定性評分以及序列保守性評分,對miRNAs-CDH1之間結(jié)合的特異性和穩(wěn)定性進(jìn)行排序。

        1.5qRT-PCR

        首先采用Trizol法提取總RNA。PCR擴(kuò)增采用兩步法進(jìn)行。程序設(shè)定如下:95℃、30 s進(jìn)行1個循環(huán);95℃、30s,60℃、30s,進(jìn)行40個循環(huán)。miR-219-5p的qRT-PCR引物,正向引物:5'-CCGCCCCGGGC CGCGGCUCC-3',反向引物:5'-GCCCCCAAACCUCG AGCGGG-3',由廣州銳博生物科技有限公司技術(shù)合成。在實時熒光定量PCR儀上進(jìn)行檢測,實驗重復(fù)3管,取平均值。反應(yīng)體系如下:10μmol/L正向引物1μl;10μmol/L反向引物1μl;DNA模板2μl;ddH2O 8.5μl;2×SYBR Primix Ex TaqTMⅡ12.5μl;總反應(yīng)體系為25μl。

        1.6蛋白免疫印跡法(Western blot)

        提取細(xì)胞的總蛋白,Bradford法測蛋白濃度;蛋白樣品置于100℃水浴中變性5 min;十二烷基硫酸鈉(sodium dodecyl sulfate,SDS)電泳,設(shè)定電壓為100 V,時間約1~2 h;采用聚偏氟乙烯(polyvinylidene fluoride,PVDF)膜三明治轉(zhuǎn)移法,轉(zhuǎn)膜電壓為100 V,時間為1 h;麗春紅溶液染色3~5 min;5%脫脂奶粉封閉,1~2 h;雜交袋中,加入5%的封閉液和一抗(濃度為1︰1 500),4℃冰箱中過夜;再與二抗結(jié)合(濃度1︰1 000);暗室中壓上X線片,曝光約30~90 s,定影顯影、洗片,最后通過掃描儀進(jìn)行圖像掃描。

        1.7Trasnwell實驗

        采用Lipofectamine2000將miR-219-5p模擬子(mimic)、抑制子(inhibitor)、陰性對照組(negative contral)轉(zhuǎn)染到肝癌HepG2細(xì)胞系,獲得3個重組細(xì)胞株:HepG2-miR-219-5p mimic、HepG2-miR-NC、HepG2-miR-219-5p-inhibitor。具體步驟如下:培養(yǎng)細(xì)胞密度到3×105/孔,6孔板接種。細(xì)胞融合度達(dá)80%時,進(jìn)行脂質(zhì)體轉(zhuǎn)染。Transwell侵襲小室實驗:①侵襲小室的預(yù)處理:首先將Matrigel膠與無血清培養(yǎng)基按1︰100稀釋,將稀釋液用來浸泡侵襲小室。在侵襲小室上面加入100μl稀釋的基質(zhì)膠,紫外線消毒殺菌。②小室腔壓力的平衡:在小室的上腔中加入200μl、下腔中加入600μl的無血清培養(yǎng)基,平衡小室腔內(nèi)的壓力。③侵襲細(xì)胞計數(shù):將侵襲小室取出,對膜上的細(xì)胞加無水乙醇進(jìn)行固定,結(jié)晶紫染色。將未侵入基質(zhì)的腫瘤細(xì)胞通過棉簽輕輕擦拭掉,未除掉即為已經(jīng)成功侵入基質(zhì)膠中的腫瘤細(xì)胞,計數(shù)10個高倍視野中(×20)侵襲細(xì)胞的數(shù)目,重復(fù)3次,取均值,進(jìn)行統(tǒng)計分析。

        1.8統(tǒng)計學(xué)方法

        采用SPSS 17.0統(tǒng)計軟件進(jìn)行數(shù)據(jù)分析,計量資料用均數(shù)±標(biāo)準(zhǔn)差(±s)表示,3組均數(shù)的比較用方差分析,兩兩比較用LSD-t檢驗,P<0.05為差異有統(tǒng)計學(xué)意義。

        2 結(jié)果

        2.1miRWalk預(yù)測CDH1(E-cadherin)基因的靶miRNAs

        采用miRwalk在線程序?qū)DH1的靶miRNA進(jìn)行預(yù)測,結(jié)果發(fā)現(xiàn)10個軟件當(dāng)中有5個以上預(yù)測到有20個miRNAs可與CDH1結(jié)合(見表1)。

        2.2miRanda預(yù)測miRNA-CDH1的結(jié)合

        上述20個靶miRNAs在miRanda軟件當(dāng)中的熱動力學(xué)穩(wěn)定性分值和序列保守性分值見表2。結(jié)果發(fā)現(xiàn),符合條件的miRNAs(熱動力學(xué)穩(wěn)定性分值≤-0.1,序列保守性分值為0.5~0.7)有8個,其中,miR-219-5p與CDH1結(jié)合的熱動力學(xué)穩(wěn)定性得分最低,說明miR-219-5p-CDH1之間的結(jié)合穩(wěn)定性最高、特異性最好。

        2.3肝癌組織中miR-219-5p與E-Cadherin表達(dá)的關(guān)系

        30例肝癌組織中miR-219-5p平均表達(dá)水平低于20例癌旁肝組織,兩者表達(dá)的差異有統(tǒng)計學(xué)意義;而肝癌組織中E-Cadherin相對表達(dá)水平高于癌旁肝組織,經(jīng)t檢驗,兩者表達(dá)差異有統(tǒng)計學(xué)意義(t=10.354,P=0.000,見圖1)。說明肝癌組織中出現(xiàn)miR-219-5p低表達(dá)和E-Cadherin高表達(dá),而癌旁肝組織中出現(xiàn)miR-219-5p高表達(dá)和E-Cadherin低表達(dá)。

        2.4肝癌細(xì)胞中miR-219-5與E-Cadherin表達(dá)的關(guān)系

        分析顯示在高轉(zhuǎn)移細(xì)胞株MHCC97-H中miR-219-5p相對水平低于低轉(zhuǎn)移細(xì)胞株MHCC97-L,高轉(zhuǎn)移細(xì)胞株MHCC97-H中E-Cadherin相對表達(dá)水平低于低轉(zhuǎn)移細(xì)胞株MHCC97-L,高轉(zhuǎn)移細(xì)胞株MHCC97-H中N-cadherin相對表達(dá)水平高于低轉(zhuǎn)移細(xì)胞株MHCC97-L(見圖2)。說明肝癌細(xì)胞中miR-219-5p表達(dá)下調(diào),導(dǎo)致E-Cadherin表達(dá)下調(diào),miR-219-5p可能通過調(diào)控E-Cadherin/N-cadherin的表達(dá)水平,與肝癌細(xì)胞侵襲轉(zhuǎn)移有關(guān)。

        2.5miR-219-5p與E-Cadherin結(jié)合驗證

        3個重組細(xì)胞株HepG2-miR-219-5p mimic、HepG2-miR-NC、HepG2-miR-219-5p-inhibitor中,HepG2-miR-219-5p mimic細(xì)胞株中miR-219-5p水平表達(dá)增高,而E-Cadherin表達(dá)水平下調(diào),N-cadherin高表達(dá);HepG2-miR-219-5p-inhibitor細(xì)胞株中miR-219-5p水平表達(dá)下調(diào),而E-Cadherin表達(dá)水平上調(diào),N-cadherin低表達(dá)(見圖3)。說明轉(zhuǎn)染miR-219-5p mimic后,miR-219-5p表達(dá)水平增高,下調(diào)E-Cadherin而N-cadherin上調(diào);而轉(zhuǎn)染miR-219-5p inhibitor后,miR-219-5p表達(dá)水平下調(diào),上調(diào)E-Cadherin而N-cadherin下調(diào)。說明miR-219-5p能和E-Cadherin的發(fā)生結(jié)合下調(diào)其表達(dá)水平,與EMT有關(guān)。

        表1 預(yù)測的20個miRNAs可與CDH1結(jié)合

        表2 預(yù)測的12個miRNAs的熱動力學(xué)穩(wěn)定性和序列保守性得分

        圖1 肝癌和癌旁肝組織中miR-219-5p與E-Cadherin表達(dá)的關(guān)系

        圖2 肝癌細(xì)胞中miR-219-5p與E-Cadherin、N-cadherin表達(dá)關(guān)系

        2.6miR-219-5p下調(diào)E-Cadherin與肝癌侵襲能力變化

        Transwell實驗檢測轉(zhuǎn)染miR-219-5p mimic下調(diào)E-Cadherin后,結(jié)果顯示,HepG2-miR-219-5p mimic細(xì)胞株中侵襲細(xì)胞計數(shù)為(24±3)個/高倍鏡視野,明顯低于HepG2-miR-NC的(47±5)個/高倍鏡視野、HepG2的(51±5)個/高倍鏡視野,經(jīng)t檢驗,兩者差異有統(tǒng)計學(xué)意義(t=9.952,P=0.003,見表3和圖4);HepG2-miR-219-5p-inhibitor細(xì)胞株中侵襲細(xì)胞計數(shù)為(97±8)個/高倍鏡視野,均明顯高于其他3組,經(jīng)t檢驗,差異有統(tǒng)計學(xué)意義(t=8.856,P= 0.005,見表3和圖4)。

        圖34 個細(xì)胞系中miR-219-5p與E-Cadherin表達(dá)關(guān)系

        表3 不同肝癌細(xì)胞系的侵襲細(xì)胞計數(shù)

        圖4 侵襲實驗檢測不同肝癌細(xì)胞系的侵襲能力(×200)

        3 討論

        惡性腫瘤最顯著的生物學(xué)特征是能夠發(fā)生轉(zhuǎn)移,腫瘤細(xì)胞的轉(zhuǎn)移過程是一個多基因、多步驟、多環(huán)節(jié)的動態(tài)變化過程[10]。該過程中涉及到復(fù)雜的細(xì)胞生物學(xué)過程:腫瘤細(xì)胞的黏附、侵襲、胞外基質(zhì)的重塑、腫瘤血管的發(fā)生以及機(jī)體的免疫狀態(tài)發(fā)生改變等環(huán)節(jié)[11]。肝細(xì)胞癌的轉(zhuǎn)移涉及到許多基因和蛋白分子以及轉(zhuǎn)移相關(guān)信號通路的改變[12-14]。該基因和蛋白的調(diào)控受許多因素的調(diào)控,近年來,有關(guān)miRNA調(diào)控基因表達(dá)與腫瘤侵襲轉(zhuǎn)移的關(guān)系研究已成為腫瘤研究當(dāng)中的熱點(diǎn)和重點(diǎn)。

        EMT是腫瘤發(fā)生轉(zhuǎn)移的主要分子事件之一,是一個復(fù)雜的動態(tài)發(fā)生的過程。其中,細(xì)胞骨架的重構(gòu)是EMT發(fā)生的早期事件,主要表現(xiàn)為上皮細(xì)胞通過EMT獲得了間質(zhì)細(xì)胞的表型[15]。E-cadherin主要功能是維持細(xì)胞間緊密連接的完整性,阻止細(xì)胞發(fā)生侵襲和轉(zhuǎn)移擴(kuò)散,因此,低表達(dá)E-cadherin的細(xì)胞往往可以誘導(dǎo)EMT的發(fā)生[16]。N-cadherin是細(xì)胞間黏連的主要結(jié)構(gòu)性成分,其主要功能是介導(dǎo)細(xì)胞間的黏附和遷移[17]。

        EMT與多種腫瘤的浸潤和轉(zhuǎn)移相關(guān),EMT是腫瘤侵襲轉(zhuǎn)移的一個重要步驟,在該過程當(dāng)中,許多miRNAs分子參與EMT的調(diào)節(jié)[18-20]。EMT也在肝癌轉(zhuǎn)移中具有重要作用,然而EMT對肝癌轉(zhuǎn)移的影響是多方面的。宋瑩等[21]發(fā)現(xiàn),肝細(xì)胞生長因子可能通過誘導(dǎo)Snail的表達(dá)促進(jìn)肝癌細(xì)胞EMT的發(fā)生,與肝癌發(fā)生、發(fā)展有關(guān)。隋承光等[22]發(fā)現(xiàn),過表達(dá)miR-100通過抑制mTOR的表達(dá)抑制肝癌細(xì)胞的遷移及侵襲能力,主要是通過上調(diào)E-cadherin的表達(dá),阻抑EMT過程的發(fā)生。

        miRNA-mRNA之間的相互作用具有嚴(yán)格的互補(bǔ)結(jié)合的特點(diǎn),通過相關(guān)軟件預(yù)測miRNA-mRNA結(jié)合,尋找特定的靶基因具有較好的可行性。本研究首先采用miRwalk在線程序,通過多個生物信息學(xué)軟件進(jìn)行聯(lián)合檢測,結(jié)合多個軟件的檢測結(jié)果具有“交集”性,則說明預(yù)測的可靠性較好[23]。結(jié)果發(fā)現(xiàn),miRwalk在線程序?qū)DH1的靶miRNA進(jìn)行預(yù)測,結(jié)果發(fā)現(xiàn)10個軟件當(dāng)中有5個以上預(yù)測到有20個miRNAs可與CDH1結(jié)合,符合條件的miRNAs有8個,其中,miR-219-5p與CDH1結(jié)合的熱動力學(xué)穩(wěn)定性得分最低,說明miR-219-5p-CDH1之間的結(jié)合穩(wěn)定性最高、特異性最好。

        對于生物信息學(xué)結(jié)果需要進(jìn)行實驗驗證,本研究發(fā)現(xiàn),30例肝癌組織中miR-219-5p平均表達(dá)水平低于20例癌旁肝組織中;而肝癌組織中E-Cadherin相對表達(dá)水平高于癌旁肝組織中。同時在肝癌細(xì)胞系中,研究發(fā)現(xiàn)在肝癌細(xì)胞中miR-219-5p表達(dá)下調(diào),導(dǎo)致E-Cadherin表達(dá)下調(diào),miR-219-5p可能通過調(diào)控E-Cadherin/N-cadherin的表達(dá)水平,與肝癌細(xì)胞侵襲轉(zhuǎn)移有關(guān)。

        miR-219-5p是與E-Cadherin(CDH1)是否具有靶向結(jié)合,為本研究合成miR-miR-219-5p inhibitor和miR-miR-219-5p mimic以及陰性對照組,分別轉(zhuǎn)染到肝癌細(xì)胞,以觀察miR-miR-219-5p對CDH1的靶向調(diào)控作用。結(jié)果發(fā)現(xiàn),HepG2-miR-219-5p mimic細(xì)胞株中miR-219-5p水平表達(dá)增高,而E-Cadherin表達(dá)水平下調(diào),N-cadherin高表達(dá);HepG2-miR-219-5p-inhibitor細(xì)胞株中miR-219-5p水平表達(dá)下調(diào),而E-Cadherin表達(dá)水平上調(diào),N-cadherin低表達(dá)。說明轉(zhuǎn)染mimic后,miR-219-5p表達(dá)水平增高,下調(diào)E-Cadherin而N-cadherin上調(diào);而轉(zhuǎn)染inhibitor后,miR-219-5p表達(dá)水平下調(diào),上調(diào)E-Cadherin而N-cadherin下調(diào)。miR-219-5p可靶向結(jié)合E-Cadherin并下調(diào)其表達(dá)水平,與EMT有關(guān)。

        E-cadherin能夠維持細(xì)胞間的緊密連接的完整性,阻止細(xì)胞發(fā)生侵襲和轉(zhuǎn)移擴(kuò)散,其表達(dá)下調(diào)被證明是腫瘤細(xì)胞發(fā)生侵襲轉(zhuǎn)移的早期事件,低表達(dá)E-cadherin細(xì)胞往往可以誘導(dǎo)EMT的發(fā)生[24]。N-cadherin是細(xì)胞間黏連的主要結(jié)構(gòu)性成分,介導(dǎo)細(xì)胞間的黏附和遷移,N-cadherin在上皮惡性腫瘤中的表達(dá)往往上調(diào),其高表達(dá)往往通過調(diào)控EMT而促進(jìn)腫瘤侵襲和轉(zhuǎn)移[25]。近年來研究發(fā)現(xiàn)E-cadherin/N-cadherin在許多腫瘤中都存在異常表達(dá),說明E-cadherin/N-cadherin在腫瘤的發(fā)生、發(fā)展以及侵襲轉(zhuǎn)移中起著重要的作用[26-27]。

        本研究發(fā)現(xiàn),轉(zhuǎn)染miR-219-5p mimic后,miR-219-5p表達(dá)水平增高,下調(diào)E-Cadherin而N-cadherin上調(diào);而轉(zhuǎn)染miR-219-5p inhibitor后,miR-219-5p表達(dá)水平下調(diào),上調(diào)E-Cadherin而N-cadherin下調(diào)。同時通過transwell實驗發(fā)現(xiàn),轉(zhuǎn)染miR-219-5p mimic后侵襲細(xì)胞計數(shù)明顯低于HepG2-miR-NC、HepG2;轉(zhuǎn)染miR-219-5p inhibitor后細(xì)胞株中侵襲細(xì)胞計數(shù)明顯高于HepG2-miR-NC、HepG2。說明miR-219-5p上調(diào),下調(diào)E-Cadherin而N-cadherin表達(dá)增加,抑制肝癌細(xì)胞侵襲轉(zhuǎn)移;而miR-219-5p下調(diào),上調(diào)E-Cadherin而N-cadherin表達(dá)減少,抑制肝癌細(xì)胞侵襲轉(zhuǎn)移。miR-219-5p靶向E-Cadherin調(diào)控EMT在肝癌侵襲轉(zhuǎn)移中有重要作用,針對E-Cadherin的分子靶向治療在臨床肝癌治療中可能有重要作用。

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        [15]PORTO L P,DOS SANTOS J N,RAMALHO L M,et al.E-cadherin regulators are differentially expressed in the epithelium and stroma of keratocystic odontogenic tumors[J].J Oral Pathol Med, 2016,45(4):302-311.

        [16]PIOTROWSKI-DASPIT A S,TIEN J,NELSON C M.Interstitial fluid pressure regulates collective invasion in engineered human breast tumors via Snail,vimentin,and E-cadherin[J].Integr Biol (Camb),2016,8(3):319-331.

        [17]WANG M,REN D,GUO W,et al.N-cadherin promotes epithelial-mesenchymal transition and cancer stem cell-like traits via ErbB signaling in prostate cancer cells[J].Int J Oncol,2016, 48(2):595-606.

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        [19]TANG J,LI Y,WANG J,et al.Molecular mechanisms of microRNAs in regulating epithelial-mesenchymal transitions in human cancers[J].Cancer Lett,2016,371(2):301-313.

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        [21]宋瑩,劉浩,尹江,等.肝細(xì)胞生長因子誘導(dǎo)人肝癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化[J].中國生物化學(xué)與分子生物學(xué)報,2015,31(7):716-722.

        [22]隋承光,孟凡東,付立業(yè).MicroRNA-100調(diào)節(jié)mTOR表達(dá)對肝癌細(xì)胞侵襲轉(zhuǎn)移及上皮間質(zhì)轉(zhuǎn)化的影響及機(jī)制研究[J].現(xiàn)代腫瘤醫(yī)學(xué),2015,23(1):15-19.

        [23]DWEEP H,GRETZN,STICHTC.miRWalk database for miRNA-target interactions[J].Methods Mol Biol,2014,1182: 289-305.

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        [25]MARTíNEZ-RAMíREZ AS,GARAY E,GARCíA-CARRANCá A,et al.The P2RY2 receptor induces carcinoma cell migration and EMT through cross-talk with epidermal growth factor receptor[J].J Cell Biochem,2016,117(4):1016-1026.

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        (張蕾編輯)

        MicroRNA-219-5p regulates EMT and inhibits invasion and metastasis of hepatoma cells by targeting E-cadherin

        Li-hui Zhu1,Yong Luo2,Wen-qiu Liao1,Li Zhang1,Guo-qing Li1
        (1.Department of Gastroenterology,2.Intensive Care Unit,the Second Affiliated Hospital of Nanhua University,Hengyang,Hunan 421001,China)

        Objective To investigate the molecular mechanism of microRNA-219-5p(miR-219-5p)targeting E-cadherin(CDH1)in the regulation of epithelial mesenchymal trasition(EMT),thus inhibiting the invasion and metastasis of hepatoma cells.Methods Bioinformatics methods were used to determine miRNAs with the best specificity and stability of binding to E-cadherin.The correlation between E-cadherin expression detected by Western blot,and miR-219-5p level by qRT-PCR in 30 hepatoma tissues and 20 normal tissues,respectively,was analyzed. miR-219-5p level was detected by qRT-PCR.E-cadherin and N-cadherin levels were detected by Western blot in high and low metastatic hepatoma cell lines.miR-219-5p mimic,inhibitor and negative control were transfected into HepG2 cell line by Lipofectamine 2000,miR-219-5p expression was detected by qRT-PCR and the expressions of E-cadherin and N-cadherin were detected by Western blot.And the effects of miR-219-5p expression change oninvasive and metastatic abilities were also tested by the transwell method.Results Bioinformatics methods showed that miR-219-5p was the best target miRNA with the highest specificity and stability for binding to E-cadherin. miR-219-5p had low expression and E-cadherin had high expression in the HCC,while miR-219-5p had high expression and E-cadherin had low expression in the paracancerous tissues.There were high expression of miR-219-5p,low expression of E-cadherin and high expression of N-cadherin in highly metastatic cell line MHCC97-H. There were low expression of miR-219-5p,high expression of E-cadherin and low expression of N-cadherin in low metastatic cell line MHCC97-L(P<0.05).Increased miR-219-5p caused E-cadherin down-regulation and N-cadherin up-regulation.miR-219-5p down-regulation caused E-cadherin up-regulation and N-cadherin down-regulation in HepG2-miR-219-5p-inhibitor cell line(P<0.05).The invasion cell count was(24±3)/HP in the HepG2-miR-219-5p mimic cells which was significantly lower than that in the control group(P<0.05). Conclusions miRNA-219-5p can specifically bind to E-Cadherin and regulate the EMT signaling pathway,thus suppress the invasion and metastasis of hepatoma cells.

        miRNA;hepatoma;invasion;metastasis;E-cadherin;EMT

        R 573.2

        A

        10.3969/j.issn.1005-8982.2016.18.005

        1005-8982(2016)18-0022-08

        2016-04-18

        羅勇,E-mail:fordluo@qq.com

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