田偉，張娟，王利娜，張?zhí)┟?，賈萬良，高巖，馬文群

(1 邯鄲市中心醫(yī)院，河北邯鄲056000；2 邯鄲市傳染病醫(yī)院；3 邯鄲市第二醫(yī)院)

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·論著·

miR-181c在膠質(zhì)母細(xì)胞瘤組織中的表達(dá)變化及其對(duì)腫瘤細(xì)胞侵襲、遷移能力的影響

田偉1，張娟1，王利娜2，張?zhí)┟?，賈萬良3，高巖1，馬文群1

(1 邯鄲市中心醫(yī)院，河北邯鄲056000；2 邯鄲市傳染病醫(yī)院；3 邯鄲市第二醫(yī)院)

目的觀察微小RNA181c(miR-181c)在膠質(zhì)母細(xì)胞瘤(GBM)組織中的表達(dá)及其對(duì)腫瘤細(xì)胞侵襲和遷移的影響。方法選擇41例份GBM組織(GBM組)、15例份正常腦組織(對(duì)照組)，采用實(shí)時(shí)定量PCR法檢測組織中miR-181c表達(dá)。取膠質(zhì)母細(xì)胞瘤T98G細(xì)胞，分為A、B、C、D組，A、B、C組分別轉(zhuǎn)染過表達(dá)的miR-181c、空白對(duì)照miRNA、抑制表達(dá)的miR-181c，D組為不做任何處理的空白組。轉(zhuǎn)染48 h觀察各組腫瘤細(xì)胞的侵襲及遷移能力。結(jié)果GBM組組織中miR-181c的表達(dá)明顯低于對(duì)照組，P<0.05。轉(zhuǎn)染48 h時(shí)B、C、D組細(xì)胞侵襲數(shù)均多于A組，P<0.05；B、C、D組細(xì)胞相對(duì)遷移距離均長于A組，P<0.05。結(jié)論GBM組織中miR-181c呈低表達(dá)；miR-181c低表達(dá)與GBM細(xì)胞的高侵襲和遷移能力密切相關(guān)。

微小RNA181c；膠質(zhì)瘤；膠質(zhì)母細(xì)胞瘤；細(xì)胞遷移；細(xì)胞侵襲

膠質(zhì)瘤是最常見的中樞神經(jīng)系統(tǒng)惡性腫瘤[1]，膠質(zhì)母細(xì)胞瘤(GBM)是其中惡性程度最高的一種，預(yù)后極差。有報(bào)道稱手術(shù)切除腫瘤及放、化療聯(lián)合治療的GBM患者的中位生存期仍<15個(gè)月[2,3]。高侵襲性和易遷移是GBM術(shù)中難以徹底切除、術(shù)后容易復(fù)發(fā)的主要原因[4],但其具體機(jī)制目前尚不明確。微小RNA(miRNA)在腫瘤的發(fā)生發(fā)展中起重要作用重要作用[5～7]，miR-181是新發(fā)現(xiàn)的一種miRNA。研究[8,9]發(fā)現(xiàn)，上調(diào)miR-181的表達(dá)可抑制腫瘤細(xì)胞的增殖、侵襲及遷移。但目前miR-181c在GBM侵襲和遷移的影響尚未見報(bào)道。2013年3月～2015年3月我們觀察了miR-181c在GBM組織的表達(dá)情況，并觀察其對(duì)腫瘤細(xì)胞侵襲和遷移的影響，旨在為GBM的治療提供新的思路。

1　材料與方法

1.1標(biāo)本、材料、試劑及儀器人GBM組織標(biāo)本來自2013年3月～2015年3月邯鄲市中心醫(yī)院保存的GBM標(biāo)本41例?；颊咧心?1例、女20例，年齡27～63歲；均經(jīng)病理確診為GBM；均為初次手術(shù)，術(shù)前未行放化療治療。正常腦組織15例由頭部外傷手術(shù)內(nèi)減壓獲得。標(biāo)本使用均經(jīng)患者本人或家屬同意，實(shí)驗(yàn)過程經(jīng)邯鄲市倫理委員會(huì)批準(zhǔn)。人GBM細(xì)胞株T98G購自中國科學(xué)院生物物理研究所，置于含10%胎牛血清、青鏈霉素雙抗的DMEM培養(yǎng)基中，37 ℃、5% CO2的恒溫培養(yǎng)箱培養(yǎng)。細(xì)胞生長至70%～80%進(jìn)行傳代操作后備用。儀器和試劑：胎牛血清、青鏈霉素雙抗、DMEM培養(yǎng)基購自美國Gibco公司，過表達(dá)的miR-181c(miR-181c mimics)、空白對(duì)照miRNA(miR-nc)、抑制表達(dá)的miR-181c(anti-miR-181c)由上海吉瑪生物技術(shù)有限公司合成，Lipofectamine 2000 購自美國Invitrogen公司,Olympus IX70顯微鏡購自日本奧利巴斯影像公司。

1.2miR-181c在人GBM組織及正常腦組織中的表達(dá)檢測采用實(shí)時(shí)定量PCR法。取41例份人GBM組織(GBM組)、15例份正常腦組織(對(duì)照組)，將標(biāo)本磨碎后置于TRIzol中,按照使用說明書步驟提取總RNA,瓊脂糖凝膠電泳檢測RNA完整性，應(yīng)用TaqmanTMmicroRNA進(jìn)行miRNA反轉(zhuǎn)錄，隨后TaqMan探針法進(jìn)行miRNA檢測及分析，同時(shí)選取U6內(nèi)參作為對(duì)照從而得出miR-181c的相對(duì)表達(dá)量。

1.3miR-181c對(duì)T98G細(xì)胞侵襲及遷移的影響觀察取適量T98G細(xì)胞，分為A、B、C、D組，A、B、C組分別用Lipofectamine 2000試劑轉(zhuǎn)染miR-181c mimics、miR-nc、anti-miR-181c，所有操作均嚴(yán)格按照使用說明書進(jìn)行；D組為不做任何處理的空白組。采用Transwell法觀察細(xì)胞的侵襲能力：轉(zhuǎn)染48 h時(shí)取各組細(xì)胞，胰酶消化后以2×104/孔接種于matrigel-Transwell小室內(nèi)，小室內(nèi)采用無血清DMEM培養(yǎng)基進(jìn)行培養(yǎng)，下層應(yīng)用10%胎牛血清的DMEM培養(yǎng)基培養(yǎng)。20 h后棉簽擦去上層未發(fā)生侵襲細(xì)胞，甲醇固定后，1 %結(jié)晶紫染色數(shù)分鐘，PBS清洗后顯微鏡下觀察計(jì)數(shù)侵襲細(xì)胞，以侵襲細(xì)胞數(shù)代表細(xì)胞的侵襲能力。采用細(xì)胞劃痕遷移試驗(yàn)觀察細(xì)胞的侵襲能力：轉(zhuǎn)染48 h時(shí)取各組細(xì)胞，胰酶消化后將細(xì)胞接種至6孔板，90%融合后，應(yīng)用10 μL比照直尺進(jìn)行細(xì)胞劃線，劃線后赤壁采用無血清DMEM培養(yǎng)基進(jìn)行培養(yǎng)并應(yīng)用顯微鏡采集照片，24 h后再次進(jìn)行圖像采集，6孔板內(nèi)做好標(biāo)記使每次采集圖像為相同視野，應(yīng)用Image Pro Plus軟件進(jìn)行圖像分析，按照細(xì)胞相對(duì)遷移距離=0 h劃痕寬度-24 h劃痕寬度/0 h劃痕寬度×痕寬度%計(jì)算各組的相對(duì)遷移距離。實(shí)驗(yàn)重復(fù)3次，取平均值。

2　結(jié)果

2.1兩組組織miR-181c表達(dá)比較觀察組和對(duì)照組miRNA181c的相對(duì)表達(dá)量分別為0.60±0.68、3.34±1.48，觀察組與對(duì)照組相比，P<0.05。

2.2各組細(xì)胞侵襲數(shù)及遷移距離比較轉(zhuǎn)染48 h時(shí)各組細(xì)胞侵襲數(shù)及遷移距離比較見表1。轉(zhuǎn)染48 h時(shí)各組細(xì)胞侵襲情況見圖1，各組細(xì)胞遷移情況見圖2。

表1　轉(zhuǎn)染48 h時(shí)各組細(xì)胞侵襲數(shù)及遷移距離比較±s)

注：與A組比較，*P<0.01。

圖1　各組細(xì)胞侵襲情況

3　討論

目前研究認(rèn)為MiR-181家族含有8個(gè)成熟序列，分別是hsa-miR-181a-5p、hsa-miR-181a-3p、hsa-miR-181a-2-3p、hsa-miR-181b-5p、hsa-miR-181b-3p、hsa-miR-181c-5p、hsa-miR-181c-3p、hsa-miR-181d-5p他們之間的堿基序列差異甚微，常成簇排列在染色體上，協(xié)同表達(dá)，協(xié)同發(fā)揮功能。miR-181家族成員的作用機(jī)理與其他miRNA成員一樣，通過與靶標(biāo)的mRNA(信使RNA)的3′-UTR堿基互補(bǔ)結(jié)合，抑制其翻譯功能或降低其穩(wěn)定性，進(jìn)而達(dá)到精確調(diào)控基因表達(dá)的作用，既能調(diào)控癌基因的表達(dá)，又能調(diào)控抑癌基因的表達(dá)。近年來有關(guān)microRNA-181家族的研究十分活躍，并且發(fā)現(xiàn)其在多種惡性腫瘤的發(fā)生發(fā)展中發(fā)揮重要作用[8～10]。在肝癌中，Syamal等[11]發(fā)現(xiàn)miR-181a對(duì)OPN表達(dá)具有負(fù)調(diào)控作用，能通過降低OPN表達(dá)而抑制肝癌細(xì)胞的黏附侵襲轉(zhuǎn)移功能。Ji等[12]在研究肝細(xì)胞癌的腫瘤干細(xì)胞時(shí)，發(fā)現(xiàn)miR-181家族成員可以直接靶向調(diào)控細(xì)胞分化相關(guān)的轉(zhuǎn)錄因子如CDX2，以及Wnt信號(hào)通路抑制因子NLK的表達(dá)，從而維持其腫瘤干細(xì)胞特性。Wang等[13]發(fā)現(xiàn)TGF-b介導(dǎo)的miR-181b和miR-181d在肝癌組織中表達(dá)顯著升高，而作為癌癥抑制因子靶點(diǎn)的TIMP3表達(dá)顯著降低。Gao等[14]在肺癌研究中發(fā)現(xiàn)miR-181a低表達(dá)與患者不良預(yù)后顯著相關(guān)，miR-181a可能成為肺癌的臨床診療標(biāo)志物。Zhu等[15]在研究人肺癌的多藥耐藥時(shí)發(fā)現(xiàn)miR-181b涉及了多藥耐藥的形成并且提示其有潛力成為逆轉(zhuǎn)多藥耐藥的治療靶標(biāo)。Wang等[16]在乳腺癌干細(xì)胞的研究中發(fā)現(xiàn)miR-181通過抑制腫瘤抑制基因ATM的表達(dá)，進(jìn)而促進(jìn)乳腺癌干細(xì)胞的增殖。Pekarsky等[17]在研究慢性B淋巴細(xì)胞白血病(B-CLL)時(shí)發(fā)現(xiàn)白血病相關(guān)癌基因TCL1是miR-181的直接調(diào)控靶標(biāo)，miR-181有可能充當(dāng)慢性B淋巴細(xì)胞白血病的治療藥物。Shin等[18]在研究口腔癌細(xì)胞中發(fā)現(xiàn)miR-181a能逆轉(zhuǎn)腫瘤的惡性行為，并有潛力成為治療癌癥的新型藥物。Yang等[19]發(fā)現(xiàn)miR-181a及miR-181b與口腔癌淋巴結(jié)轉(zhuǎn)移血管侵襲及不良預(yù)后等密切相關(guān)。

圖2　各組細(xì)胞劃痕實(shí)驗(yàn)結(jié)果

研究[7]證實(shí),microRNA可以作為評(píng)價(jià)GBM生物學(xué)行為及患者預(yù)后的分子生物學(xué)標(biāo)記。Shi等[20]將攜帶miR-181a及miR-181b基因的質(zhì)粒表達(dá)載體同時(shí)轉(zhuǎn)染多種膠質(zhì)母細(xì)胞瘤細(xì)胞株，發(fā)現(xiàn)兩者均能顯著抑制所有細(xì)胞株的增殖及侵襲轉(zhuǎn)移能力，并能促進(jìn)凋亡，其中miR-181b的作用比miR-181a更為顯著，這些提示miR-181a及miR-181b在膠質(zhì)瘤發(fā)生發(fā)展中均充當(dāng)抑癌基因角色。Li等[21]發(fā)現(xiàn)miR-181b在膠質(zhì)瘤干細(xì)胞U87的表達(dá)顯著低于正常膠質(zhì)瘤細(xì)胞U87，miR-181b過表達(dá)可抑制膠質(zhì)瘤干細(xì)胞U87的增殖及其體外成瘤能力，并增加膠質(zhì)瘤干細(xì)胞U87對(duì)化療藥物替莫唑胺的敏感性，這些說明miR-181b在膠質(zhì)瘤干細(xì)胞U87的無限增殖中也扮演抑癌基因作用，而且可能在針對(duì)膠質(zhì)瘤干細(xì)胞的治療中充當(dāng)重要的治療藥物。Radek等[22]在研究多發(fā)性膠質(zhì)母細(xì)胞瘤的臨床預(yù)后時(shí)，對(duì)腫瘤組織進(jìn)行miR-181c和miR-21表達(dá)水平聯(lián)合檢測，可以預(yù)測確診后6個(gè)月內(nèi)的疾病進(jìn)展情況，而且特異性達(dá)81%，敏感性達(dá)92%，說明miR-181c對(duì)識(shí)別術(shù)后早期復(fù)發(fā)的高危膠質(zhì)瘤患者具有重要意義。Conti等[23]報(bào)道在Ⅱ～Ⅳ期膠質(zhì)瘤的臨床組織標(biāo)本中，miR-181b表達(dá)顯著降低，而miR-21和miR-221表達(dá)顯著升高，并提示miRNA可能在腦膠質(zhì)瘤的臨床分期診斷中發(fā)揮重要作用。然而，miR-181c在膠質(zhì)母細(xì)胞瘤中的表達(dá)及生物學(xué)作用尚不清楚。在本研究中，我們發(fā)現(xiàn)與正常腦組織相比，miR-181c在GBM組織中的表達(dá)均顯著降低，這種差異表達(dá)提示miR-181c可能在膠質(zhì)母細(xì)胞瘤中發(fā)揮重要的作用。

侵襲和遷移是惡性腫瘤顯著地生物學(xué)特征，膠質(zhì)瘤的侵襲和遷移能力是決定患者預(yù)后的主要因素[24,25]。影響腦膠質(zhì)瘤侵襲性生長的因素主要包括：膠質(zhì)瘤自身的生物學(xué)特性、血管生成、膠質(zhì)瘤細(xì)胞所處微環(huán)境及膠質(zhì)瘤與機(jī)體免疫系統(tǒng)間的相互作用[4]等。缺氧、代謝、占位效應(yīng)、趨化、手術(shù)刺激等外界因素均可誘導(dǎo)腫瘤脫離瘤體向正常腦組織侵襲[5]。目前研究認(rèn)為microRNA與腫瘤侵襲的關(guān)系十分密切，在肝癌、肺癌、乳腺癌、口腔癌的侵襲轉(zhuǎn)移過程中均發(fā)現(xiàn)了存在microRNA的參與[13～17]。在本研究中，我們發(fā)現(xiàn)在膠質(zhì)瘤細(xì)胞株T98G中上調(diào)miR-181c的表達(dá)能夠顯著抑制膠質(zhì)瘤細(xì)胞株的遷移和侵襲能力。這說明miR-181c能夠在影響GBM侵襲性生長中發(fā)揮重要的作用。結(jié)合既往文獻(xiàn)報(bào)道，我們推測其可能作用機(jī)制為:①miR-181c通過影響TGF-b、Notch等信號(hào)通路促進(jìn)膠質(zhì)瘤的侵襲性生長;②miR181c可能通過囊泡在腫瘤細(xì)胞與基質(zhì)細(xì)胞、免疫細(xì)胞間相互傳遞，從而影響腫瘤微環(huán)境導(dǎo)致腫瘤的侵襲性生長。

綜上所述，GBM組織中存在miR-181c低表達(dá)，miR-181c低表達(dá)與GBM細(xì)胞的高侵襲和遷移能力密切相關(guān)。但其具體作用機(jī)制尚有待于進(jìn)一步研究。

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Expression changes of miR-181c in glioblastoma and its effects on invasion and migration of tumor cells

TIAN Wei1,ZHANG Juan,WANG Lina,ZHANG Taimin,JIA Wanliang,GAO Yan,MA Wenqun

(1 Handan Central Hospital,Handan 056000,China)

ObjectiveTo observe the expression changes of microRNA181c(miR-181c)in glioblastoma(GBM)and its effects on invasion and migration of tumor cells.Methodsreal-time PCR was used to detect the miR-181c expression in 41 GBM tissues(GBM group)and 15 normal brain tissues(control group).GBM cell line T98G was divided into four groups: groups A,B,C and D.In the groups A,B and C,the T98G was transfected with miR-181c with overexpression,control miRNA and miR-181c,respectively,but the group D without any treatment.After transfection 48 h,the invasion and migration in each group was investigated.ResultsCompared with the control group,the miR-181c expression was down-regulated in the GBM group,P<0.05.After transfection 48 h,the invasive cells in the groups B,C and D were more than those of the group A,P<0.05.Moreover,cell relative migration distance in the groups B,C and D was also longer than that of the group A,P<0.05.ConclusionThe miR-181c is lowly expressed in GBM tissues,and the low miR-181c expression is closely related with GBM cell invasion and migration.

microRNA181c;glioma;glioblastoma;cell migration;cell invasion

河北省衛(wèi)生廳青年基金資助項(xiàng)目(20150036)。

田偉(1980-)，男，碩士，主要研究方向?yàn)槟X腫瘤、腦血管病的神經(jīng)外科治療。E-mail：tianwei2003happy@163.com

10.3969/j.issn.1002-266X.2016.27.001

R739.41

A

1002-266X(2016)27-0001-04

2016-02-11)