上調(diào)miR-200c表達(dá)對(duì)人肝癌干細(xì)胞化療耐藥性的影響

丁　紅，千年松，楊　凡，張鵬飛，王艷榮，戴廣海

(1.解放軍總醫(yī)院，北京 100853；2.第四軍醫(yī)大學(xué)西京醫(yī)院，陜西 西安 710032)

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上調(diào)miR-200c表達(dá)對(duì)人肝癌干細(xì)胞化療耐藥性的影響

丁紅1，千年松1，楊凡2，張鵬飛1，王艷榮1，戴廣海1

(1.解放軍總醫(yī)院，北京 100853；2.第四軍醫(yī)大學(xué)西京醫(yī)院，陜西 西安 710032)

目的觀察人肝癌干細(xì)胞中miR-200c的表達(dá)改變對(duì)化療藥物敏感性的影響。方法利用流式細(xì)胞分選技術(shù)從MHCC97人肝癌細(xì)胞系中分離出肝癌干細(xì)胞。應(yīng)用脂質(zhì)體介導(dǎo)轉(zhuǎn)染miR-200c mimics到肝癌干細(xì)胞；采用實(shí)時(shí)熒光定量RT-PCR對(duì)轉(zhuǎn)染miR-200c mimics前后肝癌干細(xì)胞中miR-200c表達(dá)水平進(jìn)行檢測(cè)；通過噻唑藍(lán)(MTT)法檢測(cè)miR-200c對(duì)肝癌干細(xì)胞藥物敏感性的影響。結(jié)果肝癌干細(xì)胞中miR-200c的相對(duì)表達(dá)量為0.17±0.02，轉(zhuǎn)染miR-200c mimics后肝癌干細(xì)胞中miR-200c的相對(duì)表達(dá)量為1.24±0.28，轉(zhuǎn)染前后比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。轉(zhuǎn)染前5-氟尿嘧啶 (5-Fu)和阿霉素(ADM)對(duì)肝癌干細(xì)胞的半數(shù)抑制濃度(IC50)分別為22.74 mg/L和9.56 mg/L，轉(zhuǎn)染miR-200c mimics后5-Fu和ADM對(duì)肝癌干細(xì)胞的IC50分別為12.63 mg/L和3.51 mg/L，轉(zhuǎn)染前后比較差異均有統(tǒng)計(jì)學(xué)意義(P均<0.05)。結(jié)論上調(diào)人肝癌干細(xì)胞中miR-200c的表達(dá)可以增強(qiáng)肝癌干細(xì)胞的化療敏感性，具有逆轉(zhuǎn)其化療耐藥的作用。

肝癌；腫瘤干細(xì)胞；miR-200c；化療耐藥性

腫瘤干細(xì)胞(cancer stem cell,CSC)學(xué)說認(rèn)為，腫瘤干細(xì)胞自身所具有的多藥耐藥特性是導(dǎo)致惡性腫瘤對(duì)化療藥物治療敏感性降低，并引起腫瘤復(fù)發(fā)甚至轉(zhuǎn)移的“罪魁禍?zhǔn)住盵1-2]。microRNA(miRNA)為在真核細(xì)胞生物體中廣泛存在的非編碼小分子單鏈RNA，其能夠調(diào)控腫瘤細(xì)胞中多個(gè)耐藥基因的表達(dá)，因而其參與腫瘤多藥耐藥性的具體機(jī)制也成為目前腫瘤治療領(lǐng)域的研究熱點(diǎn)[3]。miR-200c是近年來發(fā)現(xiàn)的一個(gè)與腫瘤形成、腫瘤進(jìn)展和化療耐藥有關(guān)的miRNA[4]。然而miR-200c是否參與調(diào)控肝癌干細(xì)胞的化療耐藥性，目前尚未見相關(guān)研究報(bào)道。本研究從肝癌細(xì)胞中分選出肝癌干細(xì)胞，通過轉(zhuǎn)染miR-200c mimics上調(diào)肝癌干細(xì)胞miR-200c的表達(dá)，觀察miR-200c對(duì)肝癌干細(xì)胞耐藥性的影響，現(xiàn)將結(jié)果報(bào)道如下。

1　實(shí)驗(yàn)資料

1.1主要材料與試劑人肝癌細(xì)胞MHCC97購自上海復(fù)旦大學(xué)中山醫(yī)院肝癌研究所；四甲基偶氮唑藍(lán)(MTT)和熒光染料Hoechst33342購自Sigma公司；DMEM F12培養(yǎng)基、小牛血清購于Gibco公司；TaqMan MicroRNA Assay、mirVana RNA Isolation Kit和mirVanaTMmiRNA Isolation Kit購自美國Ambion公司；Prism?7300實(shí)時(shí)熒光定量PCR儀購自美國ABI公司。miR-200c、U6引物及miR-200c mimics購自上海吉瑪制藥技術(shù)有限公司。

1.2方法

1.2.1細(xì)胞培養(yǎng)將MHCC97細(xì)胞接種于DMEM F12 培養(yǎng)液(10%小牛血清+100 IU/mL青霉素+100 IU/mL鏈霉素)中，放入細(xì)胞培養(yǎng)箱中于37 ℃、5% CO2條件下培養(yǎng)；0.25%胰蛋白酶消化、傳代，取對(duì)數(shù)生長期細(xì)胞進(jìn)行實(shí)驗(yàn)。

1.2.2肝癌干細(xì)胞的分選將MHCC97細(xì)胞制成單細(xì)胞懸液并調(diào)整細(xì)胞密度為1×109L-1，分為2組，分別加入熒光染料Hoechst33342和維拉帕米至終濃度分別為6 mg/L和50 mmol/L。在Advantage II型流式細(xì)胞儀上，選擇355 nmUV激光發(fā)射源進(jìn)行檢測(cè)，根據(jù)細(xì)胞二維散點(diǎn)圖形態(tài)及參數(shù)分選出邊緣群細(xì)胞即為肝癌干細(xì)胞。

1.2.3細(xì)胞轉(zhuǎn)染將分選后的肝癌干細(xì)胞接種于6孔板中，培養(yǎng)24 h(細(xì)胞融合度達(dá)60%左右)后，按脂質(zhì)體Lipofectamine2000說明進(jìn)行SP細(xì)胞miR-200c mimics的轉(zhuǎn)染。待細(xì)胞轉(zhuǎn)染48 h后采用RT-PCR檢測(cè)細(xì)胞miR-200c表達(dá)水平。miR-200c mimics序列： 5’-UAAUACUGCCGGGUAAUGAUGA-3’。

1.2.4實(shí)時(shí)熒光定量RT-PCR檢測(cè)轉(zhuǎn)染48 h采用mirVana RNA Isolation Kit和mirVanaTMmiRNA Isolation Kit并按說明進(jìn)行細(xì)胞總RNA及miRNA的提取。逆轉(zhuǎn)錄反應(yīng)體系：1×mirVanaRT Primer 1 μL，mirVana5×RT Buffer 2 μL，Arrayseript Enzme Mix 0.4 μL，25 ng RNA，加無RNA酶水至總體積10 μL。PCR擴(kuò)增體系：Taqman MicroRNA Assay(20×) 1 μL，Product from RT reaction 1.33 μL，Taqman 2×Universal PCR Master Mix 10 μL，加無RNA酶水7.67 μL至總體積20 μL。反應(yīng)條件：95 ℃ 10 min；95 ℃ 15 s，60 ℃ 30 s，70 ℃ 30 s共40個(gè)PCR循環(huán)。RT- PCR數(shù)據(jù)結(jié)果采用2-ΔΔCT法進(jìn)行分析。U6 snRNA引物序列：上游為5’-CTCGCTTCGGCAGCACA-3’，下游為5’-AACGCTTCACGAATTTGCGT-3’；miR-200c引物序列：上游為5’-GGTAATACTGCCGGGTAAT-3’，下游為5’-CAGTGCGTGTCGTGGAGT-3’。

1.2.5MTT實(shí)驗(yàn)將分選后的肝癌干細(xì)胞按5×104/孔接種于96孔板中，每孔100 μL。細(xì)胞培養(yǎng)箱中培養(yǎng)24 h后，分為5-氟尿嘧啶 (5-Fu)組、阿霉素(ADM)組和空白對(duì)照組。配制不同濃度的含5-Fu和ADM化療藥物的細(xì)胞培養(yǎng)基，然后每孔分別加入相應(yīng)培養(yǎng)基100 μL，每個(gè)藥物不同濃度分別設(shè)置6個(gè)復(fù)孔；同時(shí)設(shè)置無藥物干預(yù)的細(xì)胞對(duì)照組和無細(xì)胞的空白對(duì)照組。繼續(xù)孵育細(xì)胞24 h 后，加入20 μL MTT(5 g/L)孵育4 h后去除培養(yǎng)基，每個(gè)培養(yǎng)孔加入二甲基亞砜150 μL，振蕩反應(yīng)10 min。置于全自動(dòng)酶標(biāo)儀中，設(shè)定波長為490 nm，讀取各孔吸光度(A)值，重復(fù)3次，計(jì)算半數(shù)抑制劑量(IC50)。

2　結(jié)　　果

2.1肝癌干細(xì)胞分選情況利用流式細(xì)胞分選技術(shù)分離純化肝癌干細(xì)胞，流式細(xì)胞儀二維參數(shù)圖左下角兩種熒光均陰性或很弱的區(qū)域即為肝癌干細(xì)胞，結(jié)果顯示肝癌干細(xì)胞約占肝癌細(xì)胞總數(shù)的1.94%。

2.2轉(zhuǎn)染miR-200c mimics前后肝癌干細(xì)胞miR-200c表達(dá)量轉(zhuǎn)染前，肝癌干細(xì)胞中miR-200c的相對(duì)表達(dá)量為0.17±0.02，轉(zhuǎn)染miR-200c mimics后肝癌干細(xì)胞中miR-200c的相對(duì)表達(dá)量為1.24±0.28，轉(zhuǎn)染前后比較差異有統(tǒng)計(jì)學(xué)意義 (P<0.05)。

2.3轉(zhuǎn)染miR-200c mimics前后肝癌干細(xì)胞耐藥性情況轉(zhuǎn)染前5-Fu和ADM對(duì)肝癌干細(xì)胞的 IC50分別為22.74 mg/L和9.56 mg/L，轉(zhuǎn)染miR-200c mimics后5-Fu和ADM對(duì)肝癌干細(xì)胞的IC50分別為12.63 mg/L和3.51 mg/L，轉(zhuǎn)染前后比較差異均有統(tǒng)計(jì)學(xué)意義(P均<0.05)，提示增強(qiáng)肝癌干細(xì)胞miR-200c的表達(dá)能夠增加肝癌干細(xì)胞對(duì)化療藥物的敏感性。

3　討　　論

miRNA是廣泛存在于真核細(xì)胞生物體中的具有高度保守序列的小RNA。其作用機(jī)制主要在于抑制靶基因的翻譯或直接降解靶基因mRNA。目前的研究證實(shí)，miRNA可以調(diào)節(jié)約30%人類基因的表達(dá)和翻譯[5]。尤其值得關(guān)注的是，研究人員在miRNA研究過程中發(fā)現(xiàn)miRNA能夠通過調(diào)控耐藥相關(guān)相關(guān)靶基因的表達(dá)，進(jìn)而影響其信號(hào)通路，從而導(dǎo)致腫瘤細(xì)胞的耐藥性發(fā)生改變。如miR-328作用于轉(zhuǎn)運(yùn)蛋白ABCG2的3’-UTRs，通過抑制ABGG2的表達(dá)來提高腫瘤細(xì)胞的化療敏感性[6]；miR-125b通過調(diào)控腫瘤耐藥基因Bcl-2 和ABCC4的表達(dá)來影響腫瘤細(xì)胞的耐藥性[7]。由于miRNA在調(diào)控腫瘤細(xì)胞耐藥性上的作用至關(guān)重要，且miRNA具有內(nèi)源性和調(diào)控多個(gè)下游靶基因表達(dá)的特性，因而，通過調(diào)控腫瘤細(xì)胞中相關(guān)耐藥miRNAs的表達(dá)，理論上具有比調(diào)控單個(gè)普通耐藥基因具有更好的效能。

隨著腫瘤領(lǐng)域中干細(xì)胞機(jī)制相關(guān)研究的不斷深入，CSCs是腫瘤形成和進(jìn)展的始作俑者這個(gè)觀點(diǎn)已得到醫(yī)學(xué)界的廣泛認(rèn)同。大量的研究證實(shí)，CSCs具有天然耐藥性是導(dǎo)致目前化療藥物治療腫瘤失敗的關(guān)鍵所在[8]。CSCs對(duì)化療藥物產(chǎn)生耐藥性的相關(guān)機(jī)制十分復(fù)雜，尚待進(jìn)一步研究。目前已知與其腫瘤細(xì)胞膜表達(dá)ABC轉(zhuǎn)運(yùn)蛋白、增強(qiáng)的抗DNA損傷及修復(fù)能力、抗凋亡基因表達(dá)增強(qiáng)等機(jī)制有關(guān)[9-10]。雖然隨著臨床上新型化療藥物的不斷研發(fā)和應(yīng)用，能夠很大程度上殺死腫瘤細(xì)胞。但是由于CSCs具有的天然耐藥性使其很難被化療藥物完全消滅，殘存的CSCs則會(huì)導(dǎo)致化療后腫瘤復(fù)發(fā)[11]。因此，針對(duì)CSC的化療耐藥特性機(jī)制進(jìn)行靶向性的研究對(duì)于提高化療效果至關(guān)重要。本次研究采用流式熒光分選技術(shù)這一目前公認(rèn)的腫瘤干細(xì)胞分選技術(shù)從肝癌細(xì)胞系中分選出的肝癌干細(xì)胞，總體約占所有肝癌細(xì)胞總數(shù)的1.94%，這一結(jié)果與相關(guān)文獻(xiàn)報(bào)道數(shù)據(jù)是一致的[12]。

miR-200c屬于miR-200家族成員，通過調(diào)控細(xì)胞的上皮-間充質(zhì)轉(zhuǎn)化(EMT)，以及KRAS、ZEBl等基因的功能和表達(dá)，進(jìn)而調(diào)控腫瘤細(xì)胞增殖、轉(zhuǎn)移和凋亡[13-14]。此外，有研究數(shù)據(jù)顯示[15]，在培養(yǎng)的針對(duì)多柔比星耐藥乳腺癌細(xì)胞株中miR-200c的表達(dá)較敏感株中明顯下調(diào)，其可能的機(jī)制在于miR-200c能調(diào)控酪氨酸激酶受體B(TrkB)和Bmi-1的表達(dá)，進(jìn)而影響乳腺癌細(xì)胞對(duì)多柔比星的敏感性。本次研究我們通過人工合成miR-200c mimics并成功轉(zhuǎn)染肝癌干細(xì)胞后能導(dǎo)致肝癌干細(xì)胞miR-200a表達(dá)明顯升高。進(jìn)一步通過耐藥實(shí)驗(yàn)發(fā)現(xiàn)：上調(diào)人肝癌干細(xì)胞中miR-200c的表達(dá)后，肝癌干細(xì)胞對(duì)5-Fu和ADM的IC50較干預(yù)前明顯降低。以上結(jié)果表明，上調(diào)肝癌干細(xì)胞中miR-200c的表達(dá)能夠提高其對(duì)化療藥物的敏感性。后續(xù)實(shí)驗(yàn)中，我們可通過生物學(xué)實(shí)驗(yàn)確定miR-200c的靶基因，進(jìn)一步研究其在腫瘤耐藥中的具體調(diào)控機(jī)制，為克服和逆轉(zhuǎn)腫瘤細(xì)胞的多藥耐藥研究提供實(shí)驗(yàn)基礎(chǔ)和依據(jù)。

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Influence of miR-200c up-regulation on chemotherapy resistance of liver cancer stem cells

DING Hong1, QIAN Niansong1, YANG Fan2, ZHANG Pengfei1, WANG Yanrong1, DAI Guanghai1

(1.General Hospital of PLA, Beijing 100853, China; 2.Xijing Hospital of The Fourth Military Medical University, Xi’an 710032, Shaanxi, China)

Objective It is to observe the influence of miR-200c up-regulation on the characteristics of chemotherapy resistance of liver cancer stem cells.Methods Liver cancer stem cells were isolated by fluorescence activated cell sorting (FACS) from MHCC97 cell line of human hepatocellular carcinoma.The miR-200c mimics were transfected into liver cancer stem cells by liposome.The expression levels of miR-200c in liver cancer stem cells were measured by reverse transcription polymerase chain reaction (RT-PCR).MTT method was used to evaluate the chemotherapy resistance of liver cancer stem cells.Results The expression of miR-200c in liver cancer stem cells was 0.17±0.02 before transfection, and was 1.24±0.28 after transfection, there was significant difference between before and after transfection (P<0.05).The half inhibition concentration (IC50) of 5-fluorouracil (5-Fu) and adriamycin (ADM) were 22.74 mg/L and 9.56 mg/L respectively before transfection on liver cancer stem cells, and were 12.63 mg/L and 3.51 mg/L respectively after transfection, there was significant difference between before and after transfectin (all P<0.05).Conclusion Up-regulation of the miR-200c expression in liver cancer stem cells can enhance the chemotherapy sensitivity on liver cancer stem cells; it has the effect of reversing the chemotherapy resistance.

hepatocellular carcinoma; cancer stem cell; miR-200c; chemotherapy resistance

丁紅，女，醫(yī)師，主要從事惡性腫瘤的臨床治療與基礎(chǔ)研究。

戴廣海，E-mail:daigh60@sohu.com

北京市科技新星資助計(jì)劃(xx2013107)

10.3969/j.issn.1008-8849.2016.24.002

R-33

A

1008-8849(2016)24-2626-03

2016-03-30