抑制白血病K562細(xì)胞FoxM1表達(dá)可增強(qiáng)細(xì)胞對(duì)高三尖杉酯堿的敏感性

抑制白血病K562細(xì)胞FoxM1表達(dá)可增強(qiáng)細(xì)胞對(duì)高三尖杉酯堿的敏感性*

陳謹(jǐn)1,周敏然2,孫婷2,秦雪梅2,陳忠敏3,陳春燕2，于媛2△

(1安徽醫(yī)學(xué)高等專(zhuān)科學(xué)校，安徽 合肥 230601；2山東大學(xué)齊魯醫(yī)院血液科，山東 濟(jì)南 250012；3重慶理工大學(xué)藥學(xué)與生物工程學(xué)院， 重慶400054)

[摘要]目的： 探討抑制白血病K562細(xì)胞叉頭框蛋白M1(FoxM1)是否增強(qiáng)細(xì)胞對(duì)高三尖杉酯堿(HHT)的敏感性。方法: HHT以不同濃度(0、0.015、0.030和0.045 μmol/L)和最低起效濃度不同時(shí)間(0.015 μmol/L，0、24、48和72 h)作用于K562細(xì)胞，real-time PCR和Western blot檢測(cè)FoxM1 mRNA和蛋白表達(dá)；以0.015 μmol/L HHT作用K562細(xì)胞后轉(zhuǎn)染FoxM1 siRNA，觀察沉默K562細(xì)胞FoxM1后細(xì)胞對(duì)HHT的敏感性、細(xì)胞增殖和凋亡效應(yīng)以及FoxM1相關(guān)靶分子c-Myc和Sp1表達(dá)狀況。結(jié)果: 隨著HHT濃度增加和時(shí)間延長(zhǎng)FoxM1表達(dá)逐漸降低，說(shuō)明HHT抑制K562細(xì)胞FoxM1表達(dá)；HHT處理K562細(xì)胞后轉(zhuǎn)染FoxM1 siRNA，細(xì)胞生長(zhǎng)和克隆形成顯著下降，細(xì)胞凋亡增加，因此抑制FoxM1可增加K562細(xì)胞對(duì)HHT的敏感性；FoxM1 siRNA組c-Myc和Sp1表達(dá)顯著降低，表明FoxM1可正性調(diào)控c-Myc和Sp1表達(dá)。結(jié)論: HHT可以抑制白血病K562細(xì)胞FoxM1表達(dá)，干擾FoxM1可增強(qiáng)細(xì)胞對(duì)HHT的敏感性。

[關(guān)鍵詞]三尖杉酯堿； 叉頭框蛋白M1； K562細(xì)胞； 藥物敏感性

[中圖分類(lèi)號(hào)]R363.2[文獻(xiàn)標(biāo)志碼]A

doi:10.3969/j.issn.1000-4718.2015.11.002

[文章編號(hào)]1000-4718(2015)11-1933-10

[收稿日期]2014-12-29[修回日期] 2015-08-14

[基金項(xiàng)目]*廣州市屬高校重點(diǎn)學(xué)科建設(shè)經(jīng)費(fèi)資助項(xiàng)目(穗教高教[2011]34號(hào))

通訊作者△Tel: 020-84271652; E-mail: xuxia503@126.com

Inhibition of FoxM1 sensitizes leukemia K562 cells to homoharringtonineCHEN Jin1, ZHOU Min-ran2, SUN Ting2, QIN Xue-mei2, CHEN Zhong-min3, CHEN Chun-yan2, YU Yuan2

(1AnhuiMedicalCollege,Hefei230601,China;2DepartmentofHematology,QiluHospital,ShandongUniversity,Jinan250012,China;3SchoolofPharmacyandBioengineering,ChongqingUniversityofTechnology,Chongqing400054,China.E-mail:yuyuandoctor@163.com)

ABSTRACT[]AIM: To study whether inhibition of forkhead box protein M1(FoxM1) sensitizes leukemia K562 cells to homoharringtonine (HHT). METHODS: K562 cells were incubated with HHT at different concentrations (0 μmol/L, 0.015 μmol/L, 0.030 μmol/L and 0.045 μmol/L) for different time (0 h, 24 h, 48 h and 72 h). The mRNA and protein levels of FoxM1 were detected by real-time PCR and Western blot. FoxM1 siRNA was transfected into K562 cells with 0.015 μmol/L HHT after 6 h. After 72 h incubation, the cell proliferation was detected by cell counting and soft agar assay, and the proportion of apoptotic K562 cells was determined by flow cytometry. The expression of c-Myc and Sp1 were detected by real-time PCR and Western blot. RESULTS: FoxM1 expression was reduced time-dependently and dose-dependently, suggesting that HHT mediated the downregulation of FoxM1 in K562 cells. In K562 cells, treatment with FoxM1 siRNA and HHT inhibited the cell proliferation and promoted the apoptosis significantly. Therefore, inhibition of FoxM1 sensitized leukemia K562 cells to HHT. The expression of c-Myc and Sp1 was positively regulated by FoxM1. CONCLUSION: HHT inhibits Forkhead box protein M1 expression in K562 cells. Inhibition of FoxM1 sensitizes K562 cells to HHT.

[KEY WORDS]Homoharringtonine; Forkhead box protein M1; K562 cells; Drug sensitivity

慢性粒細(xì)胞白血病(chronic myelogenous leukemia，CML)是起源于多能造血干細(xì)胞的惡性克隆增殖性疾病，包含惡性程度遞增的慢性期、加速期和急變期。CML一旦進(jìn)入急變期，細(xì)胞增殖加快、分化受阻，骨髓和外周血中不成熟細(xì)胞大量積聚，對(duì)靶向抑制BCR/ABL酪氨酸激酶活性的伊馬替尼等治療反應(yīng)差，病情惡化，預(yù)后不佳。研究表明CML在急變演進(jìn)過(guò)程中分子調(diào)控方式發(fā)生了改變，其中涉及不依賴(lài)BCR/ABL的復(fù)雜調(diào)控過(guò)程[1-2]。

高三尖杉酯堿(homoharringtonine, HHT)是我國(guó)從三尖杉屬植物中分離出的抗腫瘤生物堿， HHT通過(guò)不依賴(lài)于BCR/ABL機(jī)制，對(duì)于CML加速期和急變期，以及對(duì)伊馬替尼等酪氨酸激酶抑制劑耐藥的CML患者也有效[3-6]。然而，HHT有較強(qiáng)的心臟、骨髓抑制、高血糖等毒副作用，臨床用藥受限，因此發(fā)現(xiàn)與HHT具有協(xié)同作用的增敏分子可降低HHT的臨床有效用藥劑量，是提高HHT藥物效應(yīng)的重要策略。HHT屬于細(xì)胞周期特異性藥物，叉頭框蛋白M1(forkhead box protein M1, FoxM1)是叉頭基因轉(zhuǎn)錄因子家族成員，主要通過(guò)調(diào)控細(xì)胞周期相關(guān)分子轉(zhuǎn)錄介導(dǎo)細(xì)胞增殖，參與腫瘤發(fā)生[7]，抑制FoxM1可提高實(shí)體瘤細(xì)胞對(duì)化療藥物敏感性[8-11]。FoxM1和HHT都可以調(diào)控細(xì)胞周期，靶向干預(yù)FoxM1表達(dá)是否可以協(xié)同增敏HHT抗白血病效應(yīng)未見(jiàn)報(bào)道，我們以慢性髓系白血病急變期細(xì)胞系K562為模型，研究抑制K562細(xì)胞FoxM1表達(dá)是否可以增強(qiáng)HHT的藥物敏感性，為提高HHT治療CML急變的臨床療效提供初步實(shí)驗(yàn)性線索。

材料和方法

1主要試劑

HHT購(gòu)自山東大學(xué)齊魯醫(yī)院，生產(chǎn)廠家杭州民生藥業(yè)有限公司。白血病細(xì)胞系K562購(gòu)自山東省醫(yī)學(xué)科學(xué)院。FoxM1 siRNA和對(duì)照siRNA購(gòu)自Sigma。FoxM1 siRNA序列為5’-GACAACUGUCAAGUGUACCACUCUU-3’，對(duì)照 siRNA序列為5’-CCUACAUCCCGAUCGAUGAUGUUGA-3’。siRNA轉(zhuǎn)染試劑LipofectamineTM2000 reagent和RNA提取相關(guān)試劑TRIzol購(gòu)自Invitrogen；RT試劑盒購(gòu)自Fermentas；實(shí)時(shí)熒光定量PCR試劑盒購(gòu)自TaKaRa；PCR引物由上海博尚生物技術(shù)有限公司合成。Western blot試劑包括30%丙烯酰胺溶液、5×上樣緩沖液、SDS、Tris、甘氨酸等購(gòu)自北京索萊寶科技有限公司。FoxM1抗體購(gòu)自美國(guó)Santa Cruz。ECL化學(xué)發(fā)光檢測(cè)試劑盒購(gòu)自Millipore。凋亡檢測(cè)試劑盒購(gòu)自南京百奧生物科技有限公司。

2細(xì)胞培養(yǎng)

K562細(xì)胞系用10% FBS和RPMI-1640培養(yǎng)基，置于37 ℃、5%CO2細(xì)胞培養(yǎng)箱中培養(yǎng)。鋪板前用多聚賴(lài)氨酸處理培養(yǎng)板24 h，鋪板18～24 h后進(jìn)行實(shí)驗(yàn)。

3siRNA轉(zhuǎn)染實(shí)驗(yàn)

接種1.0×105cells/well于6孔板中，過(guò)夜培養(yǎng)至30%～50%的細(xì)胞融合度。取脂質(zhì)體LipofectimineTM2000 5 μL及siRNA 5 μL分別溶于250 μL Opti-MEM中，混勻后室溫靜置。5 min后將上述2種混合液均勻混合構(gòu)成轉(zhuǎn)染復(fù)合物，室溫靜置20 min。將轉(zhuǎn)染復(fù)合物加入每孔細(xì)胞中孵育72 h，收集細(xì)胞，進(jìn)行檢測(cè)。

4實(shí)時(shí)熒光定量PCR檢測(cè)FoxM1 mRNA水平

4.1引物的設(shè)計(jì)與合成FoxM1上游引物5’-TGCAGCTAGGATGTGAATCTTC-3’，下游引物5’-GGAGCCCAGTCCATCAGAACT-3’；β-actin上游引物5’-AGTTGCGTTACACCCTTTCTTG-3’，下游引物5’-CACCTTCACCGTTCCAGTTTT-3’。

4.2RNA提取與逆轉(zhuǎn)錄反應(yīng)0.5 mL TRIzol處理K562細(xì)胞，提取RNA。將1 μg 總RNA、1 μL隨機(jī)六聚體引物在離心管中混合，補(bǔ)焦碳酸二乙酯(DEPC)水至12 μL，65 ℃保溫5 min，置于冰上；每管中再加入4 μL 5×RT緩沖液，2 μL 10 mmol/L dNTP，1 μL RNA酶抑制劑和1 μL M-MLV逆轉(zhuǎn)錄酶，小心混勻，PCR儀中按“25 ℃，5 min；42 ℃，60 min；70 ℃，5 min”的程序處理樣品，獲得cDNA。

4.3實(shí)時(shí)熒光定量PCR 將cDNA按照1∶5的比例進(jìn)行稀釋?zhuān)唤CR體系后，在實(shí)時(shí)熒光定量PCR儀上按程序“95 ℃ 10 s；95 ℃ 5 s，60 ℃ 31 s，40個(gè)循環(huán)”進(jìn)行PCR。

5Western blot檢測(cè)FoxM1蛋白水平

提取細(xì)胞總蛋白并測(cè)定總蛋白濃度；進(jìn)行聚丙烯酰氨凝膠電泳，溴酚藍(lán)染料在濃縮膠階段，電壓80 V，約40 min后，染料進(jìn)入分離膠時(shí)，電泳增至120 V，約1.5 h后，當(dāng)溴酚藍(lán)指示劑剛剛移出凝膠底端時(shí)結(jié)束電泳；轉(zhuǎn)膜、封閉、加入I抗(FoxM1 I抗：1∶500；β-actin I抗：1∶8 000)；4 ℃孵育過(guò)夜、回收I抗；TBST洗膜，10 min×3次；加入II抗、 TBST洗膜，10 min×3次；PBS洗膜，10 min×1次；化學(xué)發(fā)光試劑盒顯色。

6細(xì)胞生長(zhǎng)曲線實(shí)驗(yàn)

將K562細(xì)胞用HHT作用后再轉(zhuǎn)染FoxM1 siRNA，轉(zhuǎn)染0 h、24 h、48 h和72 h時(shí)分別進(jìn)行細(xì)胞計(jì)數(shù)，繪制細(xì)胞生長(zhǎng)曲線。

7軟瓊脂克隆形成實(shí)驗(yàn)

將0.4%上層瓊脂與經(jīng)過(guò)不同處理的K562細(xì)胞懸液(1 500 cells/well)按照1∶1的比例配制7 mL，混勻后將含有細(xì)胞的上層培養(yǎng)基1 mL/well加入已含1%下層瓊脂培養(yǎng)基的6孔板內(nèi)。不同處理的細(xì)胞各做3個(gè)復(fù)孔。室溫靜置約10 min，待瓊脂凝固后，置于37 ℃、5% CO2細(xì)胞培養(yǎng)箱中孵育。約15 d后觀察白色致密不透光的細(xì)胞團(tuán)。

8流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率

收集處理后的K562細(xì)胞1×106，冷PBS清洗，將用1×binding buffer重懸的細(xì)胞重懸液100 μL放入5 mL流式管中。每管先加入PE標(biāo)記的5 μL Annexin V，輕輕吹勻后，再加入5 μL 7-AAD，室溫避光孵育15 min。每管中再加入400 μL 1×binding buf-fer。用細(xì)胞流式儀進(jìn)行凋亡率檢測(cè)。

9統(tǒng)計(jì)學(xué)處理

采用SPSS 13.0統(tǒng)計(jì)軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行分析。2組間比較用t檢驗(yàn)，多組間的比較采用單因素方差分析，數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

1HHT抑制白血病K562細(xì)胞FoxM1表達(dá)

以不同濃度(0、0.015、0.030及0.045 μmol/L)HHT作用K562細(xì)胞 72 h后， K562細(xì)胞中FoxM1表達(dá)隨著HHT作用濃度增加逐漸降低；以最低起效濃度0.015 μmol/L的HHT作用于K562細(xì)胞不同時(shí)間(0 h、24 h、48 h和72 h)，K562細(xì)胞FoxM1表達(dá)隨著HHT作用時(shí)間延長(zhǎng)而降低，見(jiàn)圖1。以上結(jié)果提示HHT可以抑制K562細(xì)胞FoxM1表達(dá)，且呈濃度與時(shí)間依賴(lài)性。

Figure 1.HHT inhibited FoxM1 expression in K562 cells. A: concentration-dependent effect (72 h); B: time-dependent effect (0.15 μmol/L). Mean±SD.n=3.*P<0.05,**P<0.01.

圖1HHT抑制K562細(xì)胞FoxM1表達(dá)

2抑制FoxM1可增強(qiáng)HHT抑制K562細(xì)胞的增殖作用

0.015 μmol/L HHT作用K562細(xì)胞6 h后再轉(zhuǎn)染FoxM1 siRNA或?qū)φ?siRNA, 0 h、24 h、48 h和72 h分別進(jìn)行細(xì)胞計(jì)數(shù)，繪制細(xì)胞生長(zhǎng)曲線，結(jié)果顯示HHT+FoxM1 siRNA組的細(xì)胞增殖速率顯著降低，見(jiàn)圖2A。轉(zhuǎn)染72 h時(shí)收集細(xì)胞進(jìn)行軟瓊脂克隆形成實(shí)驗(yàn)，克隆形成能力也顯著降低，見(jiàn)圖2B。

3抑制FoxM1可增強(qiáng)HHT促K562細(xì)胞凋亡作用

將K562細(xì)胞用0.015 μmol/L HHT處理6 h后再轉(zhuǎn)染FoxM1 siRNA或?qū)φ?siRNA，流式細(xì)胞術(shù)檢測(cè)到HHT+FoxM1 siRNA組的細(xì)胞凋亡率顯著增高，見(jiàn)圖2C。

Figure 2.Inhibition of FoxM1 enhanced the proliferation inhibition and apoptosis promotion in K562 cells induced by HHT. A: cell counting; B: soft agar assay; C: flow cytometry. Mean±SD.n=3.*P<0.05,**P< 0.01vsHHT+NC siRNA.

圖2抑制FoxM1可增強(qiáng)HHT抑制K562細(xì)胞增殖和促K562細(xì)胞凋亡的作用

4FoxM1正性調(diào)控c-Myc和Sp1表達(dá)

當(dāng)用0.015 μmol/L HHT處理K562細(xì)胞6 h后再轉(zhuǎn)染FoxM1 siRNA或?qū)φ?siRNA，HHT+FoxM1 siRNA組FoxM1、c-Myc 和Sp1 的mRNA和蛋白表達(dá)也顯著降低，見(jiàn)圖3。

Figure 3.FoxM1 combined with HHT positively regulated the expression of c-Myc and Sp1. Mean±SD.n=3.*P<0.05,**P<0.01vsHHT+NC siRNA.

圖3FoxM1聯(lián)合HHT正性調(diào)控K562細(xì)胞c-Myc和Sp1表達(dá)

討論

CML的急變發(fā)生機(jī)制復(fù)雜，不依賴(lài)BCR/ABL機(jī)制參與其中，涉及多種癌基因、抑癌基因和轉(zhuǎn)錄因子等網(wǎng)絡(luò)調(diào)控過(guò)程，分子間相互作用，共同促進(jìn)CML急變[2]。

已有文獻(xiàn)報(bào)道FoxM1與實(shí)體瘤細(xì)胞對(duì)化療藥物敏感性或耐受性相關(guān)，F(xiàn)oxM1過(guò)表達(dá)可增強(qiáng)肝癌細(xì)胞對(duì)TNF-α誘導(dǎo)的凋亡耐受性[9]，抑制FoxM1通過(guò)下調(diào)DNA修復(fù)基因Rad51增加惡性膠質(zhì)瘤細(xì)胞對(duì)抗腫瘤藥替莫唑胺的敏感性[10]，F(xiàn)oxM1敲除可增加腫瘤細(xì)胞對(duì)蛋白酶體抑制劑的敏感性，促進(jìn)細(xì)胞凋亡[11]。

K562細(xì)胞系是從慢性粒細(xì)胞白血病(急變期)患者建立的紅白血病細(xì)胞株，在本研究中，觀察到隨著HHT作用濃度增加或作用時(shí)間延長(zhǎng)，K562細(xì)胞中FoxM1表達(dá)逐漸降低，說(shuō)明HHT可以抑制K562細(xì)胞FoxM1的表達(dá)，并且呈濃度和時(shí)間依賴(lài)性。用低劑量HHT作用K562細(xì)胞后，再轉(zhuǎn)染FoxM1 siRNA，觀察到FoxM1 siRNA與HHT協(xié)同作用導(dǎo)致K562細(xì)胞生長(zhǎng)抑制、細(xì)胞凋亡率增高，說(shuō)明抑制K562細(xì)胞FoxM1表達(dá)可協(xié)同增敏HHT藥理效應(yīng)。

c-Myc是Myc轉(zhuǎn)錄因子家族的一員，F(xiàn)oxM1可以直接作用于c-Myc的啟動(dòng)子區(qū)，促進(jìn)其轉(zhuǎn)錄表達(dá)，F(xiàn)oxM1也作為c-Myc的直接下游靶基因，部分介導(dǎo)c-Myc對(duì)細(xì)胞增殖的促進(jìn)功能[11-12]。研究發(fā)現(xiàn)FoxM1可以與轉(zhuǎn)錄因子Sp1協(xié)同轉(zhuǎn)錄激活c-Myc，而Sp1也可轉(zhuǎn)錄激活FoxM1和c-Myc，在腫瘤細(xì)胞發(fā)生發(fā)展中發(fā)揮作用[12-13]。本研究中在用HHT處理K562細(xì)胞后再轉(zhuǎn)染FoxM1 siRNA，觀察到FoxM1表達(dá)顯著降低的同時(shí)，c-Myc和Sp1表達(dá)也均降低，表明FoxM1正性調(diào)控c-Myc和Sp1表達(dá)，進(jìn)而影響細(xì)胞增殖與凋亡，在其與HHT的協(xié)同增敏中發(fā)揮作用。

綜上所述，靶向干預(yù)K562細(xì)胞FoxM1則增強(qiáng)細(xì)胞對(duì)HHT藥物敏感性，提示FoxM1抑制劑或siRNA可以協(xié)同HHT藥理效應(yīng)的發(fā)揮，此為HHT應(yīng)用于CML急變治療提供了可借鑒的實(shí)驗(yàn)性初步線索。

[參考文獻(xiàn)]

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(責(zé)任編輯： 盧萍， 羅森)