低濃度亞砷酸鈉下調(diào)PML蛋白表達(dá)誘導(dǎo)肝癌干細(xì)胞分化的分子途徑

金世龍，譚智明，張鵬，匡遠(yuǎn)黎，杜波，唐華明，王鄭

低濃度亞砷酸鈉下調(diào)PML蛋白表達(dá)誘導(dǎo)肝癌干細(xì)胞分化的分子途徑

金世龍，譚智明，張鵬，匡遠(yuǎn)黎，杜波，唐華明，王鄭

目的探討低濃度亞砷酸鈉誘導(dǎo)肝癌干細(xì)胞(LCSCs)分化的分子途徑。方法采用免疫熒光、Western blotting和熒光定量PCR分析肝細(xì)胞癌(HCC)組織和細(xì)胞PML、Oct4和Sox2基因的表達(dá)情況，比較低濃度亞砷酸鈉處理后HCC細(xì)胞PML、Oct4和Sox2基因表達(dá)及LCSCs生物功能的變化，確定低濃度亞砷酸鈉對(duì)LCSCs分化的誘導(dǎo)作用及其分子途徑。結(jié)果0.5μg/ml亞砷酸鈉可明顯抑制HuH7和原代HCC細(xì)胞形成腫瘤球，增強(qiáng)HuH7細(xì)胞對(duì)吡柔吡星(THP)的敏感性(P＜0.01)，并抑制HuH7和原代HCC細(xì)胞移植成瘤的能力(P＜0.05)。HCC組織和細(xì)胞表達(dá)PML、Oct4、Sox2 mRNA和蛋白，且0.5μg/ml亞砷酸鈉可使Oct4、Sox2 mRNA(P＜0.05)和蛋白(P＜0.01)表達(dá)下調(diào)。0.5μg/ml亞砷酸鈉處理后HuH7和原代HCC細(xì)胞PML蛋白表達(dá)明顯下調(diào)(P＜0.05)，而Hep3B、HepG2、SMCC-7721、HuH7和原代HCC細(xì)胞PML mRNA表達(dá)無明顯變化。0.5μg/ml亞砷酸鈉可抑制含LCSCs的HuH7和原代HCC細(xì)胞關(guān)鍵基因Oct4、Sox2 mRNA的表達(dá)(P＜0.05)，而且能改變LCSCs對(duì)THP的敏感性、抑制腫瘤球形成和異體移植成瘤等生物學(xué)特征。結(jié)論HCC組織和細(xì)胞表達(dá)PML mRNA及蛋白，低濃度亞砷酸鈉與HCC細(xì)胞內(nèi)PML蛋白直接結(jié)合使之降解，PML核體(PML-NB)隨之崩解，PML-NB上與增殖分化相關(guān)基因的轉(zhuǎn)錄被抑制，從而下調(diào)HCC細(xì)胞Oct4、Sox2等LCSCs相關(guān)基因的表達(dá)，抑制HCC細(xì)胞生長(zhǎng)和誘導(dǎo)LCSCs分化。

癌，肝細(xì)胞；腫瘤干細(xì)胞；細(xì)胞分化

肝細(xì)胞癌(hepatocellular carcinoma，HCC)發(fā)病率居世界惡性腫瘤第五位，診斷時(shí)多數(shù)已屬進(jìn)展期[1-2]，手術(shù)切除率低于50%，即使手術(shù)切除也易復(fù)發(fā)和轉(zhuǎn)移，5年生存率只有10%～30%。肝癌干細(xì)胞(liver cancer stem cells，LCSCs)具有自我更新、多潛能分化和抵抗放化療等特性[3]，在HCC發(fā)生、發(fā)展、復(fù)發(fā)和轉(zhuǎn)移中起關(guān)鍵作用[4-6]。目前臨床治療尚不能針對(duì)性地清除LCSCs，基礎(chǔ)研究也未找到防治HCC復(fù)發(fā)和轉(zhuǎn)移的方法。盡管多項(xiàng)研究結(jié)果顯示砒霜(arsenic trioxide，As2O3)或亞砷酸鈉能明顯抑制HCC細(xì)胞增殖并誘導(dǎo)癌細(xì)胞凋亡，但多中心Ⅱ期臨床試驗(yàn)結(jié)果顯示單用As2O3治療HCC效果并不滿意[7]。近期本課題組研究發(fā)現(xiàn)HuH7和原代HCC細(xì)胞含有LCSCs，低濃度亞砷酸鈉可抑制HCC細(xì)胞Oct4、Sox2表達(dá)，誘導(dǎo)LCSCs分化[8]。本研究探討亞砷酸鈉是否可下調(diào)HCC細(xì)胞早幼粒細(xì)胞白血病(promyelocytic leukemia，PML)蛋白表達(dá)以及低濃度亞砷酸鈉是否通過PML蛋白誘導(dǎo)LCSCs分化。

1 材料與方法

1.1 主要材料及試劑 HuH7、HepG2、Hep3B、SMMC-7721等HCC細(xì)胞由第三軍醫(yī)大學(xué)西南醫(yī)院肝膽外科和病理科贈(zèng)送。DMEM培養(yǎng)基和胎牛血清為Hyclone公司產(chǎn)品，PML抗體購自Fantibody公司。HCC組織取自切除的肝癌組織標(biāo)本。LSM780激光共聚焦顯微鏡為Carl Zeiss公司產(chǎn)品?？俁NA提取試劑盒、cDNA反轉(zhuǎn)錄試劑盒為BioFlux公司產(chǎn)品，Promega GoTaq qPCR Master Mix為Promega公司產(chǎn)品。Mx3000P熒光定量PCR儀為美國STRATAGNE公司產(chǎn)品。

1.2 方法

1.2.1 亞砷酸鈉處理HCC細(xì)胞 HCC細(xì)胞在含10% FBS的DMEM培養(yǎng)液中于37℃、5%CO2、飽和濕度的培養(yǎng)箱孵育，細(xì)胞匯合度達(dá)到70%～80%時(shí)采用0.25%胰酶消化細(xì)胞。普通及原代培養(yǎng)HCC細(xì)胞經(jīng)0.1～1.0μg/ml終濃度亞砷酸鈉處理。

1.2.2 腫瘤球培養(yǎng) 以無血清DMEM/F12(1:1)基礎(chǔ)培養(yǎng)基，加入EGF(10ng/ml)、bFGF(20μg/ml)、N2、B27等制成標(biāo)準(zhǔn)培養(yǎng)基，于超低黏附6孔板加入1ml標(biāo)準(zhǔn)培養(yǎng)基及5.0×103個(gè)HuH7和原代HCC細(xì)胞，在37℃、5%CO2、飽和濕度培養(yǎng)箱中孵育，每日半量換新鮮培養(yǎng)基，觀察記錄細(xì)胞球情況。

1.2.3 CCK-8分析HuH7細(xì)胞對(duì)吡柔吡新(THP)的敏感性 將HuH7細(xì)胞接種于96孔板，每孔加1.0×103個(gè)細(xì)胞，加入100μl含10%FBS的DMEM培養(yǎng)基(每2d換液)使其貼壁生長(zhǎng)。次日細(xì)胞貼壁后分別加入不同濃度亞砷酸鈉(0.25、0.5、0.75、1.0μg/ml)，每種濃度設(shè)12個(gè)孔，設(shè)調(diào)零孔12個(gè)(不加細(xì)胞)，細(xì)胞對(duì)照孔12個(gè)(不加藥物)，每組設(shè)0.5μg/ml THP對(duì)照孔12個(gè)(加THP)，在37℃、5%CO2、飽和濕度的培養(yǎng)箱中孵育96h后吸出培養(yǎng)液。每孔分別加入90μl DMEM培養(yǎng)基和10μl CCK-8溶液，繼續(xù)培養(yǎng)2h，在酶標(biāo)儀上測(cè)定細(xì)胞在450nm處的吸光度(A)值。

1.2.4 Western blotting分析HCC細(xì)胞PML、Oct4和Sox2蛋白表達(dá)

1.2.4.1 HCC細(xì)胞總蛋白提取 將HuH7、HepG2、Hep3B和SMCC-7721這4種HCC細(xì)胞常規(guī)胰酶消化制成細(xì)胞懸液，細(xì)胞數(shù)1.0×106，1500r/min離心5min，移入1.5ml EP管，PBS洗2次。細(xì)胞懸液離心后吸盡上清液，加入含蛋白酶抑制劑的裂解液200μl，冰上裂解30min，4℃下12 000r/min離心15min，上清液移入EP管，蛋白定量后置–70℃?zhèn)溆谩?/p>

1.2.4.2 HCC組織總蛋白提取 患者接受HCC切除術(shù)，切除HCC組織樣本后立即切取癌腫組織(腫瘤邊緣)，稱取100mg HCC加入500μl預(yù)冷的細(xì)胞裂解液，置于冰上用玻璃勻漿器碾磨，裂解30min，4℃12 000r/min離心15min，上清液移入EP管，蛋白定量后置–70℃?zhèn)溆谩?/p>

1.2.4.3 Western blotting檢測(cè)HCC細(xì)胞PML蛋白表達(dá) 配制10%分離膠和4%積層膠。上樣量為每泳道90μg，體積25～30μl。電泳積層膠電壓80V，分離膠電壓100～120V，電泳2～3h后轉(zhuǎn)至PVDF膜。取下PVDF膜，5%脫脂奶粉PBS封閉，室溫振搖1～2h。將封閉的膜滴加一抗按1:100(3%BSA)，4℃濕盒內(nèi)過夜，次日PBS漂洗15min×1次，5min×4次，二抗按1:2500(3%BSA)，25℃振搖1h，PBS漂洗15min×1次，5min×4次?；旌系润w積A液和B液(各500μl，共1ml)，將顯色劑滴加到PVDF膜正面，開始顯色記錄圖像。

1.2.4.4 免疫熒光分析HCC細(xì)胞PML蛋白的表達(dá)4種HCC細(xì)胞普通培養(yǎng)在10mm蓋玻片上，待60%～80%融合時(shí)用37℃預(yù)熱PBS振搖漂洗，5min×2次，4%多聚甲醛室溫固定15min，PBS洗5min后用含0.5% Triton-X-100的PBS室溫下脫色，搖床振搖15min，PBS洗3次。取出蓋玻片放到載玻片上，10%山羊血清37℃封閉30min，PML抗體用PBS按1:50稀釋后滴加15～20μl到蓋玻片上，濕盒內(nèi)4℃冰箱過夜。有細(xì)胞的蓋玻片放進(jìn)24孔板，PBS脫色，搖床洗3次，取出蓋玻片滴加20μl用PBS按1:50稀釋的熒光抗體，37℃避光孵育60min。蓋玻片放進(jìn)24孔板內(nèi)，經(jīng)PBS洗3次，Hoechst33342染細(xì)胞核，PBS洗蓋玻片2次，取出蓋玻片，封片后激光共聚焦顯微鏡下觀察，記錄細(xì)胞PML蛋白表達(dá)情況。

1.2.5 熒光PCR分析HCC細(xì)胞PML、Oct4和Sox2 mRNA表達(dá) 用Trizol Reagent提取HCC細(xì)胞總RNA，檢測(cè)RNA含量后立即置–130℃保存。按BioKT cDNA first strand cynthesis Kit使用說明設(shè)置20μl反應(yīng)體系，置室溫10min，于42℃ 45min、95℃5min、冰浴5min的循環(huán)條件下進(jìn)行反應(yīng)。反轉(zhuǎn)錄后檢測(cè)cDNA含量，于–20℃保存。引物序列如下：GAPDH正義5'-TGCAACCGGGAAGGAAATGA-3'，反義5'-GCCCAATACGACCAAATCAGA-3；PML正義5'-GGCTCGAGAAGGATGTGGTC-3'，反義5'-GAAGTGAGGGCTCCCATAGC-3'；Oct4正義5'-GGCTCGAGAAGGATGTGGTC-3'，反義5'-GAAGTGAGGGCTCCCATAGC-3'；Sox2正義5'-CAGGAGTTGTCAAGGCAGAGA-3'，反義5'-CGCCGCCGATGATTGTTATT-3'。熒光定量PCR反應(yīng)條件：預(yù)變性95℃ 4min；95℃ 30s，55℃20s，72℃ 20s，40個(gè)循環(huán)。擴(kuò)增產(chǎn)物經(jīng)過3%瓊脂糖凝膠電泳鑒定為目標(biāo)條帶。

1.3 HCC細(xì)胞異體移植成瘤實(shí)驗(yàn) HCC細(xì)胞按每組2.0×106個(gè)細(xì)胞分為4組，分別采用PBS配成100μl細(xì)胞懸液，注射到清潔級(jí)裸鼠背部皮下。細(xì)胞移植后隔日皮下注射3～5μg亞砷酸鈉，共注射7次，每組注射10只裸鼠，每周觀察成瘤情況。

1.4 統(tǒng)計(jì)學(xué)處理 采用SPSS 13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。計(jì)量資料之間的比較采用單因素方差分析，率的比較采用χ2檢驗(yàn)。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 亞砷酸鈉對(duì)HuH7細(xì)胞THP敏感性的影響0.5μg/ml亞砷酸鈉可明顯抑制HuH7和原代HCC細(xì)胞形成腫瘤球(圖1)。CCK-8檢測(cè)結(jié)果顯示，0.5μg/ml THP、0.5μg/ml亞砷酸鈉均不能抑制HuH7細(xì)胞生長(zhǎng)(P=0.06，P=0.933)，0.75μg/ml亞砷酸鈉和0.5μg/ml亞砷酸鈉+0.5μg/ml THP可明顯抑制HuH7細(xì)胞增殖(P＜0.001，圖2)。提示亞砷酸鈉可增強(qiáng)HuH7細(xì)胞對(duì)THP的敏感性。

圖1 亞砷酸鈉對(duì)HuH7和原代HCC細(xì)胞腫瘤球形成的影響Fig.1 Effect of sodium arsenite on the formation of tumor sphere in HuH7 cells and primary HCC cellsA. HuH7 cells; B. HuH7 cells treated with 0.5μg/ml of sodium arsenite; C. Primary HCC cells; D. Primary HCC cells treated with sodium arsenite

圖2 亞砷酸鈉和THP處理96h后HuH7細(xì)胞生長(zhǎng)抑制情況(CCK-8分析)Fig.2 Growth inhibition of HuH7 cells 96h after treatment with sodium arsenite and THP (CCK-8 analysis)(1)P＜0.01 compared with THP control group; (2)P＜0.01 compared with 0.5μg/ml As group; (3)P＜0.01 compared with 0.75μg/ml As group; (4)P＜0.01 compared with 1.0μg/ml As group

2.2 亞砷酸鈉對(duì)HuH7和原代HCC細(xì)胞PML蛋白表達(dá)的影響 Western blotting檢測(cè)結(jié)果顯示，HuH7、HepG2、Hep3B、SMMC-7721表達(dá)PML蛋白(圖3A)。24例HCC組織均不同程度表達(dá)PML蛋白(圖3B)。免疫熒光分析結(jié)果顯示，PML蛋白表達(dá)于HuH7、HepG2、SMCC-7721和Hep3B細(xì)胞核，在核內(nèi)呈點(diǎn)狀顆粒分布(圖3E)。熒光定量PCR分析結(jié)果顯示，HCC組織及Hep3B、HepG2、SMCC-7721、HuH7和原代HCC細(xì)胞都表達(dá)PML mRNA(圖3C、F)。經(jīng)亞砷酸鈉處理96h后HuH7和原代HCC細(xì)胞PML蛋白表達(dá)明顯下調(diào)(P＜0.05，圖3D)，HuH7細(xì)胞核PML蛋白熒光顆粒明顯減少(P＜0.01，圖3E)，隨0.5μg/ml亞砷酸鈉處理HuH7細(xì)胞時(shí)間延長(zhǎng)，PML蛋白逐漸減少，最后消失(圖3G)。亞砷酸鈉處理后Hep3B、HepG2和SMCC-7721細(xì)胞核PML蛋白熒光顆粒減少更快。熒光定量PCR分析顯示，亞砷酸鈉處理Hep3B、HepG2、SMCC-7721、HuH7和原代HCC細(xì)胞后，其PML mRNA表達(dá)無明顯變化(圖3F)。提示亞砷酸鈉直接下調(diào)HuH7和原代HCC細(xì)胞表達(dá)PML蛋白，早期對(duì)HCC細(xì)胞PML mRNA表達(dá)無明顯作用。

圖3 亞砷酸鈉對(duì)HuH7和原代HCC細(xì)胞PML蛋白表達(dá)的影響Fig.3 Effect of sodium arsenite on the expression of PML protein in HuH7 and HCC primary cellsA.PML protein expression in HuH7,HepG2,Hep3B and SMMC-7721 cells (Western blotting,NB4 and HL60 cells were utilized as positive controls,Lo2 cells were used as human hepatic cell control); B. PML protein expression in HCC tissue samples; C. PML gene expression in HCC tissues; D. PML protein expression in HuH7 and primary HCC cells (treatment with 0.5μg/ml of sodium arsenite for 5 days; E. PML protein expression in the nuclei of HuH7,HepG2,SMCC-7721 and Hep3B cells [×400,the number of fluorescent PML protein particles was markedly decreased in the nuclei of HuH7 cells treated with 0.5μg/ml of sodium arsenite for 5 days (HuH7+As)]; F. PML gene expression in five sort of HCC cells (no significant change when treated with sodium arsenite for 5 days,P>0.05); G. PML protein expression in HuH7 cells (down-regulated gradually when treated with 0.5μg/ml sodium arsenite after 24,48,72,96 and 120h cultivation)

2.3 亞砷酸鈉對(duì)HuH7和原代HCC細(xì)胞Oct4、Sox2 mRNA表達(dá)的影響 24例HCC組織和4種HCC細(xì)胞均表達(dá)Oct4蛋白(圖4A)，22例HCC組織和4 種HCC細(xì)胞表達(dá)Sox2蛋白(圖4B)。24例HCC組織均存在Oct4(圖4C)和Sox2(圖4D)mRNA表達(dá)。經(jīng)亞砷酸鈉處理后，HuH7和原代HCC細(xì)胞Oct4、Sox2 mRNA(P＜0.05)，Oct4蛋白(P＜0.05)和Sox2蛋白(P＜0.01)表達(dá)均下調(diào)(圖4A、B)。

2.4 HCC細(xì)胞異體移植成瘤實(shí)驗(yàn) 結(jié)果顯示，10只裸鼠皮下移植HuH7細(xì)胞，9只裸鼠成瘤(9/10)，10只裸鼠皮下移植HCC原代細(xì)胞，7只裸鼠成瘤(7/10)。HCC細(xì)胞移植后隔日皮下注射3～5μg亞砷酸鈉，連續(xù)用7次，HuH7組僅1只裸鼠成瘤(1/10)，原代HCC細(xì)胞組沒有成瘤(0/10)，顯示亞砷酸鈉具有抑制HuH7和原代HCC細(xì)胞移植成瘤的能力(P＜0.05)，推測(cè)亞砷酸鈉已誘導(dǎo)LCSCs發(fā)生分化。

圖4 HCC組織及細(xì)胞Oct4、Sox2 mRNA(熒光PCR)和蛋白(Western blotting)表達(dá)Fig.4 Expression of genes and proteins of Oct4 and Sox2 in HCC tissue and cellsA. Oct4 and Sox2 protein expression (24 HCC tissues); B. Oct4 and Sox2 protein expression (HepG2,SMCC-7721,Hep3B,HuH7 cells); C,D. Oct4 and Sox2 gene expression (24 HCC tissues); 1,3,5,7. HepG2,SMCC-7721,Hep3B and HuH7 cells; 2,4,6,8. HepG2,SMCC-7721,Hep3B and HuH7 cell treated with 0.5μg/ml sodium arsenite for 5 days

3 討 論

LCSCs具有自我更新、多潛能分化和抵抗放化療等特性，在HCC的發(fā)生、發(fā)展、復(fù)發(fā)和轉(zhuǎn)移中起關(guān)鍵作用。近期研究證實(shí)亞砷酸鈉具有抑制HepG2、Hep3B、SMCC-7721和HuH7細(xì)胞生長(zhǎng)的作用，HuH7細(xì)胞耐受能力最強(qiáng)，HuH7和原代HCC細(xì)胞含LCSCs，亞砷酸鈉可誘導(dǎo)LCSCs分化[8]。本研究結(jié)果顯示，0.5μg/ml亞砷酸鈉可明顯抑制HuH7和原代HCC細(xì)胞形成腫瘤球，且明顯增強(qiáng)HuH7細(xì)胞對(duì)THP的敏感性，具有抑制HuH7和原代HCC細(xì)胞移植成瘤的能力，提示亞砷酸鈉可抑制或改變LCSCs特有的生物學(xué)功能，LCSCs能被亞砷酸鈉誘導(dǎo)分化或清除。

本課題組前期研究發(fā)現(xiàn)HuH7和原代HCC細(xì)胞可表達(dá)Oct4、Sox2蛋白，且亞砷酸鈉可以下調(diào)這兩種基因及蛋白的表達(dá)[8]，本研究進(jìn)一步證實(shí)HCC組織和細(xì)胞均表達(dá)Oct4、Sox2等干細(xì)胞轉(zhuǎn)錄因子，且亞砷酸鈉具有抑制或下調(diào)HCC細(xì)胞Oct4、Sox2等LCSCs關(guān)鍵基因及蛋白表達(dá)的作用。

本課題組前期研究還發(fā)現(xiàn)HCC組織和HuH7細(xì)胞可表達(dá)PML mRNA和蛋白[8]，為明確亞砷酸鈉是否直接靶向HCC細(xì)胞下調(diào)PML蛋白的表達(dá)，我們首先觀察是否所有HCC組織和細(xì)胞株都表達(dá)PML蛋白，其次用0.5μg/ml亞砷酸鈉處理HuH7和原代HCC細(xì)胞5d，檢測(cè)HCC細(xì)胞PML蛋白和mRNA的表達(dá)變化。結(jié)果顯示，亞砷酸鈉處理HuH7和原代HCC細(xì)胞后PML蛋白表達(dá)均明顯下調(diào)，HuH7細(xì)胞核PML蛋白熒光顆粒明顯減少，而Hep3B、HepG2、SMCC-7721和HuH7細(xì)胞PML mRNA表達(dá)無明顯變化。提示HCC細(xì)胞和組織表達(dá)PML蛋白和基因，具有亞砷酸鈉直接靶向作用的分子基礎(chǔ)，且亞砷酸鈉可直接下調(diào)HuH7和原代HCC細(xì)胞PML蛋白的表達(dá)。

PML基因可維持造血干細(xì)胞(hematopoietic stem cekks，HSCs)、急性粒細(xì)胞白血病(acute promyelocytic leukemia，APL)及慢性粒細(xì)胞白血病(chronic myelogenous leukemia，CML)的白血病起始細(xì)胞(leukemia initiating cells，LICs)穩(wěn)定[9]，PML蛋白表達(dá)下調(diào)或缺失可導(dǎo)致其分化。PML蛋白可與Daxx，Sp100，CBP/p300，SUMO-1，pRb，rfp，ZSG20等形成PML核體(PML nuclear bodies，PMLNB)，PML-NB募集轉(zhuǎn)錄因子并參與染色體構(gòu)型調(diào)控轉(zhuǎn)錄，PML蛋白表達(dá)減少或缺失可使PML-NB結(jié)構(gòu)解體，多種核轉(zhuǎn)錄因子失去發(fā)揮功能的平臺(tái)[10-13]。PML蛋白和PML-NB決定Oct4的表達(dá)，Oct4基因表達(dá)和維持干細(xì)胞染色體開放構(gòu)型也需要PML蛋白，PML蛋白表達(dá)缺失會(huì)導(dǎo)致細(xì)胞失去穩(wěn)定性而發(fā)生分化[12-17]。Chuang等[14]研究證實(shí)，PML蛋白是Oct4表達(dá)的陽性調(diào)節(jié)子，PML蛋白下調(diào)可抑制Oct4基因表達(dá)。Sox2基因在維持LCSCs多能性和決定其是否走向分化方面具有關(guān)鍵作用[18-19]。即PML蛋白不僅決定干細(xì)胞關(guān)鍵基因Oct4、Sox2的表達(dá)，而且還維持著HSCs、APL及CML的LICs穩(wěn)定。我們的結(jié)果顯示，亞砷酸鈉不僅使Oct4、Sox2 mRNA和蛋白表達(dá)下調(diào)，而且可改變HuH7和HCC原代細(xì)胞中LCSCs的功能特征，表明Oct4蛋白下調(diào)使LCSCs難以維持多能性，LCSCs可能已經(jīng)分化。Zhang等[11]證實(shí)As2O3治療APL的機(jī)制在于As2O3中砷直接與癌蛋白PML-RAR結(jié)合，使癌蛋白降解。本研究發(fā)現(xiàn)經(jīng)亞砷酸鈉處理后HuH7和原代HCC細(xì)胞PML蛋白表達(dá)明顯下調(diào)，HuH7細(xì)胞核PML蛋白熒光顆粒明顯減少，隨著0.5μg/ml亞砷酸鈉處理HuH7細(xì)胞時(shí)間的延長(zhǎng)，PML蛋白逐漸減少，最后消失，但熒光定量PCR分析發(fā)現(xiàn)亞砷酸鈉處理Hep3B、HepG2、SMCC-7721、HuH7和原代HCC細(xì)胞后，其PML mRNA表達(dá)無明顯變化，提示亞砷酸鈉直接下調(diào)HuH7和原代HCC細(xì)胞PML蛋白的表達(dá)，進(jìn)一步肯定了砷直接與HCC或APL細(xì)胞PML蛋白結(jié)合，使PML蛋白降解。

綜上所述，我們認(rèn)為HCC組織和細(xì)胞株表達(dá)PML基因及蛋白，低濃度亞砷酸鈉與HCC細(xì)胞內(nèi)PML蛋白直接結(jié)合使之降解，PML-NB隨之崩解，PML-NB上與增殖分化相關(guān)的基因轉(zhuǎn)錄被抑制，從而下調(diào)HCC細(xì)胞Oct4、Sox2等LCSCs相關(guān)基因的表達(dá)，抑制HCC細(xì)胞生長(zhǎng)和誘導(dǎo)LCSCs分化。

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The molecular pathway of low concentration of sodium arsenite in inducing differentiation of liver cancer stem cells by down-regulating promyelocytic leukemia protein expression

JIN Shi-long,TAN Zhi-ming*,ZHANG Peng,KUANG Yuan-li,DU Bo,TANG Hua-ming,WANG Zheng
Department of Hepatobiliary Surgery,Kai Xian People’s Hospital of Chongqing City,Chongqing 405400,China
*< class="emphasis_italic">Corresponding author,E-mail: 605346554@qq.com

,E-mail: 605346554@qq.com
This work was supported by the Chinese Medicine Technological Item of Municipal Health Bureau of Chongqing City (ZY20132047)

ObjectiveTo study the molecular pathway of low concentration of sodium arsenite in inducing differentiation of liver cancer stem cells.MethodsWestern blotting analysis,immunofluorescence assay and quantitative PCR were used to examine the gene and protein expression of promyelocytic leukemia (PML),Oct4 and Sox2 in HCC tissue and cell lines,and the molecule pathway of low concentration of sodium arsenite inducing differentiation of liver cancer stem cells was confirmed by comparing the changes in the gene and protein expression of PML,Oct4 and Sox2 in HCC cells and biological function of LCSCs after the treatment with low concentration of sodium arsenite.Results0.5μg/ml of sodium arsenite was shown to alter the biological characteristics of LCSCs in HuH7 and primary HCC cells,including the ability to form tumor spheres,resistance to pirarubicin (P＜0.01),and the capability of forming tumors after allogeneic transplantation (P＜0.05). Both HCC cells and tissues expressed the gene and protein of PML,Oct4 and Sox2,and 0.5μg/ml of sodium arsenite not only downregulated the gene and protein expression of Oct4 (P＜0.05) and Sox2 in HCC cells (P＜0.05),but also downregulated the protein expression of PML (P＜0.05). In contrast,sodium arsenite did not inhibit the gene expression of PML in Hep3B,HepG2,SMCC-7721,HuH7 and primary HCC cells. Furthermore,through down-regulated PML protein expression with arsenite,the biological characteristics of HuH7 and primary HCC cells containing LCSCs was simultaneously altered,and the expression of stem gene Oct4 and Sox2 was downregulated (P＜0.05),while HCC cells proliferation was inhibited as well.ConclusionsBoth HCC tissues and cells canexpress the PML gene and PML protein. Low concentrations of sodium arsenite would directly bind to PML protein in HCC cells,resulting in degradation of the PML protein,followed by collapse of PML-NBs,inhibition of transcription of the proliferation- and differentiation-related genes in PML-NBs,and down-regulation of expressions of Oct4,Sox2 and other CSC-related genes in HCC cells,thus inhibiting HCC cell growth and inducing LCSC differentiation.

carcinoma,hepatocellular; neoplastic stem cells; cell differentiation

R735.7

A

0577-7402(2015)12-0966-06

10.11855/j.issn.0577-7402.2015.12.06

2015-06-25；

2015-08-27)

(責(zé)任編輯：熊曉然)

重慶市衛(wèi)生局中醫(yī)藥科技項(xiàng)目(ZY20132047)

金世龍，醫(yī)學(xué)博士，主任醫(yī)師，副教授。主要從事肝癌手術(shù)后復(fù)發(fā)與轉(zhuǎn)移機(jī)制的研究

405400 重慶 重慶市開縣人民醫(yī)院肝膽外科(金世龍、譚智明、張鵬、匡遠(yuǎn)黎、杜波、唐華明、王鄭)

譚智明，E-mail: 605346554@qq.com