AR-V7在前列腺癌細(xì)胞耐藥中的作用

馬 淵， 李紀(jì)夫， 王玉杰△

(新疆醫(yī)科大學(xué) 1第一附屬醫(yī)院泌尿外科， 2第五附屬醫(yī)院泌尿外科， 新疆 烏魯木齊 830011)

AR-V7在前列腺癌細(xì)胞耐藥中的作用

馬 淵1， 李紀(jì)夫2， 王玉杰1△

(新疆醫(yī)科大學(xué)1第一附屬醫(yī)院泌尿外科，2第五附屬醫(yī)院泌尿外科， 新疆 烏魯木齊 830011)

目的探討雄激素受體剪接變異體7(AR-V7)在前列腺癌細(xì)胞耐藥中的作用及分子機(jī)制。方法運(yùn)用Lipofectamine 2000轉(zhuǎn)染法將AR-V7 siRNA(siAR-V7)轉(zhuǎn)染至4株前列腺癌細(xì)胞中，命名為PC3-siAR-V7、DU145-siAR-V7、LNCaP-siAR-V7和ArCaP-siAR-V7細(xì)胞，以轉(zhuǎn)染無關(guān)序列(NC siRNA)為陰性對照。運(yùn)用real-time PCR和Western blot實(shí)驗(yàn)分別檢測轉(zhuǎn)染前后細(xì)胞中AR-V7的mRNA和蛋白表達(dá)水平；運(yùn)用MTT法和Transwell法分別檢測細(xì)胞活力和細(xì)胞遷移率；運(yùn)用螢光素酶報(bào)告基因?qū)嶒?yàn)和Western blot實(shí)驗(yàn)分別檢測AR的啟動(dòng)子活性及下游靶基因前列腺特異性抗原(PSA)和FK506結(jié)合蛋白5(FKBP5)的蛋白表達(dá)。構(gòu)建耐比卡魯胺的前列腺癌細(xì)胞系LNCaP-DR，運(yùn)用免疫熒光觀察LNCaP、LNCaP-siAR-V7和LNCaP-DR細(xì)胞中AR和AR-V7的亞細(xì)胞定位；運(yùn)用蛋白質(zhì)免疫共沉淀實(shí)驗(yàn)檢測AR-V7與熱休克蛋白90(HSP90)的相互作用。結(jié)果4株前列腺癌細(xì)胞中的AR-V7 mRNA水平顯著高于正常前列腺上皮細(xì)胞RWPE-1(P<0.05)；PC3-siAR-V7、DU145-siAR-V7、LNCaP-siAR-V7和ArCaP-siAR-V7細(xì)胞中AR-V7蛋白水平、細(xì)胞活性及細(xì)胞遷移率顯著低于NC siRNA轉(zhuǎn)染的細(xì)胞(P<0.05)。隨著比卡魯胺劑量的增加，所有細(xì)胞活力逐漸降低，而下調(diào)AR-V7表達(dá)顯著增強(qiáng)前列腺癌細(xì)胞對比卡魯胺的敏感性(P<0.05)。Western blot結(jié)果顯示下調(diào)AR-V7表達(dá)顯著抑制AR啟動(dòng)子活性，其下游PSA和FKBP5蛋白水平明顯降低(P<0.05)。免疫熒光結(jié)果發(fā)現(xiàn)AR和AR-V7主要存在于前列腺癌細(xì)胞核內(nèi)，AR少量存在于細(xì)胞質(zhì)中；下調(diào)AR-V7表達(dá)則抑制AR的核轉(zhuǎn)運(yùn)；AR存在于耐藥細(xì)胞核中，AR-V7在耐藥細(xì)胞中高表達(dá)。免疫共沉淀結(jié)果顯示內(nèi)源性AR-V7與HSP90相互作用。結(jié)論AR-V7在前列腺癌細(xì)胞中高表達(dá)，下調(diào)AR-V7的表達(dá)顯著抑制前列腺癌細(xì)胞活力和遷移；前列腺癌細(xì)胞耐藥與AR-V7高表達(dá)相關(guān)，其機(jī)制可能通過AR-V7與HSP90相互作用介導(dǎo)AR-V7的核轉(zhuǎn)運(yùn)，激活A(yù)R信號通路調(diào)控下游靶基因的轉(zhuǎn)錄活性最終導(dǎo)致細(xì)胞耐藥。

雄激素受體剪接變異體7； 前列腺癌； 耐藥性； 免疫共沉淀

前列腺癌是男性泌尿系統(tǒng)常見惡性腫瘤之一，其發(fā)病率和死亡率位居男性惡性腫瘤的第2位和第5位，每年有24萬新發(fā)病例[1-2]。資料顯示我國前列腺癌的發(fā)病率和死亡率呈逐年上升且年輕化趨勢[3-5]。目前前列腺癌的治療手段包括手術(shù)治療早期前列腺癌、單純?nèi)葜委?、去勢和抗雄激素?lián)合治療以及內(nèi)分泌治療或雄激素剝奪療法(androgen deprivation therapy，ADT)治療中晚期前列腺癌和轉(zhuǎn)移性前列腺癌。但是，在長期的治療過程中幾乎所有的患者均產(chǎn)生耐藥性并發(fā)展成為難治性去勢抵抗性前列腺癌(castration-resistant prostate cancer，CRPC)。

目前對前列腺癌耐藥的分子機(jī)制尚不清楚，雄激素受體(androgen receptor，AR)信號通路的激活在前列腺癌的發(fā)生、發(fā)展以及耐藥過程中發(fā)揮重要作用[6-8]。研究發(fā)現(xiàn)雄激素受體剪接變異體7(androgen receptor splice variant 7，AR-V7)在CRPC細(xì)胞或組織中高表達(dá)，并與患者預(yù)后不良有關(guān)[9-10]。最新研究發(fā)現(xiàn)AR-V7在紫杉烷化療過程中具有抗藥性作用，首次提出AR-V7與前列腺癌耐藥性密切相關(guān)[11]。AR-V7與前列腺癌細(xì)胞耐藥的關(guān)系及AR-V7誘導(dǎo)前列腺癌細(xì)胞耐藥的分子機(jī)制尚不清楚。本研究旨在闡明AR-V7在前列腺癌細(xì)胞生長、轉(zhuǎn)移及耐藥中的作用，探討AR-V7誘導(dǎo)前列腺癌細(xì)胞耐藥的分子機(jī)制，為以AR-V7為新靶點(diǎn)研發(fā)內(nèi)分泌治療增敏劑提供理論依據(jù)。

材 料 和 方 法

1細(xì)胞株與試劑

人前列腺癌細(xì)胞系PC3、DU145、LNCaP、ArCaP及正常前列腺上皮細(xì)胞株RWPE-1購自ATCC；DMEM培養(yǎng)基購自Gibco、胎牛血清(fetal bovine serum，F(xiàn)BS)、青霉素和鏈霉素購自北京鼎國昌盛生物技術(shù)有限責(zé)任公司；比卡魯胺、胰蛋白酶、MTT和二甲基亞砜(DMSO)購自Sigma；Transwell板購自Corning；Lipofectamine 2000購自Invitrogen；抗AR和AR-V7抗體購自Cell Signaling Technology；抗熱休克蛋白90(heat shook protein 90， HSP90)、FK506結(jié)合蛋白5(FK506-binding protein 5，F(xiàn)KBP5)和前列腺特異性抗原(prostate-specific antigen，PSA)抗體購自Abcam；抗tubulin抗體購自北京中杉金橋生物技術(shù)有限公司；反轉(zhuǎn)錄試劑盒 (PrimeScript? RT reagent kit with gDNA Eraser)和real-time PCR 試劑盒 (SYBR? Premix Ex TaqTMⅡ) 購于TaKaRa；BCA蛋白濃度檢測試劑盒購自Thermo。

2方法

2.1細(xì)胞培養(yǎng) PC3、DU145、LNCaP、ArCaP和RWPE-1細(xì)胞培養(yǎng)于DMEM培養(yǎng)基(10% FBS、1%青霉素、1%鏈霉素)中，置于37 ℃、5% CO2及飽和濕度中培養(yǎng)至細(xì)胞融合度達(dá)到90%左右時(shí)進(jìn)行細(xì)胞傳代。

2.2siRNA合成和細(xì)胞轉(zhuǎn)染 針對AR-V7基因開放閱讀框序列，運(yùn)用Ambion公司的網(wǎng)上在線軟件(http://www.ambion.com/techlib/misc/siRNA_fin-der.html)設(shè)計(jì)AR-V7 siRNA (siAR-V7)，同時(shí)設(shè)計(jì)陰性對照(negative control， NC)siRNA，由上海生工合成。將生長良好的PC3、LNCaP、DU145和ArCaP細(xì)胞以細(xì)胞密度為每孔2.5×105個(gè)接種到6孔板中，當(dāng)細(xì)胞融合度為80%～90%時(shí)按照Lipofectamine 2000說明書轉(zhuǎn)染，轉(zhuǎn)染的細(xì)胞命名為PC3-siAR-V7、DU145-siAR-V7、LNCaP-siAR-V7和ArCaP-siAR-V7細(xì)胞，以轉(zhuǎn)染NC siRNA為陰性對照細(xì)胞。培養(yǎng)4 h后換成完全培養(yǎng)基繼續(xù)培養(yǎng)用于后續(xù)實(shí)驗(yàn)。

2.3MTT實(shí)驗(yàn)檢測細(xì)胞活力 轉(zhuǎn)染的細(xì)胞經(jīng)消化計(jì)數(shù)后，以細(xì)胞密度為2.5×107/L接種于96孔板中，培養(yǎng)24 h后加入不同終濃度的比卡魯胺(0、2、5、10和20 μmol/L)作用72 h進(jìn)行MTT實(shí)驗(yàn)，同時(shí)設(shè)空白對照組。每孔加入20 μL 5 g/L MTT溶液，37 ℃繼續(xù)培養(yǎng)4 h，棄掉培養(yǎng)液，每孔加入150 μL DMSO，置于搖床上室溫振蕩5 min，用酶標(biāo)儀測定492 nm處的吸光度(A)值。細(xì)胞活力抑制率(%)=(對照組A均值-實(shí)驗(yàn)組A均值)/實(shí)驗(yàn)組A均值×100%。

2.4細(xì)胞遷移實(shí)驗(yàn) siRNA轉(zhuǎn)染細(xì)胞48 h后經(jīng)胰酶消化，然后在Transwell每個(gè)培養(yǎng)孔上室中加入2×103個(gè)細(xì)胞，下室為無血清培養(yǎng)基，設(shè)置空白對照組；37 ℃繼續(xù)培養(yǎng)6 h后經(jīng)4%多聚甲醛固定、乙醇脫水、結(jié)晶紫染色、洗滌。用棉簽輕輕擦去上室的貼壁細(xì)胞，在顯微鏡下拍照并計(jì)數(shù)從Transwell上室遷移至微孔膜下層的細(xì)胞，每組設(shè)3個(gè)重復(fù)孔，通過每組Transwell的遷移細(xì)胞數(shù)比較細(xì)胞的遷移能力。

2.5構(gòu)建耐比卡魯胺的前列腺癌細(xì)胞系 LNCaP細(xì)胞先給予終濃度為0.5 μmol/L比卡魯胺作用3 d；更換培養(yǎng)基，加入終濃度為1 μmol/L比卡魯胺作用3 d；換液加入終濃度為2 μmol/L比卡魯胺作用3 d，以此類推，之后分別給予4、6、8和10 μmol/L的比卡魯胺分別作用3 d；運(yùn)用MTT法檢測細(xì)胞活力抑制率，直到細(xì)胞對比卡魯胺不敏感時(shí)表明耐比卡魯胺細(xì)胞系LNCaP-DR構(gòu)建成功，用于后續(xù)實(shí)驗(yàn)。

2.6免疫熒光觀察 LNCaP、LNCaP-siAR-V7和LNCaP-DR細(xì)胞經(jīng)細(xì)胞爬片、4%多聚甲醛固定、0.1% Triton X-100透化、5% BSA封閉、 I 抗AR-V7孵育、II抗FITC孵育、DAPI核染色，共聚焦顯微鏡拍照，觀察LNCaP耐藥性細(xì)胞中AR-V7是否發(fā)生核轉(zhuǎn)位以及AR-V7發(fā)生核轉(zhuǎn)位是否依賴于AR通路。

2.7螢光素酶報(bào)告基因?qū)嶒?yàn) LNCaP-siAR-V7細(xì)胞和LNCaP-NC siRNA細(xì)胞經(jīng)胰酶消化后接種于24孔板中，待細(xì)胞融合度為80%左右時(shí)進(jìn)行細(xì)胞轉(zhuǎn)染。按照脂質(zhì)體2000說明書將100 ng TOPFlash報(bào)告質(zhì)粒和10 ng FOPFlash海腎螢光素酶對照質(zhì)粒共轉(zhuǎn)染細(xì)胞，48 h后收集細(xì)胞。加入1×被動(dòng)裂解液重懸細(xì)胞，4 ℃裂解24 h，12 000 r/min、4 ℃離心10 min，收集上清并測定細(xì)胞上清中螢光素酶的活性。以海腎螢光素酶活性為內(nèi)參照計(jì)算樣品中螢光水平，從螢光素酶的活性判斷抑制AR-V7表達(dá)對AR啟動(dòng)子活性的影響。

2.8Real-time PCR實(shí)驗(yàn) siRNA轉(zhuǎn)染細(xì)胞48 h后收集細(xì)胞，按照Trizol試劑說明書提取總RNA，濃度測定，保存于-80 ℃。按照real-time PCR試劑盒說明書合成cDNA。反應(yīng)條件為： 42 ℃ 1 h， 70 ℃ 5 min， 4 ℃放置10 min。cDNA置于-80 ℃保存。取2 ng cDNA進(jìn)行real-time PCR反應(yīng)。AR-V7上游引物序列為5’-TTTCCACCTTGCGGGGTATG-3’，下游引物序列為5’-TGATTGGTTCAGACTTATCGTCG-3’。反應(yīng)條件為： 95 ℃ 5 min； 95 ℃ 25 s， 58 ℃ 30 s， 72 ℃ 30 s， 35個(gè)循環(huán)； 72 ℃ 10 min。運(yùn)用 SYBR Green Ⅱ 熒光染料法和IQ5TMReal-Time PCR Detection System(Bio-Rad)進(jìn)行real-time PCR數(shù)據(jù)分析, 結(jié)果經(jīng)內(nèi)參照基因tubulin校正，各基因相對含量用 2-ΔΔCt表示。

2.9Western blot實(shí)驗(yàn) 細(xì)胞正常培養(yǎng)48 h后，收集細(xì)胞，加入RIPA重懸細(xì)胞，超聲破碎后12 000×g4 ℃離心10 min，BCA法測定蛋白濃度。每個(gè)樣本取30 μg進(jìn)行SDS-PAGE，將蛋白轉(zhuǎn)移到硝酸纖維素膜上，5%脫脂奶粉室溫封閉1 h，分別孵育 I 抗(AR、AR-V7、HSP90、FKBP5、PSA和tubulin)，4 ℃過夜。洗膜、孵育 II 抗，室溫1 h。ECL顯影后掃描，蛋白相對表達(dá)量經(jīng)內(nèi)參校正后用Quantity One軟件分析。

2.10蛋白質(zhì)免疫共沉淀(co-immunoprecipitation，Co-IP) 細(xì)胞經(jīng)預(yù)冷PBS洗3遍，用細(xì)胞刮刮下細(xì)胞，4 000 r/min、4 ℃離心收集細(xì)胞；加入200 μL IP 裂解液，冰上裂解1 h，12 000×g、4 ℃離心5 min，收集上清，用IP裂解液補(bǔ)齊2 mL，取100 μL作為input；剩下的樣品平均分為2份，分別加入IgG和AR-V7抗體各2 μg，4 ℃孵育過夜。次日加入30 μL protein A，4 ℃孵育4 h，1 000×g、4 ℃離心5 min，收集沉淀，加入PBS洗滌，每次5 min，共5次。加入50 μL 1×loading buffer，100 ℃煮沸10 min，制備好的樣品進(jìn)行Western blot，步驟同方法2.9。

3統(tǒng)計(jì)學(xué)處理

應(yīng)用SPSS 17.0統(tǒng)計(jì)軟件進(jìn)行相關(guān)數(shù)據(jù)分析，結(jié)果用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，兩組之間的比較采用t檢驗(yàn)，多組間的比較采用單因素方差分析(one-way ANOVA)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1AR-V7在前列腺癌細(xì)胞中的表達(dá)水平

Real-time PCR結(jié)果顯示AR-V7的mRNA在4種前列腺癌細(xì)胞中高表達(dá)，而在正常前列腺上皮細(xì)胞RWPE-1中低表達(dá)，兩兩比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)，見圖1A；siAR-V7轉(zhuǎn)染后，AR-V7在前列腺癌細(xì)胞中低表達(dá)，AR-V7 的mRNA水平顯著低于陰性對照(P<0.05)，見圖1B；Western blot結(jié)果進(jìn)一步證實(shí)siAR-V7明顯下調(diào)AR-V7的蛋白表達(dá)水平，見圖1C。

Figure 1. AR-V7 down-regulated by siRNA in prostate cancer cells. A: the expression of AR-V7 mRNA was detected using real-time PCR in prostate cancer cells and RWPE-1 cells; B: the level of AR-V7 mRNA was detected using real-time PCR in prostate cancer cells transfected with siAR-V7; C: the level of AR-V7 protein was detected using Western blot in prostate cancer cells transfected with siAR-V7. Mean±SD.n=3.**P<0.01vsRWPE-1 cells；##P<0.01vsNC siRNA group.

圖1siRNA在前列腺癌細(xì)胞中下調(diào)AR-V7表達(dá)

2下調(diào)AR-V7表達(dá)對前列腺癌細(xì)胞內(nèi)分泌治療敏感性的影響

MTT結(jié)果顯示，AR-V7低表達(dá)的4種前列腺癌細(xì)胞活性顯著低于NC siRNA轉(zhuǎn)染的細(xì)胞；細(xì)胞經(jīng)不同劑量比卡魯胺(2、5、10和20 μmol/L)作用后，隨著比卡魯胺劑量的增加細(xì)胞活性逐漸降低，呈劑量依賴效應(yīng)；相同條件下，siAR-V7轉(zhuǎn)染細(xì)胞的活力顯著低于NC siRNA轉(zhuǎn)染細(xì)胞，兩兩比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)，見圖2。

3下調(diào)AR-V7表達(dá)對前列腺癌細(xì)胞遷移能力的影響

Transwell結(jié)果顯示，下調(diào)AR-V7表達(dá)使細(xì)胞遷移數(shù)明顯降低，顯著低于NC siRNA轉(zhuǎn)染細(xì)胞，差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)，見圖3。

4下調(diào)AR-V7表達(dá)對AR信號通路的影響

螢光素酶報(bào)告基因結(jié)果顯示，siAR-V7介導(dǎo)AR-V7低表達(dá)后，AR啟動(dòng)子活性均顯著低于NC siRNA轉(zhuǎn)染細(xì)胞(P<0.05)，見圖4A。Western blot結(jié)果發(fā)現(xiàn)下調(diào)AR-V7表達(dá)后AR蛋白水平明顯降低，AR通路下游PSA和FKBP5蛋白水平顯著下降(P<0.05)，見圖4B、C。

5AR-V7低表達(dá)或比卡魯胺耐受的前列腺癌細(xì)胞中AR亞細(xì)胞定位

免疫熒光結(jié)果發(fā)現(xiàn)AR主要位于細(xì)胞核內(nèi)，細(xì)胞質(zhì)中存在少量AR表達(dá)；而下調(diào)AR-V7表達(dá)明顯降低細(xì)胞質(zhì)中的AR的表達(dá)水平，細(xì)胞質(zhì)核中的AR表達(dá)量無明顯變化，提示下調(diào)AR-V7的表達(dá)抑制了AR的核轉(zhuǎn)位；同時(shí)在耐比卡魯胺的LNCaP細(xì)胞中發(fā)現(xiàn)，AR主要表達(dá)在細(xì)胞核中，細(xì)胞質(zhì)中沒有檢測到AR表達(dá)，見圖5A。此外，AR-V7主要存在前列腺癌細(xì)胞核內(nèi)，當(dāng)細(xì)胞產(chǎn)生比卡魯胺耐受后AR-V7的表達(dá)量明顯增加，見圖5B。

6蛋白質(zhì)免疫共沉淀結(jié)果

蛋白質(zhì)免疫共沉淀結(jié)果發(fā)現(xiàn)，HSP90蛋白與AR-V7相互作用；比卡魯胺耐受的LNCaP細(xì)胞中AR-V7的表達(dá)水平顯著增加(input)，而HSP90蛋白水平無明顯變化，HSP90與AR-V7蛋白相互作用明顯增強(qiáng)(IP:AR-V7)，見圖6。

Figure 2. The effect of AR-V7 low expression on the sensitivity of prostate cancer cells to bicalutamide. Mean±SD.n=3.*P<0.05,**P<0.01vsNC siRNA group.

圖2AR-V7低表達(dá)對前列腺癌細(xì)胞卡比魯胺敏感性的影響

Figure 3. The effect of AR-V7 low expression on the migration ability of prostate cancer cells (×40). Mean±SD.n=3.**P<0.01vsNC siRNA group.

圖3下調(diào)AR-V7表達(dá)對前列腺癌細(xì)胞遷移能力的影響

討 論

ADT是中晚期前列腺癌和轉(zhuǎn)移性前列腺癌的一線治療方案，通過阻斷雄激素的合成或阻止雄激素受體的轉(zhuǎn)錄激活抑制前列腺癌細(xì)胞生長達(dá)到治療目的。但是，幾乎所有患者在治療過程中會產(chǎn)生抗藥性并發(fā)展為難治性CRPC，目前CRPC的耐藥機(jī)制仍不清楚。AR-V7是一種AR剪切變異體，具有調(diào)控AR信號通路關(guān)鍵靶基因轉(zhuǎn)錄活性、蛋白表達(dá)和AR核轉(zhuǎn)運(yùn)等功能[12-13]。臨床研究發(fā)現(xiàn)AR-V7在前列腺癌細(xì)胞或組織中高表達(dá)，并與CRPC的發(fā)生、發(fā)展以及耐藥密切相關(guān)[9,11,14]，下調(diào)AR-V7表達(dá)顯著抑制前列腺癌細(xì)胞增殖[15]以及通過阻滯細(xì)胞周期抑制前列腺癌的生長[16]。本研究發(fā)現(xiàn)AR-V7在4種前列腺癌細(xì)胞中高表達(dá)，而在正常上皮前列腺細(xì)胞中低表達(dá)，與文獻(xiàn)報(bào)道[17]相符。進(jìn)一步研究發(fā)現(xiàn)，用siRNA干擾AR-V7表達(dá)可顯著抑制4種前列腺癌細(xì)胞的細(xì)胞活性和遷移率，以及增強(qiáng)細(xì)胞對比卡魯胺的敏感性，提示AR-V7異常表達(dá)在前列腺癌細(xì)胞化療耐受中發(fā)揮著重要作用。

Figure 4. The effect of AR-V7 low expression on AR signal pathway in the prostate cancer cells. A: the promoter activity ofARwas monitored by luciferase reporter gene assay in prostate cancer cells transfected with siAR-V7 or NC siRNA; B: the protein levels of AR, PSA and FKBP5 were monitored by Western blot in prostate cancer cells transfected with siAR-V7 or NC siRNA; C: statistical analysis of relative protein expression levels. Mean±SD.n=3.**P<0.01vsNC siRNA group.

圖4下調(diào)AR-V7表達(dá)對AR信號通路的影響

Figure 5. The effects of AR-V7 low expression (A) and bicalutamide resistance (A, B) on AR subcellular localization in the prostate cancer cells. A: ×40; B: ×100.

圖5下調(diào)AR-V7表達(dá)和內(nèi)分泌治療耐受對AR亞細(xì)胞定位的影響

Figure 6. The interaction of AR-V7 and HSP90 proteins was monitored by co-immunoprecipitation. A: the interaction of AR-V7 and HSP90 proteins was monitored by co-immunoprecipitation in LNCaP cells; B: the interaction of AR-V7 and HSP90 proteins was monitored by co-immunoprecipitation in LNCaP and LNCaP-DR cells.

圖6蛋白質(zhì)免疫共沉淀檢測AR-V7與HSP90相互作用

目前CRPC內(nèi)分泌耐受的機(jī)制仍不清楚，研究認(rèn)為其耐藥性與AR基因的擴(kuò)增和過表達(dá)以及AR信號通路再激活有關(guān)[18-20]。多項(xiàng)研究證實(shí)，AR-V7參與了CRPC對ADT治療抗藥性的產(chǎn)生[21-22]；Zhang等[23]發(fā)現(xiàn)經(jīng)紫杉醇和多西他賽治療的前列腺癌22Rv1和LNCaP95細(xì)胞中AR-V7的表達(dá)水平異常升高，最終對化療產(chǎn)生耐藥性。新近的研究發(fā)現(xiàn)在前列腺癌22Rv1細(xì)胞中AR-V7表達(dá)的增強(qiáng)與AR基因擴(kuò)增有關(guān)[24]。本研究發(fā)現(xiàn)下調(diào)AR-V7的表達(dá)能降低AR啟動(dòng)子活性，進(jìn)而抑制其下游靶基因PSA和FKBP5表達(dá)，提示AR-V7異常表達(dá)激活A(yù)R信號通路進(jìn)而促進(jìn)AR靶基因的轉(zhuǎn)錄活性。在雄激素缺失的前列腺癌細(xì)胞中的研究發(fā)現(xiàn)AR-V7通過調(diào)控經(jīng)典AR信號通路靶基因的表達(dá)抑制PC細(xì)胞的增殖[12, 15-16, 25]，與本研究結(jié)果類似。

Zhang等[23]用熒光顯微法分析了增強(qiáng)綠色免疫熒光標(biāo)記的AR和AR-V7在COS-7細(xì)胞中的表達(dá)和亞細(xì)胞定位，發(fā)現(xiàn)AR的核轉(zhuǎn)運(yùn)依賴雄激素，而AR-V7可以自主地轉(zhuǎn)移至細(xì)胞核，且紫杉醇處理后不影響AR-V7的亞細(xì)胞定位。研究證實(shí)AR-V7能阻斷恩雜魯胺介導(dǎo)的AR核轉(zhuǎn)運(yùn)，在雄激素缺乏的條件下AR-V7促進(jìn)AR的核轉(zhuǎn)運(yùn)[26]，但其分子機(jī)制仍不清楚。本研究結(jié)果顯示大量AR存在于細(xì)胞核內(nèi)，少量存在于細(xì)胞質(zhì)中，而卡比魯胺耐受的前列腺癌細(xì)胞中AR存在于細(xì)胞核內(nèi)，細(xì)胞質(zhì)中的AR完全消失，提示化療耐受后AR的出核通路受到抑制，AR主要富集在細(xì)胞核中可能與AR靶基因轉(zhuǎn)錄激活導(dǎo)致細(xì)胞耐藥有關(guān)。此外AR-V7存在于細(xì)胞核中，細(xì)胞耐藥產(chǎn)生后AR-V7的表達(dá)量明顯增加；免疫共沉淀結(jié)果發(fā)現(xiàn)AR-V7與HSP90內(nèi)源性相互作用，耐藥細(xì)胞中這種相互作用明顯增強(qiáng)；有研究發(fā)現(xiàn)AR與HSP90相互作用介導(dǎo)AR的入核，在大量共調(diào)節(jié)因子的幫助下激活A(yù)R通路并調(diào)控其下游基因的轉(zhuǎn)錄[27]。本研究結(jié)果提示AR-V7與HSP90相互作用可能介導(dǎo)AR-V7的入核激活A(yù)R信號通路促進(jìn)AR靶基因PSA和FKBP5的表達(dá)，這可能是AR-V7通過激活A(yù)R通路最終導(dǎo)致細(xì)胞耐藥的機(jī)制之一。

綜上所述，AR-V7在前列腺癌細(xì)胞中高表達(dá)，下調(diào)AR-V7的表達(dá)顯著抑制細(xì)胞活性和遷移；AR-V7參與CRPC的耐藥，其機(jī)制可能通過激活A(yù)R信號通路促進(jìn)AR靶基因轉(zhuǎn)錄活性而實(shí)現(xiàn)。本研究為以AR-V7為新靶點(diǎn)研發(fā)化療增敏劑提供理論依據(jù)。

[1] Bray F, Jemal A, Grey N, et al. Global cancer transitions according to the Human Development Index (2008-2030): a population-based study[J]. Lancet Oncol, 2012, 13(8):790-801.

[2] Siegel R, Ma J, Zou Z, et al. Cancer statistics, 2014[J]. CA Cancer J Clin, 2014, 64(1):9-29.

[3] Du LB, Li HZ, Wang XH, et al. Analysis of cancer incidence in Zhejiang cancer registry in China during 2000 to 2009[J]. Asian Pac J Cancer Prev, 2014, 15(14):5839-5843.

[4] Qu M, Ren SC, Sun YH. Current early diagnostic biomarkers of prostate cancer[J]. Asian J Androl, 2014, 16(4):549-554.

[5] 韓蘇軍，張思維，陳萬清，等. 中國前列腺癌發(fā)病現(xiàn)狀和流行趨勢分析[J]．臨床腫瘤學(xué)雜志, 2013, 18(4):330-334.

[6] Migliaccio A, Castoria G, Di Domenico M, et al. Steroid-induced androgen receptor-oestradiol receptor beta-Src complex triggers prostate cancer cell proliferation[J]. EMBO J, 2000, 19(20):5406-5417.

[7] Di Donato M, Bilancio A, D'Amato L, et al. Cross-talk between androgen receptor/filamin A and TrkA regulates neurite outgrowth in PC12 cells[J]. Mol Biol Cell, 2015, 26(15):2858-2872.

[8] Brawley OW. Prostate cancer epidemiology in the United States[J]. World J Urol, 2012, 30(2):195-200.

[9] 方習(xí)武, 夏術(shù)階, 唐孝達(dá). 雄激素受體在人類前列腺癌發(fā)展中的作用研究[J].中國病理生理雜志, 2004, 20(7):1275-1279.

[10] Hu DG, Hickey TE, Irvine C, et al. Identification of androgen receptor splice variant transcripts in breast cancer cell lines and human tissues[J]. Horm Cancer, 2014, 5(2):61-71.

[11] Thadani-Mulero M, Portella L, Sun S, et al. Androgen receptor splice variants determine taxane sensitivity in prostate cancer[J]. Cancer Res, 2014, 74(8):2270-2282.

[12] Hu R, Isaacs WB, Luo J. A snapshot of the expression signature of androgen receptor splicing variants and their distinctive transcriptional activities[J]. Prostate, 2011, 71(15):1656-1667.

[13] Antonarakis ES, Lu C, Luber B, et al. Androgen receptor splice variant 7 and efficacy of taxane chemotherapy in patients with metastatic castration resistant prostate cancer[J]. JAMA Oncol, 2015, 1(8):582-591.

[14] Li Y, Chan SC, Brand LJ, et al. Androgen receptor splice variants mediate enzalutamide resistance in castration resistant prostate cancer cell lines[J]. Cancer Res, 2013, 73(2):483-489.

[15] Guo Z, Yang X, Sun F, et al. A novel androgen receptor splice variant is up-regulated during prostate cancer progression and promotes androgen depletion-resistant growth[J]. Cancer Res, 2009, 69(6):2305-2313.

[16] Hu R, Lu C, Mostaghel EA, et al. Distinct transcriptional programs mediated by the ligand dependent full length androgen receptor and its splice variants in castration resistant prostate cancer[J]. Cancer Res, 2012, 72(14):3457-3462.

[17] Hu R, Dunn TA, Wei S, et al. Ligand independent androgen receptor variants derived from splicing of cryptic exons signify hormone refractory prostate cancer[J]. Cancer Res, 2009, 69(1):16-22.

[18] 顏艷靈, 鐘雪云, 鐘振宇. 利用RNA干涉技術(shù)研究雄激素受體在人前列腺癌細(xì)胞增殖中的作用[J]. 中國病理生理雜志, 2004, 20(4):580-583.

[19] Ryan CJ, Smith MR, de Bono JS, et al. Abiraterone in metastatic prostate cancer without previous chemotherapy[J]. N Engl J Med, 2013, 368(6):138-148.

[20] de Bono JS, Logothetis CJ, Molina A, et al. Abiraterone and increased survival in metastatic prostate cancer[J]. N Engl J Med, 2011, 364(6):1995-2005.

[21] Efstathiou E, Titus M, Wen S, et al. Molecular characterization of enzalutamide-treated bone metastatic castration-resistant prostate cancer[J]. Eur Urol, 2015, 67(1):53-60.

[22] Antonarakis ES, Lu C, Wang H, et al. AR-V7 and resis-tance to enzalutamide and abiraterone in prostate cancer[J]. N Engl J Med, 2014,371(11)：1028-1038.

[23] Zhang G, Liu X, Li J, et al. Androgen receptor splice variants circumvent AR blockade by microtubule-targeting agents[J]. Oncotarget, 2015, 6(27):23358-23371.

[24] Li Y, Alsagabi M, Fan D, et al. Intragenic rearrangement and altered RNA splicing of the androgen receptor in a cell-based model of prostate cancer progression[J]. Can-cer Res, 2011, 71(6):2108-2117.

[25] Krause WC, Shafi AA, Nakka M, et al. Androgen receptor and its splice variant, AR-V7, differentially regulate FOXA1 sensitive genes in LNCaP prostate cancer cells[J]. Int J Biochem Cell Biol, 2014, 54(1):49-59.

[26] Cao B, Qi Y, Zhang G, et al. Androgen receptor splice variants activating the full-length receptor in mediating resistance to androgen-directed therapy[J]. Oncotarget, 2014, 5(6):1646-1656.

[27] Culig Z, Santer FR. Androgen receptor signaling in prostate cancer[J]. Cancer Metastasis Rev, 2014, 33(2-3):413-427.

(責(zé)任編輯： 林白霜， 羅 森)

EffectofAR-V7onendocrinetherapyresistanceofprostatecancer

MA Yuan1, LI Ji-fu2, WANG Yu-jie1

(1DepartmentofUrology,TheFirstAffiliatedHospital，2DepartmentofUrology,TheFifthAffiliatedHospital,XinjiangMedicalUniversity,Wulumuqi830011,China.E-mail:wangyujie@163.com)

AIM: To explore the effect of androgen receptor splice variant 7 (AR-V7) on endocrine therapy resistance of prostate cancer cells and the resistance mechanisms.METHODSFour prostate cancer cell lines were transfected with AR-V7 siRNA (siAR-V7) using Lipofectamine 2000 kit， and the transfected cells were named as PC3-siAR-V7, DU145-siAR-V7, LNCaP-siAR-V7 and ArCaP-siAR-V7 cells. The prostate cancer cells transfected with negative control (NC) siRNA served as negative controls. The expression of AR-V7 at mRNA and protein levels was detected by real-time PCR and Western blot， respectively. The cell viability and cell migration rate were measured by MTT assay and Transwell method, respectively. The promoter activity ofARand the protein levels of targeted prostate-specific antigen (PSA) and FK506-binding protein 5 (FKBP5) were monitored by luciferase reporter gene assay and Western blot， respectively. The bicalutamide-resistant cell line, LNCaP-DR, was constructed， and the subcellular localization of AR and AR-V7 proteins in LNCaP, LNCaP-siAR-V7 and LNCaP-DR cells was observed by the method of immunofluorescence. The protein interaction of AR-V7 and heat shock protein 90 (HSP90) was determined by co-immunoprecipitation.RESULTSThe mRNA level of AR-V7 in the 4 prostate cancer cell lines was significantly higher than that in normal prostate epithelial cell line RWPE-1 (P<0.05). The AR-V7 level, cell viability and cell migration rate in the cells transfected with siAR-V7 were notablely lowered compared with the NC siRNA-transfected cells (P<0.05). The cell viability was gradually decreased following with the increase in bicalutamide dose， and down-regulation of AR-V7 expression significantly enhanced the sensitivity to bicalutamide (P<0.05). Down-regulation of AR-V7 expression significantly inhibitedARpromoter activity and reduced the protein levels of PSA and FKBP5 (P<0.05). The results of immunofluorescence observation showed that most AR and AR-V7 were mainly located in the nucleus， a few AR was located in the cytoplasm, and down-regulation of AR-V7 expression inhibited AR nuclear transport. AR was entirely located in the nucleus and the protein levels of AR-V7 was significantly increased in the bicalutamide-resistant cells. The interaction of endogenous AR-V7 with HSP90 was found in the prostate cancer cells.CONCLUSIONHigh AR-V7 level is found in the prostate cancer cells, and down-regulation of AR-V7 expression inhibits the cell viability and migration. High AR-V7 level is related to the bicalutamide resistance. The possible mechanism is that AR nuclear transport is mediated by the interaction of AR-V7 with HSP90 to activate AR signal pathway and regulate targeted gene transcriptional activity, thus resulting in drug resistance.

Androgen receptor splice variant 7; Prostate cancer; Drug resistance; Co-immunoprecipitation

R730.23; R737.25

A

10.3969/j.issn.1000- 4718.2017.10.023

1000- 4718(2017)10- 1874- 08

2017- 02- 16

2017- 03- 12

△通訊作者 Tel: 0991-7921184; E-mail: wangyujie@163.com

雜志網(wǎng)址： http://www.cjpp.net