馮繼紅,馮冬冬,傅仲學(xué)
(1.重慶醫(yī)科大學(xué)附屬第一醫(yī)院 胃腸外科,重慶 400016;2.安徽省六安市壽縣中醫(yī)院 心內(nèi)科,安徽 六安 232200)
臨床醫(yī)學(xué)研究
PRDX1通過(guò)上皮間質(zhì)轉(zhuǎn)化促進(jìn)結(jié)腸癌SW480細(xì)胞侵襲轉(zhuǎn)移
馮繼紅1,馮冬冬2,傅仲學(xué)1
(1.重慶醫(yī)科大學(xué)附屬第一醫(yī)院 胃腸外科,重慶 400016;2.安徽省六安市壽縣中醫(yī)院 心內(nèi)科,安徽 六安 232200)
目的 探討過(guò)氧化還原酶1 (Peroxiredoxin 1,PRDX1)在結(jié)腸癌上皮間質(zhì)轉(zhuǎn)化( epithelialmesenchymal transition,EMT) 發(fā)生中的作用及其參與結(jié)腸癌侵襲轉(zhuǎn)移的可能機(jī)制。方法 通過(guò)慢病毒轉(zhuǎn)染使結(jié)腸癌SW480細(xì)胞PRDX1過(guò)表達(dá)并通過(guò)Western blot檢測(cè)驗(yàn)證;采用Transwell方法和細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲及轉(zhuǎn)移能力的變化;免疫細(xì)胞熒光檢測(cè)結(jié)腸癌SW480細(xì)胞EMT相關(guān)蛋白Twist1、E-cadherin及Vimentin的表達(dá);Western blot方法檢測(cè)EMT轉(zhuǎn)錄因子Twist1、EMT相關(guān)蛋白E-cadherin、N-cadherin及Vimentin及轉(zhuǎn)移相關(guān)蛋白MMP-9的表達(dá)。結(jié)果 轉(zhuǎn)染PRDX1組SW480細(xì)胞中PRDX1呈過(guò)表達(dá);Transwell法和細(xì)胞劃痕實(shí)驗(yàn)顯示過(guò)表達(dá)PRDX1組細(xì)胞的侵襲及轉(zhuǎn)移能力較對(duì)照組明顯增強(qiáng);免疫細(xì)胞熒光顯示轉(zhuǎn)染PRDX1組細(xì)胞其上皮細(xì)胞標(biāo)記E-cadherin明顯下調(diào),間質(zhì)標(biāo)記Vimentin及EMT轉(zhuǎn)錄因子Twist1表達(dá)明顯上調(diào);Western blot結(jié)果表明過(guò)表達(dá)PRDX1組E-cadherin較對(duì)照組顯著降低(P<0.01),而Twist1、MMP-9、N-cadherin及Vimentin較對(duì)照組顯著升高(P<0.05)。結(jié)論 PRDX1可促進(jìn)人結(jié)腸癌SW480細(xì)胞EMT的發(fā)生,從而增強(qiáng)結(jié)腸癌SW480細(xì)胞的侵襲、轉(zhuǎn)移能力。
過(guò)氧化還原酶1;結(jié)腸癌;侵襲;轉(zhuǎn)移;上皮間質(zhì)轉(zhuǎn)化
結(jié)腸癌是世界上最常見(jiàn)惡性腫瘤之一,其發(fā)生率和死亡率位居惡性腫瘤前列[1]。我國(guó)結(jié)腸癌發(fā)病率呈穩(wěn)步上升趨勢(shì)[2],近年以8.4%的速度上升,預(yù)計(jì)在2015年,我國(guó)結(jié)腸癌新發(fā)病例將達(dá)到31.2萬(wàn)例,其中男性17.3萬(wàn)例,女性13.9萬(wàn)例[3]。由于結(jié)腸癌手術(shù)效果不夠理想,術(shù)后復(fù)發(fā)率及轉(zhuǎn)移率均較高,因此,研究結(jié)腸癌遷移侵襲的機(jī)制和開(kāi)發(fā)高效低毒的分子靶向藥物,對(duì)結(jié)腸癌防治具有重要意義。上皮間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition, EMT)是上皮來(lái)源腫瘤細(xì)胞獲得侵襲和轉(zhuǎn)移能力的重要生物學(xué)過(guò)程,在這一過(guò)程中,上皮細(xì)胞獲得間質(zhì)細(xì)胞的特性,侵襲性增加,從而促進(jìn)腫瘤轉(zhuǎn)移[4]。
過(guò)氧化還原酶1(Peroxiredoxin 1,PRDX1)是過(guò)氧化還原蛋白酶超家族(PRDXs)中主要成員之一,又稱(chēng)為增殖相關(guān)蛋白(proliferation associated protein,PAG),在結(jié)腸癌、乳腺癌、肺癌等多種腫瘤組織細(xì)胞中高表達(dá),與細(xì)胞的增殖和分化密切相關(guān)。PRDX1表達(dá)上調(diào)可增強(qiáng)腫瘤細(xì)胞的抗氧化能力,減少活性氧自由基產(chǎn)生,是腫瘤細(xì)胞抗凋亡和化療耐藥的重要原因[5]。近年有研究表明,PRDX1可調(diào)節(jié)TGF-β1誘導(dǎo)的肺腺癌A549細(xì)胞EMT,細(xì)胞過(guò)表達(dá)PRDX1后E-cadherin表達(dá)顯著下調(diào),而下調(diào)PRDX1表達(dá)后經(jīng)TGF-β1誘導(dǎo)的EMT和細(xì)胞侵襲受到抑制[6]。本研究通過(guò)初步探討PRDX1對(duì)人結(jié)腸癌SW480細(xì)胞侵襲、轉(zhuǎn)移及EMT的作用,為臨床尋求理想結(jié)腸癌治療新靶點(diǎn)奠定理論基礎(chǔ)。
1.1 主要試劑與材料 人結(jié)腸癌細(xì)胞株SW480購(gòu)自中科院上海細(xì)胞庫(kù),胎牛血清(FBS)購(gòu)自Hyclone公司,L-15培養(yǎng)基(Leibovitz’s L-15 Medium)和嘌呤霉素(Puromycin)購(gòu)自Gibco公司,兔抗人GAPDH單抗、兔抗人PRDX1單抗購(gòu)自Epitomics公司,E-cadherin、N-cadherin、Vimentin、Twist1及MMP-9抗體購(gòu)自Santa cruz公司,“SYBR?PrimeSciptTMRT-PCR Kit”購(gòu)自中國(guó)大連寶生物工程有限公司,Lipofectamine2000購(gòu)自Invitrogen公司,Premix Taq酶、DNA marker均購(gòu)自TaKaRa公司,ECL發(fā)光試劑盒購(gòu)自Millipore公司。攜帶增強(qiáng)綠色熒光蛋白基因(EGFP) 和嘌呤霉素(Puromycin)抗性基因的PRDX1及空載對(duì)照組慢病毒載體 (Ubi-MCS-EGFP-IRES-Puromycin,GV218)由上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司構(gòu)建和包裝。
1.2 方法
1.2.1 細(xì)胞培養(yǎng) SW480細(xì)胞培養(yǎng)于含10%胎牛血清(FBS)的L-15培養(yǎng)基中,在5%的CO2飽和濕度、37 ℃恒溫培養(yǎng)箱中常規(guī)培養(yǎng)。SW480細(xì)胞呈單層貼壁生長(zhǎng),每 2~3天用0.25%胰酶消化,以1∶2傳代。SW480細(xì)胞生長(zhǎng)狀態(tài)穩(wěn)定,呈對(duì)數(shù)生長(zhǎng)期時(shí)用于實(shí)驗(yàn)。
1.2.2 細(xì)胞轉(zhuǎn)染及抗性篩選 SW480細(xì)胞準(zhǔn)確計(jì)數(shù)使其在6孔板均勻平鋪,待細(xì)胞匯合率達(dá)60%左右,按細(xì)胞數(shù)及MOI值(MOI=50)計(jì)算所加的病毒量,病毒原液用增強(qiáng)劑Eni.S稀釋?zhuān)味舛葹闃?biāo)準(zhǔn)濃度(1×108),加入終濃度為8 μg/mL Polybrene、Eni.S、L-15培養(yǎng)基及計(jì)算的標(biāo)準(zhǔn)濃度病毒液,總體積為1 mL。將含有PRDX1基因擴(kuò)增片段的慢病毒(Ubi-Prdx1-EGFP-Puromycin)與空載對(duì)照慢病毒分組感染SW480細(xì)胞,命名為轉(zhuǎn)染PRDX1組(PRDX1)及空載對(duì)照組(Vector),并留未轉(zhuǎn)染的細(xì)胞作為空白對(duì)照,即未轉(zhuǎn)染組(SW480)??蛰d對(duì)照組及轉(zhuǎn)染PRDX1組細(xì)胞培養(yǎng)26~28 h后更換為含有3 μg/mL嘌呤霉素(Puromycin)的L-15培養(yǎng)液行抗性篩選,轉(zhuǎn)染3 d后行熒光顯微鏡下觀察綠色熒光,通過(guò)觀察熒光計(jì)算轉(zhuǎn)染率達(dá)95%以上可繼續(xù)行抗性篩選培養(yǎng)數(shù)日,收獲細(xì)胞行檢測(cè)。
1.2.3 Transwell侵襲實(shí)驗(yàn) 選擇孔徑為8.0 μm的Transwell 小室,裝置于24孔板中。取對(duì)數(shù)生長(zhǎng)期各組SW480細(xì)胞(實(shí)驗(yàn)分組同上)。準(zhǔn)備基質(zhì)膠,將凍存于-80 ℃冰箱的matrigel 4 ℃過(guò)夜,變成液態(tài); 無(wú)血清培養(yǎng)基以1∶9稀釋?zhuān)?0 μL均勻鋪到Transwell 上室, 37 ℃過(guò)夜,當(dāng)出現(xiàn)“白色層”時(shí)在上室中加入100 μL 預(yù)溫的無(wú)血清的L-15培養(yǎng)基,孵育30 min,使基質(zhì)膠再水化,吸去剩余培養(yǎng)液,0.25%胰酶消化細(xì)胞,無(wú)血清培養(yǎng)基洗3 次,調(diào)整細(xì)胞為1×106個(gè)/mL;取細(xì)胞懸液100 μL加入上室,下室加500 μL 完全培養(yǎng)基;37 ℃、5% CO2孵育24 h,棄培養(yǎng)基,擦拭上室細(xì)胞;取出Transwell用PBS 洗2 次,4%多聚甲醛固定細(xì)胞10 min,倒置,風(fēng)干;加0.1% 結(jié)晶紫染色10 min,PBS 洗3次,擦凈上室細(xì)胞;倒置顯微鏡下隨機(jī)計(jì)數(shù)10個(gè)視野的細(xì)胞,取均數(shù)。每組設(shè)3個(gè)復(fù)孔,實(shí)驗(yàn)重復(fù)3次。侵襲抑制率( %) = (對(duì)照組穿膜細(xì)胞-轉(zhuǎn)染組穿膜細(xì)胞) /對(duì)照組穿膜細(xì)數(shù)×100%。
1.2.4 細(xì)胞劃痕實(shí)驗(yàn) 取狀態(tài)良好的3組細(xì)胞(未轉(zhuǎn)染組、空載對(duì)照組及轉(zhuǎn)染PRDX1組)制備成單細(xì)胞懸液,按1×105個(gè)細(xì)胞(500 μL)/孔將細(xì)胞加入6 孔板,5% CO2培養(yǎng)箱內(nèi)于37℃孵育培養(yǎng)。采用劃痕法用消毒過(guò)的槍頭在70%匯合的單層細(xì)胞表面劃出一無(wú)細(xì)胞的細(xì)痕,用PBS漂洗3次以除去劃下的細(xì)胞,加入新鮮無(wú)血清培養(yǎng)液繼續(xù)培養(yǎng)觀察,24 h后在倒置顯微鏡下觀察拍照。每組實(shí)驗(yàn)重復(fù)3次。
1.2.5 免疫細(xì)胞熒光檢測(cè) 取24 孔板行細(xì)胞爬片,觀察細(xì)胞在玻片上種植均勻、生長(zhǎng)良好,吸去各孔內(nèi)培養(yǎng)基,PBS清洗3次,4%多聚甲醛固定20 min后再次 PBS沖洗玻片3次,每次5 min;每孔中加入含 0.1% Triton X-100及3%山羊血清的PBS約0.5 mL,在室溫下封閉約1 h;加入待測(cè)蛋白的一抗稀釋液,Twist1、E-cadherin及Vimentin稀釋比例均為1∶2000,4℃過(guò)夜孵育;過(guò)夜后取出24孔板,PBS 輕洗玻片2次,每次5 min;每孔玻片滴加50μL 稀釋比例為1∶2000的DyLight 549熒光二抗,37℃條件下避光孵育1 h,PBS輕洗玻片3次,每次5 min;DAPI滴加玻片上染核10 min;PBS輕洗玻片3次,每次5 min;吸干PBS殘液,室溫下風(fēng)干;勾取爬片,注意細(xì)胞爬片的一面朝下,放置載玻片上,少量中性樹(shù)封片,熒光顯微鏡下觀察拍照。
1.2.6 Western blot檢測(cè) 收集各組細(xì)胞,采用PBS洗滌2次,離心棄上清,加入200 μL預(yù)冷的RIPA細(xì)胞裂解液,置冰上30 min,4 ℃、14 000 rpm離心16 min,取上清并進(jìn)行蛋白定量(BCA法)。取30 μg蛋白加入上樣緩沖液,經(jīng)100 ℃、10 min變性后用100 g/L的SDS-PAGE分離。蛋白半干轉(zhuǎn)移至PVDF膜,用含50 g/L脫脂奶粉的TBST緩沖液室溫封閉1 h,待測(cè)蛋白一抗或抗人GAPDH的一抗,4℃過(guò)夜,TBST洗滌3次,加入HRP標(biāo)記的山羊抗小鼠二抗室溫孵育1 h,TBST洗滌3次,加入ECL顯色劑后暗室內(nèi)壓片曝光。實(shí)驗(yàn)重復(fù)3次。
2.1 轉(zhuǎn)染慢病毒后SW480細(xì)胞PRDX1蛋白的表達(dá) 以GAPDH為內(nèi)參,比較未轉(zhuǎn)染組(SW480)、空載對(duì)照組(Vector)及轉(zhuǎn)染PRDX1組(PRDX1)細(xì)胞中的PRDX1蛋白表達(dá),經(jīng)Western-blot檢測(cè),可見(jiàn)轉(zhuǎn)染PRDX1組中PRDX1顯著增加,差異具有統(tǒng)計(jì)學(xué)意義(P<0.05),說(shuō)明轉(zhuǎn)染PRDX1組SW480細(xì)胞其PRDX1蛋白過(guò)表達(dá)成功。
未轉(zhuǎn)染組:SW480;空載對(duì)照組:Vector;轉(zhuǎn)染PRDX1組:PRDX1。
2.2 Transwell細(xì)胞侵襲實(shí)驗(yàn) 未轉(zhuǎn)染組(SW480)、空載對(duì)照組(Vector)及轉(zhuǎn)染PRDX1組(PRDX1)細(xì)胞侵襲實(shí)驗(yàn)(見(jiàn)圖2)。結(jié)果顯示,穩(wěn)定轉(zhuǎn)染后的PRDX1組穿過(guò)Matrigel膠的侵襲細(xì)胞數(shù)較未轉(zhuǎn)染組和空載對(duì)照組明顯增多,統(tǒng)計(jì)學(xué)分析顯示差異有統(tǒng)計(jì)學(xué)意義(P<0.01);而其余兩組組間差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。
A:SW480;B:Vector;C:PRD×1。
2.3 細(xì)胞遷移實(shí)驗(yàn) 在3組細(xì)胞內(nèi)劃出相同寬度的痕跡后,24 h后觀察細(xì)胞劃痕處的遷移情況,可見(jiàn)轉(zhuǎn)染PRDX1組比未轉(zhuǎn)染組和空載對(duì)照組細(xì)胞遷移速度快(見(jiàn)圖3)。結(jié)果表明,轉(zhuǎn)染PRDX1組細(xì)胞遷移能力明顯高于未轉(zhuǎn)染組和空載對(duì)照組。
圖3 3組細(xì)胞24 h后侵襲轉(zhuǎn)移能力的差異(×100)
2.4 免疫細(xì)胞熒光檢測(cè)過(guò)表達(dá)PRDX1后SW480細(xì)胞Twist1、E-cadherin及Vimentin的表達(dá) 通過(guò)免疫細(xì)胞熒光檢測(cè)3組細(xì)胞中EMT標(biāo)志物的表達(dá),與空載對(duì)照組(Vector)比較,發(fā)現(xiàn)轉(zhuǎn)染PRDX1組細(xì)胞中上皮細(xì)胞標(biāo)記E-cadherin明顯下調(diào),而間質(zhì)標(biāo)記Vimentin及EMT轉(zhuǎn)錄因子Twist1的表達(dá)明顯上調(diào)(見(jiàn)圖4)。說(shuō)明過(guò)表達(dá)PRDX1后其細(xì)胞EMT標(biāo)志物發(fā)生了較明顯變化。
A
B
C
A:Twist1;B:E-cadherin;C: Vimentin。
2.5 Western-blot法檢測(cè)過(guò)表達(dá)PRDX1后細(xì)胞Twist1、E-cadherin、N-cadherin、Vimentin及MMP-9的表達(dá) 通過(guò)Western-blot法檢測(cè)3組細(xì)胞中EMT標(biāo)志物的表達(dá),從圖5中可發(fā)現(xiàn),與未轉(zhuǎn)染組(SW480)及空載對(duì)照組(Vector)比較,轉(zhuǎn)染PRDX1組細(xì)胞中上皮細(xì)胞標(biāo)記E-cadherin蛋白表達(dá)明顯下調(diào)(P<0.01),間質(zhì)標(biāo)記N-cadherin、Vimentin、EMT轉(zhuǎn)錄因子Twist1以及轉(zhuǎn)移相關(guān)蛋白MMP-9的表達(dá)均出現(xiàn)不同程度上調(diào)(P<0.05)。說(shuō)明PRDX1過(guò)表達(dá)可促進(jìn)細(xì)胞EMT的發(fā)生及其轉(zhuǎn)移能力。
圖5 Western-blot檢測(cè)3組細(xì)胞Twist1、E-cadherin、N-cadherin、Vimentin及MMP-9的表達(dá)
目前我國(guó)結(jié)直腸癌的發(fā)病率僅次于肺癌和胃癌,位居第3位,且發(fā)病率每年仍以一定速度增長(zhǎng)。大部分結(jié)腸癌患者早期沒(méi)有癥狀,超過(guò)75%的患者確診時(shí)已屬于晚期[7],臨床上雖采取包括手術(shù)、化療、放療為主的綜合治療方法,但預(yù)后仍不理想,其重要原因是結(jié)腸癌趨于局部侵襲和發(fā)生肝轉(zhuǎn)移,進(jìn)一步研究和探討結(jié)腸癌侵襲轉(zhuǎn)移的作用機(jī)制仍是結(jié)腸癌治療研究的難點(diǎn)和熱點(diǎn)。
過(guò)氧化還原蛋白(peroxiredoxins,PRDXs)是近年新發(fā)現(xiàn)的相對(duì)分子量在20~30 kDa過(guò)氧化物酶超家族,對(duì)氧化應(yīng)激反應(yīng)極為敏感,主要作用是通過(guò)硫氧還蛋白還原、清除過(guò)氧化物或超氧化物,在保持細(xì)胞內(nèi)氧化還原穩(wěn)態(tài)中發(fā)揮重要調(diào)節(jié)作用[8]。此外,PRDXs還在細(xì)胞多種分子傳導(dǎo)信號(hào)中發(fā)揮重要樞紐作用[9]。PRDX1蛋白是PRDXs家族中最受關(guān)注的抗氧化因子之一,在多種腫瘤中呈高表達(dá)或低表達(dá),與腫瘤發(fā)生、發(fā)展及侵襲轉(zhuǎn)移密切相關(guān)[10-12]。Taniuchi K等[13]報(bào)道了PRDX1在胰腺導(dǎo)管腺癌組織中高表達(dá),且與磷酸化的p38 MAPK相互作用,并發(fā)現(xiàn)PRDX1抑制后會(huì)導(dǎo)致膜波動(dòng)和突起的改變,從而促進(jìn)胰腺癌細(xì)胞的侵襲。Turner-Ivey B等[14]報(bào)道了乳腺癌中PRDX1通過(guò)其半胱氨酸Cys52位點(diǎn)與p38 MAPK磷酸化(MKP-1、MKP-5)相結(jié)合,在乳腺癌上皮細(xì)胞衰老過(guò)程中發(fā)揮重要調(diào)節(jié)作用。Riddell JR等[15]研究發(fā)現(xiàn)PRDX1通過(guò)TLR4和VEGF依賴(lài)的形式促進(jìn)前列腺癌上皮細(xì)胞的增殖、遷移和分化。此外,PRDX1還作為一種增殖相關(guān)蛋白參與細(xì)胞增殖和分化[16],還可發(fā)揮分子伴侶及免疫調(diào)節(jié)功能[17]。由此可見(jiàn),PRDX1作為促癌因子還是抑癌因子尚存在爭(zhēng)議,PRDX1在不同的腫瘤中可能發(fā)揮不同的功能和作用,且在腫瘤的發(fā)生、發(fā)展和侵襲轉(zhuǎn)移中的具體機(jī)理也不盡相同。
目前,PRDX1與結(jié)直腸癌關(guān)聯(lián)研究少見(jiàn)報(bào)道,劉峰等[18]通過(guò)基因芯片技術(shù)檢測(cè)到PRDX1在出現(xiàn)腹膜轉(zhuǎn)移的結(jié)直腸癌原發(fā)腫瘤組織中表達(dá)上調(diào)。蔣堯等[19]檢測(cè)到PRDX1在結(jié)直腸癌組織中高表達(dá),且與III期結(jié)直腸癌患者或有淋巴結(jié)轉(zhuǎn)移的患者之間有相關(guān)性。也有報(bào)道顯示,PRDX1的表達(dá)與結(jié)直腸腫瘤發(fā)展轉(zhuǎn)移及復(fù)發(fā)有關(guān),作為一種促癌因子發(fā)揮作用[20],且與E-ca呈負(fù)相關(guān),其高表達(dá)可促使EMT,從而促進(jìn)腫瘤轉(zhuǎn)移[6]。本研究采用慢病毒轉(zhuǎn)染技術(shù)使結(jié)腸癌細(xì)胞株P(guān)RDX1表達(dá)上調(diào),發(fā)現(xiàn)其侵襲和轉(zhuǎn)移能力明顯增強(qiáng),同時(shí)EMT標(biāo)志蛋白和轉(zhuǎn)錄因子也發(fā)生相應(yīng)變化。結(jié)果顯示,PRDX1可能通過(guò)調(diào)節(jié)轉(zhuǎn)錄因子Twist1的表達(dá)從而使EMT標(biāo)志蛋白E-cadherin表達(dá)發(fā)生改變,PRDX1的表達(dá)上調(diào)導(dǎo)致了E-cadherin的表達(dá)明顯降低,二者呈負(fù)相關(guān)。同時(shí),PRDX1還會(huì)引起間質(zhì)標(biāo)記N-cadherin、Vimentin及轉(zhuǎn)移相關(guān)蛋白MMP-9的表達(dá)上調(diào)。由此推斷,PRDX1的表達(dá)上調(diào)可能通過(guò)某種機(jī)制促使EMT轉(zhuǎn)錄因子Twsit1的表達(dá)增加,從而推動(dòng)了EMT進(jìn)程,導(dǎo)致腫瘤細(xì)胞的侵襲和轉(zhuǎn)移能力增強(qiáng)。結(jié)合文獻(xiàn)報(bào)道,推測(cè)PRDX1可能通過(guò)與缺氧或氧化還原相關(guān)的核心調(diào)控網(wǎng)絡(luò)來(lái)影響EMT進(jìn)程,從而賦予了腫瘤細(xì)胞更強(qiáng)大適應(yīng)環(huán)境的能力,在腫瘤細(xì)胞對(duì)抗活性氧(ROS)和清除H2O2有害刺激中發(fā)揮重要作用。至于PRDX1是否主要通過(guò)EMT來(lái)促進(jìn)結(jié)腸癌細(xì)胞侵襲和轉(zhuǎn)移,以及PRDX1在腫瘤細(xì)胞與其微環(huán)境交互網(wǎng)絡(luò)調(diào)控中所扮演的角色究竟如何尚不得而知,仍亟待深入研究。鑒于此,課題組準(zhǔn)備在后續(xù)實(shí)驗(yàn)中采用RNA干擾技術(shù)沉默PRDX1基因的表達(dá)及相關(guān)體內(nèi)實(shí)驗(yàn)進(jìn)一步探討PRDX1與EMT關(guān)聯(lián)的具體機(jī)制。由此相信,PRDX1與結(jié)腸癌EMT及侵襲轉(zhuǎn)移的關(guān)系有望成為一個(gè)新的研究方向。
綜上所述,對(duì)PRDX1在結(jié)腸癌EMT及侵襲轉(zhuǎn)移中的作用和可能機(jī)制的探討,可能對(duì)結(jié)腸癌局部浸潤(rùn)和肝臟轉(zhuǎn)移的發(fā)生及具體機(jī)制有更深入的了解,并為結(jié)腸癌轉(zhuǎn)移的治療提供新的靶點(diǎn)和方向。
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[收稿2014-12-02;修回2014-12-30]
(編輯:王福軍)
PRDX1 participates in invasion and metastasis of colon cancer through epithelial-mesenchymal transition
FengJihong1,FengDongdong2,FuZhongxue1
(1.Department of Gastroenterological Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;2.Department of Cardiology, Shouxian Hospital of Traditional Chinese Medicine,Lu′an Anhui 232200, China)
Objective To study the role of Peroxiredoxin 1 (PRDX1) in epithelial-mesenchymal transition in colon cancer and the mechanism of PRDX1 participating in the invasion and metastasis of colon cancer.Methods SW480 cells were transfected with lentivirus vectors resulting in overexpression of PRDX1 protein, and the expression of PRDX1 were validated by western blot. Transwell migration assays and wound healing assays were performed to identify the differences and changes of invasive and metastatic ability in SW480 cell lines. The expression of Twist1, E-cadherin and Vimentin were analyzed by immunocytochemistry. The expression of Twist1 (EMT related transcription factor), E-cadherin、N-cadherin and Vimentin (EMT biomarker proteins), and MMP9 (metastasis associated protein) were detected by western blot.Results PRDX1 are over-expressed in the transfection groups, as compared to that in control groups (P<0.01). The transfected cells significantly have stronger capability for invasion and metastasis as compared to those in control groups. The immunofluorescence staining revealed that the expression of E-cadherin was decreased while the expression of Vimentin and Twist1 was increased in the transfected cells. Western blot showed that the expression of E-cadherin was decreased significantly in the transfection groups in compared with control group (P<0.01), while the expression of Twist1, MMP-9, N-cadherin and Vimentin was increased significantly (P<0.05).Conclusion PRDX1 can induce EMT process of colon carcinoma line SW480 cells, and promote its ability of invasion and metastasis.
Peroxiredoxin 1 (PRDX1);colon cancer;invasion;metastasis;Epithelial-Mesenchymal Transition (EMT)
國(guó)家自然科學(xué)基金面上項(xiàng)目(NO:81172295)。
傅仲學(xué), 男,博士,教授,博士生導(dǎo)師,研究方向: 結(jié)直腸腫瘤,E-mail:fzx990521@sina.com。
R735.3
A
1000-2715(2015)01-0067-07