袁偉燕, 張 麗, 石紅琴, 龔小衛(wèi), 姜 勇
(南方醫(yī)科大學(xué)病理生理學(xué)教研室,廣東省蛋白質(zhì)組學(xué)重點(diǎn)實(shí)驗(yàn)室,廣東 廣州 510515)
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Prdx4蛋白表達(dá)改變對(duì)HeLa細(xì)胞遷移和侵襲的影響*
袁偉燕,張麗,石紅琴,龔小衛(wèi)△,姜勇△
(南方醫(yī)科大學(xué)病理生理學(xué)教研室,廣東省蛋白質(zhì)組學(xué)重點(diǎn)實(shí)驗(yàn)室,廣東 廣州 510515)
目的: 觀察過(guò)氧化物還原酶4(peroxiredoxin 4,Prdx4)蛋白表達(dá)的改變對(duì)人宮頸癌HeLa細(xì)胞遷移和侵襲的影響。方法: 采用脂質(zhì)體瞬時(shí)轉(zhuǎn)染法將表達(dá)質(zhì)粒pcDNA3.0-HA-Prdx4轉(zhuǎn)染HeLa細(xì)胞;LV-Prdx4 RNAi重組病毒感染HeLa細(xì)胞,構(gòu)建Prdx4 shRNA HeLa細(xì)胞系沉默Prdx4的表達(dá);用蛋白質(zhì)印跡法證實(shí)Prdx4蛋白表達(dá)水平的變化;應(yīng)用劃痕愈合實(shí)驗(yàn)和Transwell遷移及侵襲實(shí)驗(yàn)分別檢測(cè)細(xì)胞遷移及侵襲能力的變化。結(jié)果: pcDNA3.0-HA-Prdx4質(zhì)粒轉(zhuǎn)染組HeLa細(xì)胞的Prdx4蛋白表達(dá)水平明顯上調(diào)(P<0.05),而Prdx4 shRNA HeLa穩(wěn)定干擾細(xì)胞系Prdx4表達(dá)水平明顯下調(diào)(P<0.05)。Prdx4過(guò)表達(dá)組HeLa細(xì)胞的遷移和侵襲能力明顯增強(qiáng),與空白對(duì)照組及空載體轉(zhuǎn)染組比較,差異顯著(P<0.01)。Prdx4表達(dá)干擾組HeLa細(xì)胞的遷移和侵襲能力明顯受到抑制,與空白對(duì)照組及陰性對(duì)照組比較,差異顯著(P<0.01)。結(jié)論: 上調(diào)Prdx4蛋白表達(dá)水平可促進(jìn)HeLa細(xì)胞的遷移和侵襲,在宮頸癌的發(fā)生和發(fā)展過(guò)程中可能起重要作用;下調(diào)Prdx4蛋白表達(dá)水平可以明顯抑制HeLa細(xì)胞的遷移和侵襲能力,Prdx4有可能成為臨床治療宮頸癌的一個(gè)潛在靶點(diǎn)。
宮頸癌; 過(guò)氧化物還原酶4; 細(xì)胞遷移; 細(xì)胞侵襲
宮頸癌是最常見(jiàn)的婦科惡性腫瘤之一,在發(fā)展中國(guó)家其發(fā)病率位居首位[1],是全球女性癌癥相關(guān)死亡的第2大原因[2]。隨著人們對(duì)宮頸癌認(rèn)識(shí)程度的不斷加深、防癌普查的廣泛開(kāi)展以及治療方法的不斷改進(jìn),發(fā)病率和死亡率有所下降,但腫瘤復(fù)發(fā)和轉(zhuǎn)移仍是死亡的主要原因[3]。癌癥治療失敗和腫瘤發(fā)生侵襲的重要原因是腫瘤細(xì)胞通過(guò)破壞基底膜的完整性,穿過(guò)細(xì)胞外基質(zhì)(extracellular matrix,ECM)發(fā)生轉(zhuǎn)移[4]。有文獻(xiàn)報(bào)道,過(guò)氧化物還原酶4(peroxiredoxin 4,Prdx4)的表達(dá)水平與腫瘤密切相關(guān),Prdx1和Prdx4在膀胱癌和肺癌組織中出現(xiàn)表達(dá)量增高[5],Prdx3和Prdx4在前列腺癌組織中的表達(dá)量也增高[6-7]。同時(shí)有研究證實(shí)當(dāng)下調(diào)或敲除Prdx4的表達(dá)時(shí),會(huì)導(dǎo)致胃癌組織的生長(zhǎng)受限和凋亡增多且會(huì)導(dǎo)致精原細(xì)胞的凋亡[8]。
目前,對(duì)于已發(fā)生轉(zhuǎn)移的宮頸癌,其治療方案主要是減輕患者痛苦。因此,非常有必要進(jìn)一步研究宮頸癌的發(fā)生發(fā)展機(jī)制,以發(fā)現(xiàn)新的靶點(diǎn)和治療方法。本研究采用脂質(zhì)體瞬時(shí)轉(zhuǎn)染法使Prdx4過(guò)表達(dá)及RNA干擾(RNA interference,RNAi)技術(shù)抑制Prdx4的表達(dá),觀察其對(duì)人宮頸癌HeLa細(xì)胞遷移和侵襲的影響,進(jìn)一步探討Prdx4在細(xì)胞遷移和侵襲中的作用及其作為分子靶點(diǎn)用于腫瘤治療的潛在價(jià)值。
1細(xì)胞及試劑
人宮頸癌細(xì)胞株HeLa和pcDNA3.0-HA質(zhì)粒載體(含有HA標(biāo)簽,可與重組目的基因共表達(dá)為融合蛋白)由本實(shí)驗(yàn)室保存。LV-Prdx4 RNAi和LV-control陰性對(duì)照重組病毒及嘌呤霉素由上海吉?jiǎng)P基因有限公司制備;脂質(zhì)體轉(zhuǎn)染試劑Polyfect購(gòu)自Roche;兔抗鼠β-actin單克隆抗體和辣根過(guò)氧化物酶標(biāo)記的羊抗鼠IgG購(gòu)自CST;鼠抗人Prdx4多克隆抗體購(gòu)自Abcam;Transwell小室購(gòu)自BD;Matrigel invasion chamber購(gòu)自Corning;引物合成和DNA測(cè)序由深圳華大基因研究院完成。
2方法
2.1細(xì)胞培養(yǎng)人宮頸癌細(xì)胞株HeLa用含10%胎牛血清的DMEM完全培養(yǎng)液,置于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)[9]。待細(xì)胞融合率達(dá)到70%~80%時(shí),用0.25%胰蛋白酶消化并傳代,收集處于對(duì)數(shù)生長(zhǎng)期的細(xì)胞用于實(shí)驗(yàn)。
2.2pcDNA3.0-HA-Prdx4過(guò)表達(dá)載體的構(gòu)建和測(cè)序結(jié)果及轉(zhuǎn)染方法pcDNA3.0-HA-Prdx4過(guò)表達(dá)載體的構(gòu)建和測(cè)序結(jié)果及轉(zhuǎn)染方法在前期工作中已描述[10]。
2.3慢病毒感染HeLa細(xì)胞并構(gòu)建Prdx4 shRNA HeLa穩(wěn)定表達(dá)系針對(duì)Prdx4基因的siRNA設(shè)計(jì)與篩選參照文獻(xiàn)[10],其中hPrdx4 siRNA2的抑制效率最高,將此siRNA用于后續(xù)研究。HeLa細(xì)胞以3×103接種于96孔板,待細(xì)胞的融合率約為20%~30%進(jìn)行病毒感染,將LV-Prdx4 RNAi和LV-control陰性對(duì)照重組病毒(帶有嘌呤霉素抗性和綠色熒光報(bào)告基因)分別以復(fù)感染指數(shù)(MOI值)為10 的量感染細(xì)胞,加入polybrene(終濃度5 mg/L),37 ℃培養(yǎng)2 d后加嘌呤霉素(500 mg/L)篩選陽(yáng)性克隆,待孔板中細(xì)胞長(zhǎng)至細(xì)胞克隆時(shí),挑取單克隆擴(kuò)大培養(yǎng),直至獲得需要的穩(wěn)定感染細(xì)胞株的細(xì)胞量后,裂解細(xì)胞并收集細(xì)胞上清液,檢測(cè)目的蛋白的表達(dá)情況。
2.4蛋白質(zhì)印跡法檢測(cè)Prdx4蛋白的表達(dá)水平收集并裂解各組處理后的細(xì)胞,取總蛋白樣品各30 μg進(jìn)行SDS-PAGE。將在SDS-PAGE上分離的蛋白轉(zhuǎn)移至PVDF膜上(恒壓100 V,90 min),用5%脫脂奶粉封閉1 h,加入 I 抗鼠抗人Prdx4多克隆抗體(1∶2 000),4 ℃過(guò)夜。TBST洗膜3次,再加入II抗HRP標(biāo)記的羊抗鼠IgG(1∶5 000)1 h。TBST洗膜3次,采用化學(xué)發(fā)光法檢測(cè)目的蛋白條帶的灰度值,以目的蛋白與內(nèi)參照β-actin條帶的灰度值之比表示目的蛋白的相對(duì)表達(dá)量。
2.5細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞橫向遷移能力將處于對(duì)數(shù)生長(zhǎng)期HeLa細(xì)胞按每孔3×105個(gè)的密度接種于6孔板內(nèi),次日待細(xì)胞生長(zhǎng)至融合度約50%~60%時(shí),將pcDNA3.0-HA-Prdx4重組質(zhì)粒和pcDNA3.0-HA空載體質(zhì)粒分別轉(zhuǎn)染HeLa細(xì)胞,另外將處于對(duì)數(shù)生長(zhǎng)期的Prdx4 shRNA HeLa細(xì)胞和control shRNA HeLa細(xì)胞按每孔4×105個(gè)的密度接種于6孔板內(nèi), 以未轉(zhuǎn)染的HeLa細(xì)胞作為空白對(duì)照組。次日,收集消化以上各組細(xì)胞,取每孔3×105個(gè)的密度接種于12孔板內(nèi),待24 h后細(xì)胞融合度達(dá)到80%~90%,用200 μL槍頭垂直在細(xì)胞間橫豎2個(gè)方向各劃一道橫穿細(xì)胞的劃痕,更換培養(yǎng)基,24 h后,PBS清洗2次,顯微鏡下拍照,用Image-Pro Plus 軟件測(cè)定劃痕寬度,計(jì)算平均劃痕愈合率,以此反映細(xì)胞的橫向遷移能力。
2.6Transwell法檢測(cè)細(xì)胞縱向遷移能力及侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲能力另取以上在6孔板中收集消化的各組HeLa細(xì)胞各5×104個(gè)(用200 μL DMEM重懸成單細(xì)胞懸液),分別接種于24孔板Transwell小室中,下室加入600 μL 含10%胎牛血清的DMEM培養(yǎng)液,每組設(shè)3個(gè)復(fù)孔,12 h后,用棉簽擦去上室中的細(xì)胞,4%多聚甲醛溶液固定細(xì)胞30 min,1%的結(jié)晶紫37 ℃染色30 min,雙蒸水洗至水清,將小室晾干后,在正置光學(xué)顯微鏡下隨機(jī)選取5個(gè)視野,計(jì)數(shù)穿膜細(xì)胞數(shù)目。各組侵入小室膜下層的細(xì)胞數(shù)表示細(xì)胞的遷移能力。同時(shí)取以上各組HeLa細(xì)胞各1×105個(gè),用200 μL DMEM重懸成單細(xì)胞懸液,分別鋪入加有基質(zhì)凝膠的小室中,其余步驟同Transwell遷移實(shí)驗(yàn),計(jì)數(shù)穿膜細(xì)胞數(shù)目,取均值。
3統(tǒng)計(jì)學(xué)處理
所有實(shí)驗(yàn)均獨(dú)立重復(fù)3次以上,數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。采用SPSS 13.0和GraphPad Prism 5統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析。多組間均數(shù)比較采用單因素方差分析,組間兩兩比較采用SNK-q檢驗(yàn)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
1LV-Prdx4-RNAi及LV-control重組病毒感染HeLa細(xì)胞,構(gòu)建Prdx4 shRNA HeLa穩(wěn)定表達(dá)系
將構(gòu)建好的LV-Prdx4-RNAi及LV-control重組病毒轉(zhuǎn)染至HeLa細(xì)胞,于倒置顯微鏡下觀察可以看到帶綠色熒光的人宮頸癌HeLa細(xì)胞克隆,挑單克隆繼續(xù)傳代培養(yǎng),經(jīng)過(guò)嘌呤霉素篩選10 d后,得到了帶綠色熒光穩(wěn)定干擾的Prdx4 shRNA HeLa及control shRNA HeLa細(xì)胞,見(jiàn)圖1A。
2HeLa細(xì)胞轉(zhuǎn)染pcDNA3.0-HA-Prdx4后Prdx4表達(dá)水平顯著增高
蛋白質(zhì)印跡法檢測(cè)結(jié)果顯示,pcDNA3.0-HA-Prdx4轉(zhuǎn)染組、pcDNA3.0-HA陰性對(duì)照組和空白對(duì)照組HeLa細(xì)胞中Prdx4蛋白表達(dá)水平之間差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05);與空白對(duì)照組和陰性對(duì)照組相比,pcDNA3.0-HA-Prdx4組HeLa細(xì)胞中的Prdx4蛋白表達(dá)水平顯著增高(P<0.05),見(jiàn)圖1B。
3Prdx4 shRNA HeLa穩(wěn)定干擾細(xì)胞可顯著下調(diào)Prdx4的表達(dá)
蛋白質(zhì)印跡法檢測(cè)結(jié)果顯示,Prdx4 shRNA HeLa、control shRNA HeLa和空白對(duì)照組之間Prdx4蛋白的表達(dá)水平差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05);與空白對(duì)照及陰性對(duì)照組相比,Prdx4 shRNA HeLa組Prdx4的表達(dá)水平顯著降低(P<0.05),見(jiàn)圖1C。
Figure 1.The changes of Prdx4 protein expression in HeLa cells. A: establishment of a stablePrdx4 shRNA HeLa cell line (×100); B, C: Prdx4 protein expression in HeLa cells transfected with pcDNA3.0-HA-Prdx4 andPrdx4 shRNA HeLa cells was validated by western blotting.Mean±SD.n=3.*P<0.05vspcDNA3.0-HA-Prdx4;#P<0.05vsPrdx4 shRNA.
圖1HeLa細(xì)胞中Prdx4蛋白表達(dá)水平的變化
4過(guò)表達(dá)或下調(diào)Prdx4對(duì)HeLa細(xì)胞橫向遷移能力的影響
細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示,Prdx4過(guò)表達(dá)組之間的細(xì)胞劃痕愈合率差異有統(tǒng)計(jì)學(xué)顯著性(P<0.01);與空白對(duì)照組和陰性對(duì)照組相比,Prdx4過(guò)表達(dá)組細(xì)胞的愈合明顯高于對(duì)照組(P<0.01)。另一方面,沉默Prdx4表達(dá)組之間的細(xì)胞劃痕愈合率差異有統(tǒng)計(jì)學(xué)顯著性(P<0.01);與空白對(duì)照組及陰性對(duì)照組相比,Prdx4 shRNA組的愈合率明顯低于對(duì)照組,差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.01),見(jiàn)圖2。
Figure 2.The effects of the Prdx4 overexpression (A) and down-regulation (B) on the migration of HeLa cells detected by wound-healing assay (×100). Mean±SD.n=3.**P<0.01vspcDNA3.0-HA-Prdx4;##P<0.01vsPrdx4 shRNA.
圖2細(xì)胞劃痕實(shí)驗(yàn)分別檢測(cè)過(guò)表達(dá)或下調(diào)Prdx4對(duì)HeLa細(xì)胞遷移能力的影響
5過(guò)表達(dá)或下調(diào)Prdx4對(duì)HeLa細(xì)胞縱向遷移能力的影響
Transwell遷移實(shí)驗(yàn)結(jié)果顯示,Prdx4過(guò)表達(dá)組之間的穿膜細(xì)胞數(shù)差異有統(tǒng)計(jì)學(xué)意義(P<0.01);與空白對(duì)照組和陰性對(duì)照組相比,Prdx4過(guò)表達(dá)組的穿膜細(xì)胞數(shù)明顯增多,差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.01)。另一方面,沉默Prdx4表達(dá)組之間的穿膜細(xì)胞數(shù)差異有統(tǒng)計(jì)學(xué)意義(P<0.01);與空白對(duì)照組和陰性對(duì)照組相比,Prdx4表達(dá)沉默組的穿膜細(xì)胞數(shù)明顯減少,差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.01),見(jiàn)圖3。
6過(guò)表達(dá)或下調(diào)Prdx4對(duì)HeLa細(xì)胞侵襲能力的影響
Transwell基質(zhì)膠侵襲實(shí)驗(yàn)結(jié)果顯示,Prdx4過(guò)表達(dá)組之間的穿膜細(xì)胞數(shù)差異有統(tǒng)計(jì)學(xué)意義(P<0.01);與空白對(duì)照組和陰性對(duì)照組相比,Prdx4過(guò)表達(dá)組的細(xì)胞數(shù)明顯增多(P<0.01)。另一方面,沉默Prdx4表達(dá)之間的穿膜細(xì)胞數(shù)差異有統(tǒng)計(jì)學(xué)意義(P<0.01);與空白對(duì)照組及陰性對(duì)照組相比,Prdx4表達(dá)沉默組的穿膜細(xì)胞數(shù)明顯減少,其差異有統(tǒng)計(jì)學(xué)顯著性(P<0.01),見(jiàn)圖4。
雖然引起宮頸癌的原因很多,但目前認(rèn)為人乳頭瘤病毒(human papilloma virus,HPV)感染是宮頸癌的根本致病因素[11]。在引入了發(fā)達(dá)國(guó)家的全民普查項(xiàng)目后,盡管宮頸癌的發(fā)病率和死亡率有所下降,但其治療方法幾十年來(lái)都沒(méi)有改變[12]。宮頸癌由于對(duì)大多數(shù)抗癌藥物不敏感, 所以首選治療為手術(shù)及放射治療, 但晚期或復(fù)發(fā)轉(zhuǎn)移患者效果欠佳,而化療具有全身作用特點(diǎn), 所以尋求新的靶位基因治療晚期宮頸癌成為研究的重點(diǎn)[13]。
Figure 3.The effects of Prdx4 overexpression (A) and down-regulation (B) on the migration of the HeLa cells detected by Transwell assay (crystal violet staining, ×200). Mean±SD.n=3.**P<0.01vspcDNA3.0-HA-Prdx4;##P<0.01vsPrdx4 shRNA.
圖3Transwell法分別檢測(cè)過(guò)表達(dá)或下調(diào)Prdx4對(duì)HeLa細(xì)胞遷移能力的影響
過(guò)氧化物酶蛋白(peroxiredoxins,PRDXs)家族的酶促反應(yīng)機(jī)制是過(guò)氧化氫酶底物氧化其家族成員的一個(gè)過(guò)氧化氫特定的半胱氨酸殘基(-Cys-SPH)的過(guò)程[14]。根據(jù)Prdx4參與氧化還原反應(yīng)的半胱氨酸殘基數(shù)目,將其家族分為典型(typical)2-Cys-PRDX(PRDX1-4)、非典型(atypical)2-Cys-PRDX (PRDX5)和1-Cys-PRDX (PRDX6)3類[15]。
PRDXs家族不同的成員在參與各種生物過(guò)程:如氧化劑的解毒,細(xì)胞增殖、分化和基因表達(dá)中發(fā)揮了不同功能。Prdx1與酪氨酸激酶c-Abl相互作用,抑制其酪氨酸激酶活性,通過(guò)保持人第10號(hào)染色體缺失的磷酸酶(phosphatase and tensin homolog deleted on chromosome ten,PTEN)活性調(diào)控激活A(yù)KT信號(hào)通路[16]。在前列腺癌細(xì)胞中,Prdx1與雄激素受體相互作用,增加其反式激活活性,以及促進(jìn)癌癥表型的發(fā)展。Prdx2是血小板源生長(zhǎng)因子(platelet derived growth factor,PDGF)受體介導(dǎo)細(xì)胞信號(hào)的負(fù)調(diào)節(jié)蛋白,并在小鼠成纖維細(xì)胞中負(fù)調(diào)控核轉(zhuǎn)因子κB (nuclear factor-κB,NF-κB)的激活[17]。在人類細(xì)胞中,發(fā)現(xiàn)Prdx4在細(xì)胞質(zhì)中通過(guò)調(diào)節(jié)NF-κB 抑制蛋白α(NF-κB inhibitor α, IκBα)磷酸化以調(diào)節(jié)NF-κB信號(hào)通路的活性。Prdx4 除了在HeLa細(xì)胞中作為NF-κB激活的負(fù)調(diào)控因子外,其它在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)的功能仍知之甚少[18]。
Figure 4.The effects of Prdx4 overexpression (A) and down-regulation (B) on the invasion ability of the HeLa cells detected by Matrigel invasion chamber Transwell test (crystal violet staining, ×200). Mean±SD.n=3.**P<0.01vspcDNA3.0-HA-Prdx4;##P<0.01vsPrdx4 shRNA.
圖4Transwell基質(zhì)膠侵襲實(shí)驗(yàn)分別檢測(cè)過(guò)表達(dá)或下調(diào)Prdx4對(duì)HeLa細(xì)胞侵襲能力的影響
已有研究表明,PRDXs表達(dá)水平與腫瘤的發(fā)生和發(fā)展密切相關(guān),PRDX家族在多種腫瘤中高度表達(dá),特別是在肺癌(Prdx1、Prdx3和Prdx4)、膀胱癌(Prdx1和Prdx4)、甲狀腺癌(Prdx1)和舌癌 (Prdx1) 中高度表達(dá)[19]。另外,Prdx家族成員可以起到促進(jìn)癌癥發(fā)展的作用,其表達(dá)可以促進(jìn)細(xì)胞在氧化應(yīng)激條件下生存。例如,過(guò)表達(dá)Prdx1可以促進(jìn)細(xì)胞生長(zhǎng)和增殖,通過(guò)保護(hù)細(xì)胞免受氧化劑誘導(dǎo)的死亡。在myc基因介導(dǎo)的乳腺癌MCF-7細(xì)胞鼠成纖維細(xì)胞轉(zhuǎn)化和增殖中,Prdx3不可或缺[20]。有文獻(xiàn)報(bào)道,MAPK級(jí)聯(lián)反應(yīng)被認(rèn)為是促進(jìn)腫瘤細(xì)胞浸潤(rùn)和轉(zhuǎn)移的主要信號(hào)通路。其中,Srx促進(jìn)腫瘤進(jìn)展的一個(gè)機(jī)制是通過(guò)MAPK/AP-1/MMP9軸的調(diào)節(jié),Srx-Prdx4軸在腫瘤侵襲和轉(zhuǎn)移中發(fā)揮了很大的作用。然而, 單方面阻斷Prdx4是否能充分抑制肺癌細(xì)胞裸鼠成瘤或癌癥侵襲和轉(zhuǎn)移仍有待進(jìn)一步研究[21]。
本研究對(duì)Prdx4在調(diào)節(jié)宮頸癌細(xì)胞遷移和侵襲中的可能作用進(jìn)行了探索。成功地將pcDNA3.0-HA-Prdx4表達(dá)質(zhì)粒導(dǎo)入人宮頸癌HeLa細(xì)胞,使Prdx4蛋白表達(dá)水平上調(diào),然后采用細(xì)胞劃痕實(shí)驗(yàn)和Transwell法及Transwell基質(zhì)膠法檢測(cè)發(fā)現(xiàn),Prdx4過(guò)表達(dá)組HeLa細(xì)胞的遷移及侵襲能力明顯高于空白對(duì)照組及空載體轉(zhuǎn)染組。同時(shí),將LV-Prdx4 siRNA重組病毒感染HeLa細(xì)胞,構(gòu)建Prdx4 shRNA HeLa穩(wěn)定干擾表達(dá)系,Prdx4 shRNA HeLa細(xì)胞的遷移及侵襲能力與空白及陰性對(duì)照組比較明顯受到抑制。最后,將劃痕實(shí)驗(yàn)24 h后的各組細(xì)胞收集進(jìn)行Western blot實(shí)驗(yàn),再次驗(yàn)證其轉(zhuǎn)染效果同前。
綜上所述,Prdx4表達(dá)下調(diào)能抑制宮頸癌HeLa細(xì)胞的侵襲及轉(zhuǎn)移,由此推測(cè)Prdx4有望成為宮頸癌基因治療的一個(gè)潛在的新靶點(diǎn)。其作用機(jī)制可能與炎癥/應(yīng)激信號(hào)通路(MAPK 通路和NF-κB通路)有關(guān),其更廣泛的作用及深入的作用機(jī)制值得進(jìn)一步的研究和驗(yàn)證。
[1]Farivar TN, Johari P, Shafei S, et al. Lack of association between herpes simplex virus type 2 infection and cervical cancer: Taq Man realtime PCR assay findings[J]. Asian Pac J Cancer Prev, 2012, 13(1):339-342.
[2]Qureshi R, Arora H, Rizvi MA. EMT in cervical cancer: Its role in tumour progression and response to therapy[J]. Cancer Lett, 2015, 356(2):321-331.
[3]魏建勛,馬文君,李紅梅. 膜聯(lián)蛋白A2對(duì)人宮頸癌HeLa細(xì)胞增殖、遷移和凋亡的影響[J]. 中國(guó)病理生理雜志, 2014, 30(3):547-550.
[4]Mareel M, Leroy A. Clinical, cellular, and molecular aspects of cancer invasion[J]. Physiol Rev, 2003, 83(2):337-376.
[5]Jiang H, Wu L, Mishra M, et al. Expression of peroxiredoxin 1 and 4 promotes human lung cancer malignancy[J]. Am J Cancer Res,2014,4(5):445-460.
[6]Basu A, Banerjee H, Rojas H, et al. Differential expression of peroxiredoxins in prostate cancer: consistent upre-gulation of PRDX3 and PRDX4[J]. Prostate, 2011, 71(7):755-765.
[7]Ummanni R, Barreto F, Venz S, et al. Peroxiredoxins 3 and 4 are overexpressed in prostate cancer tissue and affect the proliferation of prostate cancer cellsinvitro[J]. J Proteome Res, 2012, 11(4):2452-2466.
[8]Iuchi Y, Okada F, Tsunoda S, et al. Peroxiredoxin 4 knockout results in elevated spermatogenic cell death via oxidative stress[J]. Biochem J, 2009, 419(1):149-158.
[9]周勤仙,邱瑜,黃建平,等. Hsa-miR-218靶向調(diào)控LASP1對(duì)宮頸癌HeLa細(xì)胞生長(zhǎng)的影響[J]. 中國(guó)病理生理雜志, 2015, 31(9):1572-1577.
[10]石紅琴,袁偉燕,張麗,等. Prdx4蛋白表達(dá)水平改變對(duì)人宮頸癌HeLa細(xì)胞增殖和凋亡的影響[J]. 腫瘤, 2015, 35(5):514-520.
[11]周莉,陳姍,張帝開(kāi). 持續(xù)性人乳頭瘤病毒感染與宮頸癌的研究進(jìn)展[J]. 中國(guó)病理生理雜志, 2010, 26(12):2482-2486.
[12]Arbyn M, Simoens C, Goffin F, et al. Treatment of cervical cancer precursors: influence of age,completeness of excision and cone depth on therapeutic failure, and on adverse obstetric outcomes[J]. BJOG, 2011, 118(10):1274-1275.
[13]顏偉,許芳根,劉安文,等. TLR4通過(guò)NF-κB信號(hào)途徑對(duì)宮頸癌細(xì)胞增殖的影響[J]. 中國(guó)病理生理雜志, 2015,31(2):301-307.
[14]Lin J, Xu J, Tian H, et al. Identification of candidate prostate cancer biomarkers in prostate needle biopsy specimens using proteomic analysis[J]. Int J Cancer, 2007, 121(12):2596-2605.
[15]Wood ZA, Schroder E, Robin HJ, et al. Structure, mechanism and regulation of peroxiredoxins[J]. Trends Biochem Sci, 2003, 28(1):32-40.
[16]Cao J, Schulte J, Knight A, et al. Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity[J]. EMBO J, 2009, 28(10):1505-1517.
[17]Choi MH, Lee IK, Kim GW, et al. Regulation of PDGF signalling and vascular remodelling by peroxiredoxin II[J]. Nature, 2005, 435(7040):347-353.
[18]Jin DY, Chae HZ, Rhee SG, et al. Regulatory role for a novel human thioredoxin peroxidase in NF-κB activation[J]. J Biol Chem, 1997, 272(49): 30952-30961.
[19]Hall A, Karplus PA, Poole LB. Typical 2-Cys peroxiredoxins: structures, mechanisms and functions[J]. FEBS J, 2009, 276(9): 2469-2477.
[20]Wonsey DR, Zeller KI, Dang CV. The c-Myc target genePRDX3 is required for mitochondrial homeostasis and neoplastic transformation[J]. Proc Natl Acad Sci U S A, 2002, 99(10): 6649-6654.
[21]Wei Q, Jiang H, Xiao Z, et al. Sulfiredoxin-Peroxiredoxin IV axis promotes human lung cancer progression through modulation of specific phosphokinase signaling[J]. Proc Natl Acad Sci U S A, 2011, 108(17): 7004-7009.
(責(zé)任編輯: 盧萍, 羅森)
Effects of Prdx4 protein expression on migration and invasion of HeLa cells
YUAN Wei-yan, ZHANG Li, SHI Hong-qin, GONG Xiao-wei, JIANG Yong
(DepartmentofPathophysiologyandKeyLaboratoryofProteomicsofGuangdongProvince,SouthernMedicalUniversity,Guangzhou510515,China.E-mail:gongxw@smu.edu.cn;jiang48231@163.com)
AIM: To investigate the effects of peroxiredoxin 4 (Prdx4) protein expression levels on the migration and invasion of human cervical cancer HeLa cells. METHODS: The plasmid pcDNA3.0-HA-Prdx4 was transfected into HeLa cells. The HeLa cells were infected with LV-Prdx4 RNAi vector to establish stablePrdx4 shRNA HeLa cells. The change in the expression of Prdx4 protein was validated by Western blotting. The wound-healing assay, and Transwell migration and invasion assays were performed to detect the migration and invasion of HeLa cells, respectively. RESULTS: The expression of Prdx4 protein was up-regulated in the HeLa cells after transfection with pcDNA3.0-HA-Prdx4 plasmid (P<0.05), whereas it was down-regulated in thePrdx4 shRNA HeLa cells (P<0.05). The abilities of migration and invasion were significantly increased in Prdx4-overexpressing HeLa cells compared with non-transfected and mock plasmid transfected control groups (P<0.01). WhenPrdx4 was knocked down by shRNA, the migration and invasion of the HeLa cells were remarkably repressed compared with blank control group and negative control group (P<0.01).CONCLUSION: The up-regulation of Prdx4 expression facilitates the migration and invasion of HeLa cells, and the down-regulation of Prdx4 expression inhibits the migration and invasion of HeLa cells, indicating that Prdx4 may be a potential molecular target for cervical cancer therapy.
Uterine cervical neoplasms; Peroxiredoxins 4; Cell migration; Cell invasion
1000- 4718(2016)04- 0637- 07
2016- 01- 11
2016- 03- 14
國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 81171958;No. 81372030)
龔小衛(wèi) Tel: 020-61648172; E-mail: gongxw@smu.edu.cn; 姜勇Tel: 020-61648231; E-mail: jiang48231@163.com
R730.23; R737.33
A
10.3969/j.issn.1000- 4718.2016.04.010
雜志網(wǎng)址: http://www.cjpp.net