PFOS暴露對(duì)肺癌細(xì)胞中信號(hào)通路的影響

于志輝,董晶熒,王亞楠,汪夏燕*

PFOS暴露對(duì)肺癌細(xì)胞中信號(hào)通路的影響

于志輝1,董晶熒1,王亞楠2,汪夏燕2*

(1.北京工業(yè)大學(xué)環(huán)境科學(xué)系,北京 100124；2.北京工業(yè)大學(xué)化學(xué)與生物系,綠色催化與分離北京市重點(diǎn)實(shí)驗(yàn)室,環(huán)境安全與生物效應(yīng)卓越中心,北京 100124)

為探討全氟辛烷磺酸鹽(PFOS)產(chǎn)生肺毒性的分子機(jī)制,采用細(xì)胞計(jì)數(shù)試劑盒(CCK-8)方法測(cè)定不同濃度PFOS對(duì)A549細(xì)胞活性的影響,并用二代測(cè)序方法測(cè)定PFOS暴露對(duì)A549細(xì)胞中miRNAs表達(dá)的影響,預(yù)測(cè)異常表達(dá)miRNAs的靶基因.通過(guò)生物信息學(xué)分析推斷靶基因參與的信號(hào)通路及潛在的生物學(xué)功能.結(jié)果顯示,低濃度PFOS(<200μmol/L)促進(jìn)A549細(xì)胞增殖,高濃度PFOS抑制細(xì)胞增殖.暴露于300μmol/L PFOS中24h的A549細(xì)胞中108個(gè)miRNAs表達(dá)量顯著上調(diào),63個(gè)miRNAs表達(dá)量顯著下調(diào).差異表達(dá)miRNAs通過(guò)Ras、Rap1、HIF-1、ErbB和VEGF等信號(hào)通路參與細(xì)胞增殖、代謝和發(fā)育等生物學(xué)過(guò)程.這表明PFOS可通過(guò)影響細(xì)胞增殖和誘發(fā)炎癥反應(yīng)對(duì)肺造成威脅.

全氟辛烷磺酸鹽(PFOS)；miRNA；信號(hào)通路

全氟烷基化合物(PFASs)由于具有良好的疏水疏油性,已被廣泛應(yīng)用于滅火劑、食品包裝、紡織品、紙張、洗發(fā)劑、表面活性劑等工商業(yè)產(chǎn)品加工過(guò)程[1-2].由于C-F鍵具有強(qiáng)極性,在強(qiáng)紫外線(xiàn)、高溫及其他化學(xué)作用的條件下具有較強(qiáng)的穩(wěn)定性,并且很難通過(guò)微生物及高等動(dòng)物的代謝作用來(lái)降解,因此PFASs可以穩(wěn)定的存在于環(huán)境中并在生物中積累[3-4].已有研究表明PFASs廣泛分布于大氣、水、土壤等多種環(huán)境介質(zhì)中,甚至在職業(yè)暴露和非職業(yè)暴露人群的血液、尿液、膽汁、母乳及臍帶血中均有檢測(cè)出PFASs[5-10].全氟辛烷磺酸鹽(PFOS)是PFASs的最終代謝產(chǎn)物,分布最為廣泛.PFOS可通過(guò)胃腸道、呼吸道和皮膚進(jìn)入人體[11],且半衰期高達(dá)5a以上[2].研究表明,PFOS具有肝[12]、肺[13]、腎[14]、免疫[15]、神經(jīng)[16]、生殖發(fā)育[17]等多種毒性,是一類(lèi)具有全身多器官和組織毒性的有機(jī)污染物.其中,PFOS對(duì)肝、神經(jīng)毒性的相關(guān)研究相對(duì)較多.PFOS的肝毒性主要表現(xiàn)為脂肪肝、肝腫大、肝細(xì)胞增生和肝細(xì)胞氧化損傷等[18-19].此外,PFOS通過(guò)誘導(dǎo)神經(jīng)細(xì)胞產(chǎn)生過(guò)量的活性氧或炎癥因子,對(duì)神經(jīng)細(xì)胞造成氧化損傷或神經(jīng)炎癥[16,20].

PFOS在非職業(yè)暴露者肺中的含量?jī)H次于肝[21],可誘發(fā)多種肺部疾病.流行病學(xué)研究表明,血清PFOS濃度與兒童哮喘病加劇具有相關(guān)性[22].Qin等[23]通過(guò)評(píng)估兒童哮喘病患者的肺功能,進(jìn)一步證明兒童哮喘病患者血清中PFOS的濃度與其肺功能呈顯著性負(fù)相關(guān).與其他組織毒性相同,PFOS也主要通過(guò)誘導(dǎo)肺組織分泌過(guò)量的炎癥因子和活性氧來(lái)造成肺毒性[24],但研究者對(duì)其中的調(diào)控機(jī)制以及涉及的信號(hào)通路知之甚少,對(duì)PFOS的肺毒性機(jī)制還沒(méi)有形成系統(tǒng)完善的認(rèn)識(shí).

microRNA(miRNA)是一類(lèi)由內(nèi)源基因編碼的長(zhǎng)度約為18～24個(gè)核苷酸的非編碼單鏈RNA分子,存在于幾乎所有的真核生物及少數(shù)病毒中,通過(guò)與mRNA的完全或不完全互補(bǔ)誘導(dǎo)mRNA降解或抑制其翻譯,實(shí)現(xiàn)轉(zhuǎn)錄后水平的基因表達(dá)調(diào)控.基于miRNA的生物學(xué)功能,已經(jīng)有很多學(xué)者從miRNA分子水平探究PFOS對(duì)生物體的毒性機(jī)制[25-27]. PFOS可引起妊娠初期人滋養(yǎng)層細(xì)胞的miR-29b含量升高,進(jìn)而使得多種蛋白的DNA甲基化和蛋白乙?；?蛋白表達(dá)量降低引起ROS含量升高[25].ROS含量的升高與子癇前期等妊娠并發(fā)癥相關(guān).此外,研究表明,PFOS通過(guò)增加SH-SY5Y細(xì)胞中miR-22的相對(duì)表達(dá)量,抑制BDNF mRNA的表達(dá),影響B(tài)DNF- ERK-CREB信號(hào)通路,為PFOS的神經(jīng)毒性提供了新的實(shí)驗(yàn)證據(jù)[26].這些研究為PFOS的肺毒性機(jī)制研究提供了新思路.然而,與miRNA相關(guān)的PFOS對(duì)肺毒性機(jī)制的研究未見(jiàn)報(bào)道.

本文采用體外細(xì)胞毒性試驗(yàn)的方法,以人非小細(xì)胞肺癌A549細(xì)胞為模型,從miRNA表觀遺傳調(diào)控角度研究PFOS對(duì)肺損傷可能的作用機(jī)制.采用細(xì)胞計(jì)數(shù)試劑盒(CCK-8)方法檢測(cè)細(xì)胞活性,探討PFOS對(duì)A549的細(xì)胞增殖毒性.二代測(cè)序篩選PFOS暴露后差異表達(dá)的miRNAs并進(jìn)行基因組百科全書(shū)(KEGG)和基因本體論(GO)富集分析,推測(cè)參與PFOS肺毒性的信號(hào)通路,深入探究PFOS肺毒性的表觀遺傳調(diào)控機(jī)制.

1 材料與方法

1.1 細(xì)胞與試劑

人非小細(xì)胞肺癌細(xì)胞A549購(gòu)自中國(guó)醫(yī)學(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所細(xì)胞資源中心(北京,中國(guó));杜氏改良Eagle培養(yǎng)基(DMEM)、胎牛血清(FBS)、磷酸鹽緩沖溶液(PBS)、青鏈霉素雙抗溶液(PS)、0.25%胰蛋白酶-乙二胺四乙酸(胰蛋白酶-EDTA)購(gòu)自美國(guó)Thermo Fisher Gibco公司;PFOS(純度98%)購(gòu)自北京百靈威科技有限公司;二甲基亞砜(DMSO)購(gòu)自上海阿拉丁生化科技股份有限公司;CCK-8試劑盒購(gòu)自北仁化學(xué)科技(北京)有限公司;TRIzol試劑購(gòu)自美國(guó)Thermo Fisher公司;miRcute miRNA提取分離試劑盒、miRcute增強(qiáng)型miRNA cDNA 第一鏈合成試劑盒、目的基因和內(nèi)參基因引物、miRcute增強(qiáng)型miRNA 熒光定量檢測(cè)試劑盒(SYBR Green)購(gòu)自天根生化科技(北京)有限公司.

1.2 細(xì)胞培養(yǎng)與處理

將A549細(xì)胞置于含10% FBS、1% PS的DMEM培養(yǎng)液中,于37℃、飽和濕度、含5% CO2的培養(yǎng)箱中培養(yǎng).將PFOS溶于DMSO中,配制500mmol/L PFOS儲(chǔ)備液儲(chǔ)存于-20°C,使用前用培養(yǎng)液進(jìn)行稀釋.為避免對(duì)細(xì)胞產(chǎn)生毒性,實(shí)驗(yàn)組中DMSO的終體積分?jǐn)?shù)不能超過(guò)0.1%,對(duì)照組為只含0.1% DMSO的培養(yǎng)液,空白組為不含細(xì)胞的0.1% DMSO的培養(yǎng)液.

1.3 CCK-8檢測(cè)細(xì)胞活性

收集對(duì)數(shù)生長(zhǎng)期的細(xì)胞制備細(xì)胞懸液.取100μL密度為4×104個(gè)/mL的A549細(xì)胞懸液接種于96孔板中培養(yǎng).孵育24h后棄去培養(yǎng)液,每孔加入200μL濃度分別為0,50,100,200,300,400,500μmol/L PFOS的培養(yǎng)液,每組6個(gè)平行.在標(biāo)準(zhǔn)條件下分別培養(yǎng)24,48,72h后棄去培養(yǎng)液,每孔加入100μL含10% CCK-8的培養(yǎng)液于37℃條件下孵育0.5h.用酶標(biāo)儀(美國(guó)Molecular Devices公司)檢測(cè)各孔在450nm處吸光度(A),吸光度與細(xì)胞活性呈正比.細(xì)胞活性的計(jì)算公式為:

1.4 RNA提取與測(cè)序

考慮到A549細(xì)胞倍增周期約為21h[28],且其在PFOS中暴露24h的半數(shù)抑制濃度(IC50)在400～ 500μmol/L之間,因此,將A549細(xì)胞在300μmol/L PFOS中暴露24h研究PFOS暴露對(duì)A549細(xì)胞中miRNAs表達(dá)的影響.取1mL密度為2×106個(gè)/mL的A549細(xì)胞懸液加入75cm2培養(yǎng)瓶中,加入適量的培養(yǎng)液,將細(xì)胞吹打均勻.待細(xì)胞貼壁生長(zhǎng)24h后,移去上清液,實(shí)驗(yàn)組加入適量PFOS濃度為300μmol/L的培養(yǎng)液,對(duì)照組加入適量含0.1% DMSO的培養(yǎng)液,每組設(shè)置3個(gè)平行.待細(xì)胞暴露24h之后,用0.25%的胰蛋白酶將細(xì)胞消化下來(lái),利用TRIzol試劑抽提總RNA.委托天根生化科技(北京)有限公司基于Illumina HiSeq 2000測(cè)序平臺(tái)對(duì)總RNA樣品進(jìn)行測(cè)序分析.

1.5 miRNAs靶基因預(yù)測(cè)和鑒定

使用R包edgeR對(duì)實(shí)驗(yàn)組和對(duì)照組樣品中所有miRNAs進(jìn)行差異分析,TMM方法歸一化.采用miRanda軟件對(duì)具有顯著性差異的miRNAs靶基因進(jìn)行預(yù)測(cè),得到miRNAs和靶基因間的對(duì)應(yīng)關(guān)系.將得到的靶基因基于topGO進(jìn)行GO功能富集分析.GO共包含3個(gè)類(lèi)群,分別描述基因的分子功能(MF)、細(xì)胞組分(CC)、參與的生物學(xué)過(guò)程(BP).本文主要對(duì)靶基因的生物學(xué)過(guò)程進(jìn)行富集分析,并對(duì)富集分析結(jié)果進(jìn)行圖形化展示.在生物體內(nèi),不同基因相互協(xié)調(diào)行使其生物學(xué)功能,通過(guò)KEGG數(shù)據(jù)庫(kù)進(jìn)行通路顯著性富集,以確定差異表達(dá)的miRNAs靶基因參與的最主要的生化代謝途徑和信號(hào)傳導(dǎo)途徑.

1.6 RT-qPCR驗(yàn)證miRNAs

參照miRcute miRNA提取分離試劑盒提取實(shí)驗(yàn)組和對(duì)照組細(xì)胞中的miRNAs.測(cè)定提取的miRNAs純度,保證所有樣品的A260/A280在1.8～2之間.使用PCR儀和熒光定量PCR儀(美國(guó)Applied Biosystems公司),結(jié)合miRcute增強(qiáng)型miRNA cDNA第一鏈合成試劑盒和miRcute增強(qiáng)型miRNA熒光定量檢測(cè)試劑盒(SYBR Green)對(duì)miRNAs樣品進(jìn)行反轉(zhuǎn)錄和實(shí)時(shí)熒光定量PCR(RT-qPCR),每組設(shè)置3個(gè)平行,具體實(shí)驗(yàn)操作參照產(chǎn)品說(shuō)明書(shū).數(shù)據(jù)處理以U6為內(nèi)參基因,對(duì)目標(biāo)基因表達(dá)量進(jìn)行標(biāo)準(zhǔn)化,計(jì)算DCt值.以對(duì)照組作為參照因子,其倍數(shù)變化為1,實(shí)驗(yàn)組基因表達(dá)差異相對(duì)于參照因子基因表達(dá)的倍數(shù)為2﹣△△Ct.分析實(shí)驗(yàn)組和對(duì)照組中miRNAs的相對(duì)表達(dá)量,并與測(cè)序結(jié)果進(jìn)行比較.

1.7 數(shù)據(jù)統(tǒng)計(jì)與分析

所有實(shí)驗(yàn)數(shù)據(jù)均采用GraphPad Prism 8軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,結(jié)果以“均值±標(biāo)準(zhǔn)差”表示.采用單因素方差分析方法比較各組之間的差異,當(dāng)<0.05時(shí)認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義.

2 結(jié)果分析

2.1 PFOS暴露對(duì)細(xì)胞活性的影響

CCK-8檢測(cè)細(xì)胞活性結(jié)果(圖1)顯示,A549細(xì)胞在高濃度(>300μmol/L)PFOS中暴露24,48,72h后細(xì)胞活性顯著降低(<0.0001),且細(xì)胞活性隨PFOS濃度增大而減小.經(jīng)過(guò)不同濃度的PFOS暴露24h后,細(xì)胞活性的變化范圍為47.9%～118.0%.其中,當(dāng)PFOS濃度為50和100μmol/L時(shí),細(xì)胞活性顯著增加(<0.001).經(jīng)過(guò)不同濃度的PFOS暴露48和72h后,細(xì)胞活性的變化范圍分別為12.1%～106.5%和2.1%～108.9%.當(dāng)PFOS濃度<300μmol/L,細(xì)胞活性與對(duì)照組相比無(wú)顯著性差異.當(dāng)PFOS濃度為400, 500μmol/L時(shí),細(xì)胞活性顯著降低.A549細(xì)胞在300μmol/L的PFOS中暴露48h后,細(xì)胞活性無(wú)顯著性變化,但暴露72h后,細(xì)胞活性顯著降低(<0.05).

圖1 PFOS暴露后的A549細(xì)胞活性

*<0.05;***<0.001; ****<0.0001

2.2 PFOS暴露對(duì)細(xì)胞中miRNAs的影響

本文利用二代測(cè)序技術(shù)篩查了PFOS暴露后miRNAs的表達(dá)情況,結(jié)果表明,在300μmol/L的PFOS中暴露24h可引起A549細(xì)胞中171個(gè)miRNAs異常表達(dá)(FC>2.0,<0.05)(圖2).其中,108個(gè)miRNAs(含miR-377-3p和miR-3199兩個(gè)已知miRNAs及106個(gè)未知miRNAs)表達(dá)量顯著上升,63個(gè)miRNAs(含已知的miR-4709-5p和62個(gè)未知miRNAs)表達(dá)量顯著下降.

2.3 miRNAs靶基因的預(yù)測(cè)

表達(dá)量顯著上升的108個(gè)miRNAs可作用于42009個(gè)靶mRNAs,表達(dá)量顯著下降的63個(gè)miRNAs對(duì)應(yīng)于30098個(gè)靶mRNAs.通過(guò)KEGG數(shù)據(jù)庫(kù)分析差異表達(dá)miRNAs靶基因的功能及其相互作用,預(yù)測(cè)到靶基因可能參與的信號(hào)通路包括——大鼠肉瘤基因(Ras)信號(hào)通路、Ras相關(guān)蛋白1(Rap1)信號(hào)通路、ErbB信號(hào)通路、缺氧誘導(dǎo)因子1(HIF-1)信號(hào)通路、血管內(nèi)皮生長(zhǎng)因子(VEGF)信號(hào)通路、磷脂酶D信號(hào)通路、神經(jīng)營(yíng)養(yǎng)因子信號(hào)通路和雷帕霉素靶蛋白(mTOR)信號(hào)通路等(圖3).GO富集分析靶基因參與的生物學(xué)過(guò)程,結(jié)果表明差異表達(dá)miRNAs靶基因參與的生物學(xué)過(guò)程包括——細(xì)胞增殖過(guò)程、生物調(diào)節(jié)過(guò)程、代謝過(guò)程、細(xì)胞過(guò)程、應(yīng)激反應(yīng)過(guò)程、多細(xì)胞生物過(guò)程、細(xì)胞組分組織或合成過(guò)程、細(xì)胞定位過(guò)程、發(fā)育過(guò)程、免疫系統(tǒng)過(guò)程、多組織過(guò)程、生物附著過(guò)程和復(fù)制過(guò)程等(圖4).

圖2 PFOS暴露后A549細(xì)胞中miRNAs的火山

圖3 差異表達(dá)miRNAs靶基因KEGG通路富集分析

圖4 差異表達(dá)miRNAs靶基因GO(BP)富集分析

2.4 RNA-Seq結(jié)果的驗(yàn)證

采用RT-qPCR方法驗(yàn)證差異表達(dá)的miR- 377-3p、miR-3199和miR-4709-5p的表達(dá)量,結(jié)果如圖5所示,miR-4709-5p與測(cè)序結(jié)果一致,在PFOS實(shí)驗(yàn)組中顯著下調(diào);miR-377-3p和miR-3199表達(dá)量無(wú)明顯變化,與測(cè)序結(jié)果不一致.

圖5 差異表達(dá)miRNAs的驗(yàn)證

3 討論

由于PFOS在環(huán)境介質(zhì)中的廣泛存在使得人們開(kāi)始關(guān)心它對(duì)人類(lèi)健康的影響.研究表明,PFOS對(duì)包括肺在內(nèi)的多種組織和系統(tǒng)均有毒性作用[29]. PFOS的肺毒性與DNA甲基化、ROS含量變化相關(guān).然而這些變化都不能充分解釋PFOS的肺毒性機(jī)理.已有研究表明PFOS可引起妊娠初期人滋養(yǎng)層細(xì)胞、大鼠肝臟和大腦組織中的miRNAs異常表達(dá).因此,本文以容易培養(yǎng)且對(duì)外加作用因子敏感的非小細(xì)胞肺癌A549細(xì)胞為研究對(duì)象,探討PFOS的肺毒性作用機(jī)制.

PFOS通過(guò)調(diào)節(jié)細(xì)胞周期影響細(xì)胞增殖,而細(xì)胞的異常增殖往往與癌癥的發(fā)生有關(guān)[30].Jabeen等[31]通過(guò)研究表觀遺傳修飾在細(xì)胞增殖和凋亡中的作用對(duì)PFOS影響A549細(xì)胞活性的機(jī)制進(jìn)行了闡述,發(fā)現(xiàn)低濃度條件下(<100μmol/L)細(xì)胞周期蛋白E和細(xì)胞周期蛋白A表達(dá)量增加,促進(jìn)A549細(xì)胞增殖,當(dāng)PFOS濃度增至400μmol/L時(shí),兩種細(xì)胞周期蛋白表達(dá)量降低,造成細(xì)胞活性顯著降低.Cui等[32]研究PFOS暴露后人正常肝細(xì)胞HL-7702的蛋白組學(xué)發(fā)現(xiàn),50μmol/L的PFOS可誘導(dǎo)HL-7702細(xì)胞中多種細(xì)胞周期蛋白及相應(yīng)的細(xì)胞周期蛋白依賴(lài)性激酶表達(dá)量增加,從而促進(jìn)細(xì)胞增殖.但當(dāng)PFOS濃度大于200μmol/L時(shí),細(xì)胞活性呈劑量依賴(lài)性降低.同樣的,本文結(jié)果顯示,當(dāng)A549細(xì)胞在50和100μmol/L PFOS中暴露24h后,細(xì)胞活性顯著增加,當(dāng)PFOS濃度大于300μmol/L時(shí)細(xì)胞活性顯著降低,且細(xì)胞活性隨PFOS濃度增大而減小.這說(shuō)明PFOS可能通過(guò)影響細(xì)胞增殖對(duì)肺產(chǎn)生毒性.

本文結(jié)果顯示PFOS暴露可引起A549細(xì)胞中多個(gè)miRNAs異常表達(dá),這些異常表達(dá)的miRNAs可作用于多個(gè)靶基因,參與Ras、Rap1、ErbB、HIF-1和VEGF等多個(gè)信號(hào)通路.Ras信號(hào)通路協(xié)同下游多個(gè)信號(hào)通路調(diào)控細(xì)胞生長(zhǎng)、增殖、分化和凋亡[33].Ras基因的異常表達(dá)與腫瘤的發(fā)生發(fā)展密切相關(guān),在30%非小細(xì)胞肺癌中發(fā)現(xiàn)Ras突變[34],因此,Ras被認(rèn)為是腫瘤發(fā)生的重要因素.Rap1是Ras通路的重要調(diào)節(jié)因子和介質(zhì),其參與的信號(hào)通路與肺癌細(xì)胞的增殖和分化相關(guān)[35].ErbB可促進(jìn)細(xì)胞增殖,激活ErbB通路可能誘發(fā)癌癥.Zhang等[36]研究發(fā)現(xiàn)持續(xù)抽煙可使人體內(nèi)多個(gè)miRNAs異常表達(dá),進(jìn)而影響ErbB通路促進(jìn)肺癌的發(fā)生. Kruspig等[37]的研究表明,ErbB通過(guò)與Ras通路相互作用促進(jìn)Kras突變肺癌細(xì)胞的增殖,因此,含ErbB抑制劑的藥物可能有利于Kras突變肺癌的治療.VEGF在肺發(fā)育及肺結(jié)構(gòu)形成和維持過(guò)程中具有重要作用,其低表達(dá)會(huì)導(dǎo)致肺組織形態(tài)結(jié)構(gòu)、功能異常[38].Zhang等[39]研究妊娠期PFOS暴露對(duì)子代大鼠肺發(fā)育的影響,發(fā)現(xiàn)PFOS能夠引起子代大鼠肺部炎癥因子白介素-1β和白介素-18的明顯增加,且與炎癥小體相關(guān)的蛋白表達(dá)也顯著升高.同時(shí),在肺泡發(fā)育和肺部血管形成過(guò)程中具有重要作用的VEGF及HIF-1的表達(dá)也受到抑制,誘發(fā)子代大鼠支氣管肺發(fā)育不良.本研究的預(yù)測(cè)結(jié)果說(shuō)明PFOS可能通過(guò)miRNAs調(diào)控Ras、Rap1、ErbB、VEGF和HIF-1等信號(hào)通路影響細(xì)胞增殖、代謝和發(fā)育等生物學(xué)過(guò)程.

測(cè)序和RT-qPCR結(jié)果均表明PFOS可引起A549細(xì)胞中miR-4709-5p表達(dá)顯著下降.miR- 4709-5p靶基因KEGG通路分析顯示,miR-4709-5p可參與促分裂原活化的蛋白激酶(MAPK)信號(hào)通路,在細(xì)胞增殖、分化和凋亡過(guò)程中具有重要作用[40]. MAPK蛋白還參與體內(nèi)多種氧化應(yīng)激和炎癥反應(yīng)過(guò)程并發(fā)揮重要調(diào)控作用[41]. Shi等[42]將斑馬魚(yú)胚胎暴露于不同濃度的PFOS中,發(fā)現(xiàn)在斑馬魚(yú)幼蟲(chóng)中出現(xiàn)氧化應(yīng)激反應(yīng),且與MAPK通路相關(guān)的基因表達(dá)異常,推測(cè)這與PFOS誘導(dǎo)的細(xì)胞凋亡有關(guān).研究表明miR-4709與結(jié)腸癌有關(guān),miR-4709作為一種致癌基因可通過(guò)作用于NR3C2促進(jìn)人結(jié)腸癌細(xì)胞的增殖和遷移[43-44].Omidi等[45]通過(guò)生物信息學(xué)分析發(fā)現(xiàn)miR-4709-5p與紅斑狼瘡疾病相關(guān),可作為一種潛在的生物標(biāo)志物.由于miR-4709-5p與多種疾病的發(fā)生有關(guān),本文推測(cè)PFOS通過(guò)下調(diào)miR- 4709-5p調(diào)控MAPK信號(hào)通路誘發(fā)肺部疾病.

4 結(jié)論

4.1 PFOS暴露可影響人非小細(xì)胞肺癌A549細(xì)胞增殖,低濃度PFOS(<200μmol/L)促進(jìn)A549細(xì)胞增殖,高濃度PFOS抑制細(xì)胞增殖,且抑制作用隨PFOS濃度增大而增大.

4.2 PFOS暴露可引起A549細(xì)胞中171個(gè)miRNAs異常表達(dá),其中,108個(gè)miRNAs表達(dá)量顯著上調(diào),63個(gè)miRNAs表達(dá)量顯著下調(diào).異常表達(dá)的miRNAs可能通過(guò)調(diào)控Ras、Rap1、ErbB、HIF-1和VEGF等信號(hào)通路影響細(xì)胞生長(zhǎng)、增殖、分化、凋亡、代謝和發(fā)育等生物學(xué)過(guò)程.通過(guò)篩選差異表達(dá)的miRNAs來(lái)預(yù)測(cè)與PFOS肺毒性相關(guān)的靶基因是第一步,還需要進(jìn)一步驗(yàn)證靶基因的準(zhǔn)確性以及作為生物標(biāo)志物進(jìn)行疾病診斷的特異性和靈敏性.

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Impacts of PFOS exposure on signaling pathways in lung cancer cells.

YU Zhi-hui1, DONG Jing-ying1, WANG Ya-nan2, WANG Xia-yan2*

(1.Department of Environmental Sciences, Beijing University of Technology, Beijing 100124, China；2.Center of Excellence for Environmental Safety and Biological Effects, Beijing Key Laboratory for Green Catalysis and Separation, Department of Chemistry and Biology, Beijing University of Technology, Beijing 100124, China)., 2021,41(10)：4878～4884

The effects of different concentrations of perfluorooctane sulfonate (PFOS) on the viability of A549 cells were determined by the CCK-8 method. The effects of PFOS exposure on miRNAs expression in A549 cells were detected by the next-generation sequencing method to investigate the molecular mechanism of pulmonary toxicity caused by PFOS. Target genes with abnormal expression of miRNAs were predicted, and their involved signaling pathways and potential biological functions were inferred through bioinformatics analysis. The results showed that a low concentration of PFOS (<200μmol/L) promoted the proliferation of A549 cells, while a high concentration of PFOS inhibited the proliferation of A549 cells. The expression levels of 108 miRNAs and 63 miRNAs in A549 cells exposed to 300 μmol/L PFOS for 24 h were significantly up-regulated and down-regulated. Differentially expressed miRNAs participate in biological processes such as cell proliferation, metabolic process, and developmental process through signaling pathways such as Ras, Rap1, HIF-1, ErbB, VEGF and so on. This study suggested that PFOS can threaten the lung by affecting cell proliferation and inducing inflammation.

perfluorooctane sulfonate (PFOS)；miRNA；signaling pathways

X503.1

A

1000-6923(2021)10-4878-07

于志輝(1961-),女,北京人,教授,博士,主要從事環(huán)境毒理學(xué)和環(huán)境電化學(xué)方面研究.發(fā)表論文10余篇.

2021-03-03

北京高校卓越青年科學(xué)家計(jì)劃項(xiàng)目(BJJWZYJH0120191000 5017)

* 責(zé)任作者, 教授, xiayanwang@bjut.edu.cn