PFOS暴露對肺癌細胞中信號通路的影響

于志輝,董晶熒,王亞楠,汪夏燕*

PFOS暴露對肺癌細胞中信號通路的影響

于志輝1,董晶熒1,王亞楠2,汪夏燕2*

(1.北京工業大學環境科學系,北京 100124；2.北京工業大學化學與生物系,綠色催化與分離北京市重點實驗室,環境安全與生物效應卓越中心,北京 100124)

為探討全氟辛烷磺酸鹽(PFOS)產生肺毒性的分子機制,采用細胞計數試劑盒(CCK-8)方法測定不同濃度PFOS對A549細胞活性的影響,并用二代測序方法測定PFOS暴露對A549細胞中miRNAs表達的影響,預測異常表達miRNAs的靶基因.通過生物信息學分析推斷靶基因參與的信號通路及潛在的生物學功能.結果顯示,低濃度PFOS(<200μmol/L)促進A549細胞增殖,高濃度PFOS抑制細胞增殖.暴露于300μmol/L PFOS中24h的A549細胞中108個miRNAs表達量顯著上調,63個miRNAs表達量顯著下調.差異表達miRNAs通過Ras、Rap1、HIF-1、ErbB和VEGF等信號通路參與細胞增殖、代謝和發育等生物學過程.這表明PFOS可通過影響細胞增殖和誘發炎癥反應對肺造成威脅.

全氟辛烷磺酸鹽(PFOS)；miRNA；信號通路

全氟烷基化合物(PFASs)由于具有良好的疏水疏油性,已被廣泛應用于滅火劑、食品包裝、紡織品、紙張、洗發劑、表面活性劑等工商業產品加工過程[1-2].由于C-F鍵具有強極性,在強紫外線、高溫及其他化學作用的條件下具有較強的穩定性,并且很難通過微生物及高等動物的代謝作用來降解,因此PFASs可以穩定的存在于環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、土壤等多種環境介質中,甚至在職業暴露和非職業暴露人群的血液、尿液、膽汁、母乳及臍帶血中均有檢測出PFASs[5-10].全氟辛烷磺酸鹽(PFOS)是PFASs的最終代謝產物,分布最為廣泛.PFOS可通過胃腸道、呼吸道和皮膚進入人體[11],且半衰期高達5a以上[2].研究表明,PFOS具有肝[12]、肺[13]、腎[14]、免疫[15]、神經[16]、生殖發育[17]等多種毒性,是一類具有全身多器官和組織毒性的有機污染物.其中,PFOS對肝、神經毒性的相關研究相對較多.PFOS的肝毒性主要表現為脂肪肝、肝腫大、肝細胞增生和肝細胞氧化損傷等[18-19].此外,PFOS通過誘導神經細胞產生過量的活性氧或炎癥因子,對神經細胞造成氧化損傷或神經炎癥[16,20].

PFOS在非職業暴露者肺中的含量僅次于肝[21],可誘發多種肺部疾病.流行病學研究表明,血清PFOS濃度與兒童哮喘病加劇具有相關性[22].Qin等[23]通過評估兒童哮喘病患者的肺功能,進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相關.與其他組織毒性相同,PFOS也主要通過誘導肺組織分泌過量的炎癥因子和活性氧來造成肺毒性[24],但研究者對其中的調控機制以及涉及的信號通路知之甚少,對PFOS的肺毒性機制還沒有形成系統完善的認識.

microRNA(miRNA)是一類由內源基因編碼的長度約為18～24個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病毒中,通過與mRNA的完全或不完全互補誘導mRNA降解或抑制其翻譯,實現轉錄后水平的基因表達調控.基于miRNA的生物學功能,已經有很多學者從miRNA分子水平探究PFOS對生物體的毒性機制[25-27]. PFOS可引起妊娠初期人滋養層細胞的miR-29b含量升高,進而使得多種蛋白的DNA甲基化和蛋白乙?；?蛋白表達量降低引起ROS含量升高[25].ROS含量的升高與子癇前期等妊娠并發癥相關.此外,研究表明,PFOS通過增加SH-SY5Y細胞中miR-22的相對表達量,抑制BDNF mRNA的表達,影響BDNF- ERK-CREB信號通路,為PFOS的神經毒性提供了新的實驗證據[26].這些研究為PFOS的肺毒性機制研究提供了新思路.然而,與miRNA相關的PFOS對肺毒性機制的研究未見報道.

本文采用體外細胞毒性試驗的方法,以人非小細胞肺癌A549細胞為模型,從miRNA表觀遺傳調控角度研究PFOS對肺損傷可能的作用機制.采用細胞計數試劑盒(CCK-8)方法檢測細胞活性,探討PFOS對A549的細胞增殖毒性.二代測序篩選PFOS暴露后差異表達的miRNAs并進行基因組百科全書(KEGG)和基因本體論(GO)富集分析,推測參與PFOS肺毒性的信號通路,深入探究PFOS肺毒性的表觀遺傳調控機制.

1 材料與方法

1.1 細胞與試劑

人非小細胞肺癌細胞A549購自中國醫學科學院基礎醫學研究所細胞資源中心(北京,中國);杜氏改良Eagle培養基(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)購自美國Thermo Fisher Gibco公司;PFOS(純度98%)購自北京百靈威科技有限公司;二甲基亞砜(DMSO)購自上海阿拉丁生化科技股份有限公司;CCK-8試劑盒購自北仁化學科技(北京)有限公司;TRIzol試劑購自美國Thermo Fisher公司;miRcute miRNA提取分離試劑盒、miRcute增強型miRNA cDNA 第一鏈合成試劑盒、目的基因和內參基因引物、miRcute增強型miRNA 熒光定量檢測試劑盒(SYBR Green)購自天根生化科技(北京)有限公司.

1.2 細胞培養與處理

將A549細胞置于含10% FBS、1% PS的DMEM培養液中,于37℃、飽和濕度、含5% CO2的培養箱中培養.將PFOS溶于DMSO中,配制500mmol/L PFOS儲備液儲存于-20°C,使用前用培養液進行稀釋.為避免對細胞產生毒性,實驗組中DMSO的終體積分數不能超過0.1%,對照組為只含0.1% DMSO的培養液,空白組為不含細胞的0.1% DMSO的培養液.

1.3 CCK-8檢測細胞活性

收集對數生長期的細胞制備細胞懸液.取100μL密度為4×104個/mL的A549細胞懸液接種于96孔板中培養.孵育24h后棄去培養液,每孔加入200μL濃度分別為0,50,100,200,300,400,500μmol/L PFOS的培養液,每組6個平行.在標準條件下分別培養24,48,72h后棄去培養液,每孔加入100μL含10% CCK-8的培養液于37℃條件下孵育0.5h.用酶標儀(美國Molecular Devices公司)檢測各孔在450nm處吸光度(A),吸光度與細胞活性呈正比.細胞活性的計算公式為:

1.4 RNA提取與測序

考慮到A549細胞倍增周期約為21h[28],且其在PFOS中暴露24h的半數抑制濃度(IC50)在400～ 500μmol/L之間,因此,將A549細胞在300μmol/L PFOS中暴露24h研究PFOS暴露對A549細胞中miRNAs表達的影響.取1mL密度為2×106個/mL的A549細胞懸液加入75cm2培養瓶中,加入適量的培養液,將細胞吹打均勻.待細胞貼壁生長24h后,移去上清液,實驗組加入適量PFOS濃度為300μmol/L的培養液,對照組加入適量含0.1% DMSO的培養液,每組設置3個平行.待細胞暴露24h之后,用0.25%的胰蛋白酶將細胞消化下來,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測序平臺對總RNA樣品進行測序分析.

1.5 miRNAs靶基因預測和鑒定

使用R包edgeR對實驗組和對照組樣品中所有miRNAs進行差異分析,TMM方法歸一化.采用miRanda軟件對具有顯著性差異的miRNAs靶基因進行預測,得到miRNAs和靶基因間的對應關系.將得到的靶基因基于topGO進行GO功能富集分析.GO共包含3個類群,分別描述基因的分子功能(MF)、細胞組分(CC)、參與的生物學過程(BP).本文主要對靶基因的生物學過程進行富集分析,并對富集分析結果進行圖形化展示.在生物體內,不同基因相互協調行使其生物學功能,通過KEGG數據庫進行通路顯著性富集,以確定差異表達的miRNAs靶基因參與的最主要的生化代謝途徑和信號傳導途徑.

1.6 RT-qPCR驗證miRNAs

參照miRcute miRNA提取分離試劑盒提取實驗組和對照組細胞中的miRNAs.測定提取的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Applied Biosystems公司),結合miRcute增強型miRNA cDNA第一鏈合成試劑盒和miRcute增強型miRNA熒光定量檢測試劑盒(SYBR Green)對miRNAs樣品進行反轉錄和實時熒光定量PCR(RT-qPCR),每組設置3個平行,具體實驗操作參照產品說明書.數據處理以U6為內參基因,對目標基因表達量進行標準化,計算DCt值.以對照組作為參照因子,其倍數變化為1,實驗組基因表達差異相對于參照因子基因表達的倍數為2﹣△△Ct.分析實驗組和對照組中miRNAs的相對表達量,并與測序結果進行比較.

1.7 數據統計與分析

所有實驗數據均采用GraphPad Prism 8軟件進行統計學分析,結果以“均值±標準差”表示.采用單因素方差分析方法比較各組之間的差異,當<0.05時認為差異具有統計學意義.

2 結果分析

2.1 PFOS暴露對細胞活性的影響

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

圖1 PFOS暴露后的A549細胞活性

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

2.2 PFOS暴露對細胞中miRNAs的影響

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

2.3 miRNAs靶基因的預測

表達量顯著上升的108個miRNAs可作用于42009個靶mRNAs,表達量顯著下降的63個miRNAs對應于30098個靶mRNAs.通過KEGG數據庫分析差異表達miRNAs靶基因的功能及其相互作用,預測到靶基因可能參與的信號通路包括——大鼠肉瘤基因(Ras)信號通路、Ras相關蛋白1(Rap1)信號通路、ErbB信號通路、缺氧誘導因子1(HIF-1)信號通路、血管內皮生長因子(VEGF)信號通路、磷脂酶D信號通路、神經營養因子信號通路和雷帕霉素靶蛋白(mTOR)信號通路等(圖3).GO富集分析靶基因參與的生物學過程,結果表明差異表達miRNAs靶基因參與的生物學過程包括——細胞增殖過程、生物調節過程、代謝過程、細胞過程、應激反應過程、多細胞生物過程、細胞組分組織或合成過程、細胞定位過程、發育過程、免疫系統過程、多組織過程、生物附著過程和復制過程等(圖4).

圖2 PFOS暴露后A549細胞中miRNAs的火山

圖3 差異表達miRNAs靶基因KEGG通路富集分析

圖4 差異表達miRNAs靶基因GO(BP)富集分析

2.4 RNA-Seq結果的驗證

采用RT-qPCR方法驗證差異表達的miR- 377-3p、miR-3199和miR-4709-5p的表達量,結果如圖5所示,miR-4709-5p與測序結果一致,在PFOS實驗組中顯著下調;miR-377-3p和miR-3199表達量無明顯變化,與測序結果不一致.

圖5 差異表達miRNAs的驗證

3 討論

由于PFOS在環境介質中的廣泛存在使得人們開始關心它對人類健康的影響.研究表明,PFOS對包括肺在內的多種組織和系統均有毒性作用[29]. PFOS的肺毒性與DNA甲基化、ROS含量變化相關.然而這些變化都不能充分解釋PFOS的肺毒性機理.已有研究表明PFOS可引起妊娠初期人滋養層細胞、大鼠肝臟和大腦組織中的miRNAs異常表達.因此,本文以容易培養且對外加作用因子敏感的非小細胞肺癌A549細胞為研究對象,探討PFOS的肺毒性作用機制.

PFOS通過調節細胞周期影響細胞增殖,而細胞的異常增殖往往與癌癥的發生有關[30].Jabeen等[31]通過研究表觀遺傳修飾在細胞增殖和凋亡中的作用對PFOS影響A549細胞活性的機制進行了闡述,發現低濃度條件下(<100μmol/L)細胞周期蛋白E和細胞周期蛋白A表達量增加,促進A549細胞增殖,當PFOS濃度增至400μmol/L時,兩種細胞周期蛋白表達量降低,造成細胞活性顯著降低.Cui等[32]研究PFOS暴露后人正常肝細胞HL-7702的蛋白組學發現,50μmol/L的PFOS可誘導HL-7702細胞中多種細胞周期蛋白及相應的細胞周期蛋白依賴性激酶表達量增加,從而促進細胞增殖.但當PFOS濃度大于200μmol/L時,細胞活性呈劑量依賴性降低.同樣的,本文結果顯示,當A549細胞在50和100μmol/L PFOS中暴露24h后,細胞活性顯著增加,當PFOS濃度大于300μmol/L時細胞活性顯著降低,且細胞活性隨PFOS濃度增大而減小.這說明PFOS可能通過影響細胞增殖對肺產生毒性.

本文結果顯示PFOS暴露可引起A549細胞中多個miRNAs異常表達,這些異常表達的miRNAs可作用于多個靶基因,參與Ras、Rap1、ErbB、HIF-1和VEGF等多個信號通路.Ras信號通路協同下游多個信號通路調控細胞生長、增殖、分化和凋亡[33].Ras基因的異常表達與腫瘤的發生發展密切相關,在30%非小細胞肺癌中發現Ras突變[34],因此,Ras被認為是腫瘤發生的重要因素.Rap1是Ras通路的重要調節因子和介質,其參與的信號通路與肺癌細胞的增殖和分化相關[35].ErbB可促進細胞增殖,激活ErbB通路可能誘發癌癥.Zhang等[36]研究發現持續抽煙可使人體內多個miRNAs異常表達,進而影響ErbB通路促進肺癌的發生. Kruspig等[37]的研究表明,ErbB通過與Ras通路相互作用促進Kras突變肺癌細胞的增殖,因此,含ErbB抑制劑的藥物可能有利于Kras突變肺癌的治療.VEGF在肺發育及肺結構形成和維持過程中具有重要作用,其低表達會導致肺組織形態結構、功能異常[38].Zhang等[39]研究妊娠期PFOS暴露對子代大鼠肺發育的影響,發現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,且與炎癥小體相關的蛋白表達也顯著升高.同時,在肺泡發育和肺部血管形成過程中具有重要作用的VEGF及HIF-1的表達也受到抑制,誘發子代大鼠支氣管肺發育不良.本研究的預測結果說明PFOS可能通過miRNAs調控Ras、Rap1、ErbB、VEGF和HIF-1等信號通路影響細胞增殖、代謝和發育等生物學過程.

測序和RT-qPCR結果均表明PFOS可引起A549細胞中miR-4709-5p表達顯著下降.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)信號通路,在細胞增殖、分化和凋亡過程中具有重要作用[40]. MAPK蛋白還參與體內多種氧化應激和炎癥反應過程并發揮重要調控作用[41]. Shi等[42]將斑馬魚胚胎暴露于不同濃度的PFOS中,發現在斑馬魚幼蟲中出現氧化應激反應,且與MAPK通路相關的基因表達異常,推測這與PFOS誘導的細胞凋亡有關.研究表明miR-4709與結腸癌有關,miR-4709作為一種致癌基因可通過作用于NR3C2促進人結腸癌細胞的增殖和遷移[43-44].Omidi等[45]通過生物信息學分析發現miR-4709-5p與紅斑狼瘡疾病相關,可作為一種潛在的生物標志物.由于miR-4709-5p與多種疾病的發生有關,本文推測PFOS通過下調miR- 4709-5p調控MAPK信號通路誘發肺部疾病.

4 結論

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

4.2 PFOS暴露可引起A549細胞中171個miRNAs異常表達,其中,108個miRNAs表達量顯著上調,63個miRNAs表達量顯著下調.異常表達的miRNAs可能通過調控Ras、Rap1、ErbB、HIF-1和VEGF等信號通路影響細胞生長、增殖、分化、凋亡、代謝和發育等生物學過程.通過篩選差異表達的miRNAs來預測與PFOS肺毒性相關的靶基因是第一步,還需要進一步驗證靶基因的準確性以及作為生物標志物進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,主要從事環境毒理學和環境電化學方面研究.發表論文10余篇.

2021-03-03

北京高校卓越青年科學家計劃項目(BJJWZYJH0120191000 5017)

* 責任作者, 教授, xiayanwang@bjut.edu.cn