转录因子ATF-7参与调控氧化石墨烯(GO)对秀丽线虫的致毒效应
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摘要
ATF-7是具有亮氨酸拉链结构的转录激活因子,为了解其是否参与调控氧化石墨烯(GO)的致毒效应,以秀丽线虫为在体研究模型,通过运动行为、肠道活性氧簇(ROS)水平和寿命分析,评价atf-7 RNA干扰对秀丽线虫应答GO毒效应的影响;同时以qRT-PCR和Patf-7::mCherry转基因秀丽线虫检测GO对atf-7表达水平的影响.结果显示,GO暴露可以降低秀丽线虫头部摆动和身体弯曲频率,且运动行为的下降与GO暴露浓度呈正相关;atf-7干扰秀丽线虫对GO致毒效应更加敏感,0.1 mg/L GO就可导致头部摆动和身体弯曲频率显著下降(P <0.01);在GO暴露条件下,atf-7干扰秀丽线虫的平均寿命由13.54 d缩短到8.8 d,寿命缩短程度大于空载干扰组(P <0.01);atf-7干扰秀丽线虫在GO暴露条件下肠道ROS水平比未暴露情况下上升了3.33倍,而空载组肠道ROS水平上升了2.66倍,atf-7干扰秀丽线虫对GO所致肠道ROS的积累更加明显(P <0.01).GO暴露可以诱导秀丽线虫atf-7的表达水平,GO暴露组atf-7在转录水平和蛋白水平较对照组分别上调了164倍和200倍.因此,atf-7是秀丽线虫应对GO致毒效应中所必需的,秀丽线虫通过上调atf-7的表达以应对GO毒效应,对秀丽线虫进行atf-7干扰降低其表达水平可使秀丽线虫对GO致毒效应更加敏感.(图4表1参24)
The atf-7 gene encodes the leucine zipper(bZIP) transcription factor, but its function in regulating the response to graphene oxide(GO) is still unclear. Caenorhabditis elegans was used as an assay system to investigate the in vivo function of atf-7 in response to GO. RNA interference in the in vivo expression of atf-7 was used to knockdown its expression.Subsequently, the locomotion behaviors, lifespan, reactive oxygen species(ROS) levels in the intestine, and the expression at the transcriptional and protein levels were analyzed in absence and presence of GO exposure. The results showed that the locomotion behaviors were reduced by GO in a concentration-dependent manner. The atf-7 RNAi strains were more susceptible to GO, resulting in a decrease of head thrashes and body bends in the concentration of 0.1 mg/L(P < 0.01). The decrease of mean lifespan was higher in the atf-7 RNAi strains(decrease from 13.54 days to 8.8 days) than those of the empty vector RNAi strains when treated with GO(P < 0.01). The atf-7 RNAi strains generated more ROS in the intestine after GO exposure, which was 3.33 times higher than without exposure, while ROS in the empty vector group increased by 2.66(P < 0.01). Under the condition of GO exposure, the expressions of atf-7 at the transcriptional and protein levels were up-regulated by 164-fold and200-fold, respectively. The transcription factor ATF-7 is vital for C. elegans to deal with the toxic effects caused by GO and the nematodes were more susceptible to GO when atf-7 was knockdown by RNAi.
The atf-7 gene encodes the leucine zipper(bZIP) transcription factor, but its function in regulating the response to graphene oxide(GO) is still unclear. Caenorhabditis elegans was used as an assay system to investigate the in vivo function of atf-7 in response to GO. RNA interference in the in vivo expression of atf-7 was used to knockdown its expression.Subsequently, the locomotion behaviors, lifespan, reactive oxygen species(ROS) levels in the intestine, and the expression at the transcriptional and protein levels were analyzed in absence and presence of GO exposure. The results showed that the locomotion behaviors were reduced by GO in a concentration-dependent manner. The atf-7 RNAi strains were more susceptible to GO, resulting in a decrease of head thrashes and body bends in the concentration of 0.1 mg/L(P < 0.01). The decrease of mean lifespan was higher in the atf-7 RNAi strains(decrease from 13.54 days to 8.8 days) than those of the empty vector RNAi strains when treated with GO(P < 0.01). The atf-7 RNAi strains generated more ROS in the intestine after GO exposure, which was 3.33 times higher than without exposure, while ROS in the empty vector group increased by 2.66(P < 0.01). Under the condition of GO exposure, the expressions of atf-7 at the transcriptional and protein levels were up-regulated by 164-fold and200-fold, respectively. The transcription factor ATF-7 is vital for C. elegans to deal with the toxic effects caused by GO and the nematodes were more susceptible to GO when atf-7 was knockdown by RNAi.
引文
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13 Zhao YL,Wu QL,Wang DY.A microRNAs-mRNAs network involved in the control of graphene oxide toxicity in Caenorhabditis elegans[J].RSC Adv,2015,5(112):92394-92405
14 Zhao YL,Zhi LT,Wu QL,Yu YL,Sun QQ,Wang DY.p38 MAPK-SKN-1/Nrf signaling cascade is required for intestinal barrier against graphene oxide toxicity in Caenorhabditis elegans[J].Nanotoxicology,2016,10(10):1469-1479
15 Ashrafi K,Shivers RP,Pagano DJ,Kooistra T,Richardson CE,Reddy KC,Whitney JK,Kamanzi O,Matsumoto K,Hisamoto N,Kim DH.Phosphorylation of the conserved transcription factor ATF-7 by PMK-1 p38 MAPK regulates innate immunity in Caenorhabditis elegans[J].PLoS Genet,2010,6(4):e1000892
16 Wu QL,Zhao YL,Fang JP,Wang DY.Immune response is required for the control of in vivo translocation and chronic toxicity of graphene oxide[J].Nanoscale,2014,6(11):5894-5906
17 Popham JD,Webster JM.Cadmium toxicity in the free-living nematode,Caenorhabditis elegans[J].Environ Res,1979,20(1):183-191
18王大勇,胡亚欧,许雪梅.铬暴露导致的秀丽线虫多重毒性的世代间比较[J].生态毒理学报,2007,2(3):297-303[Wang DY,Hu YO,Xu XM.Comparison of multi-biological toxicities between chromium exposed caenorhabditis elegans and their progeny[J].Asian JEcotoxicol,2007,2(3):297-303]
19 Zhao Y,Liu Q,Shakoor S,Gong J R,and Wang D Transgenerational safety of nitrogen-doped graphene quantum dots and the underlying cellular mechanism in Caenorhabditis elegans[J].Toxicol Res,2015,4(2):270-280
20章丛婕,王科洁,刘振亚,黄佳涛,周怀彬,吕建新.C.elegans中clk-1基因对寿命的影响[J].中国细胞生物学学报,2017,39(6):745-755[Zhang CJ,Wang KJ,Liu ZY,Huang JT,Zhou HB,LüJX.Effects of clk-1 on lifespan of Caenorhabditis elegans[J].Chin J Cell Biol,2017,39(6):745-755]
21 Peters CS,Liang X,Li S,Kannan S,Peng Y,Taub R and Diamond RH.ATF-7,a novel bZIP protein,interacts with the PRL-1 protein-tyrosine phosphatase.J Biol Chem,2001,276(17):13718-13726
22 Maekawa T,Kim S,Nakai D,Makino C,Takagi T,Ogura H,Yamada K,Chatton B,Ishii S.Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene[J].EMBO J,2010,29(1):196-208
23 Hall JA,McElwee MK,Freedman JH.Identification of ATF-7 and the insulin signaling pathway in the regulation of metallothionein in C.elegans suggests roles in aging and reactive oxygen species[J].PLoSONE,2017,12(6):e0177432
24 Li WJ,Wang DY,Wang DY.Regulation of there sponse of Caenorhabditis elegans to simulated microgravity by p38 mitogenactivated protein kinase signaling[J].Sci Rep,2018,8(1):857
2 Chang Y,Yang ST,Liu JH,Dong E,Wang Y,Cao A,Liu Y,Wang H.In vitro toxicity evaluation of graphene oxide on A549 cells[J].Toxicol Lett,2011,200(3):201-210
3 LüM,Zhang Y,Liang L,Wei M,Hu W,Li X,Huang Q.Effect of graphene oxide on undifferentiated and retinoic acid-differentiated SH-SY5Y cells line[J].Nanoscale,2012,4(13):3861-3866
4 Wa ng K,Ru a n J,Song H,Z ha ng J L,Wo Y,Guo SW,Cu i DX.Biocompatibility of graphene oxide[J].Nanoscale Res Lett,2011,6:1-8
5 Aryaei A,Jayatissa AH,Jayasuriya AC.The effect of graphene substrate on osteoblast cell adhesion and proliferation[J].J Biomed Mater Res A,2014,102(9):3282-3290
6 Zhang X,Yin J,Peng C,Hu W,Zhu Z,Li W,Fan C,Huang Q.Distribution and biocompatibility studies of graphene oxide in mice after intravenous administration[J].Carbon,2011,49(3):986-995
7 Wu Q,Yin L,Li X,Tang M,Zhang T,and Wang D Contributions of altered permeability of intestinal barrier and defecation behavior to toxicity formation from graphene oxide in nematode Caenorhabditis elegans[J].Nanoscale,2013,5(20):9934-9943
8 Chen Y,Hu X,Sun J,Zhou Q.Specific nanotoxicity of graphene oxide during zebrafish embryogenesis[J].Nanotoxicology,2016,10(1):42-52
9 Akhavan O,Ghaderi E,Akhavan A.Size-dependent genotoxicity of graphene nanoplatelets in human stem cells[J].Biomaterials,2012,33(32):8017-8025
10秦峰松,杨崇林.小线虫,大发现[J].生命科学,2006,18(5):420-425[Qin FS,Yang CL.Worm into discoveries:Caenorhabditis elegans as a model organism of life science research[J].Chin Bull Life Sci,2006,18(5):420-425]
11张燕芬,王大勇.利用模式动物秀丽线虫建立环境毒物毒性的评估研究体系[J].生态毒理学报,2008,3(4):313-322[Zhang YF,Wang DY.Establishment of toxicity evaluat ion system using model organism of Caenorhabditis elegans[J].Asian J Ecotoxicol,2008,3(4):313-322]
12王晶晶,许安,代慧,王娟,王牧笛.利用模式生物秀丽线虫评价磁场生物效应进展[J].应用与环境生物学报,2015,21(6):1003-1011[Wang JJ,Xu A,Dai H,Wang J,Wang MD.Progress in assessing biological effects of magnetic fields using model organism Caenorhabditis elegans[J].Chin J Appl Environ Biol,2015,21(6):1003-1011]
13 Zhao YL,Wu QL,Wang DY.A microRNAs-mRNAs network involved in the control of graphene oxide toxicity in Caenorhabditis elegans[J].RSC Adv,2015,5(112):92394-92405
14 Zhao YL,Zhi LT,Wu QL,Yu YL,Sun QQ,Wang DY.p38 MAPK-SKN-1/Nrf signaling cascade is required for intestinal barrier against graphene oxide toxicity in Caenorhabditis elegans[J].Nanotoxicology,2016,10(10):1469-1479
15 Ashrafi K,Shivers RP,Pagano DJ,Kooistra T,Richardson CE,Reddy KC,Whitney JK,Kamanzi O,Matsumoto K,Hisamoto N,Kim DH.Phosphorylation of the conserved transcription factor ATF-7 by PMK-1 p38 MAPK regulates innate immunity in Caenorhabditis elegans[J].PLoS Genet,2010,6(4):e1000892
16 Wu QL,Zhao YL,Fang JP,Wang DY.Immune response is required for the control of in vivo translocation and chronic toxicity of graphene oxide[J].Nanoscale,2014,6(11):5894-5906
17 Popham JD,Webster JM.Cadmium toxicity in the free-living nematode,Caenorhabditis elegans[J].Environ Res,1979,20(1):183-191
18王大勇,胡亚欧,许雪梅.铬暴露导致的秀丽线虫多重毒性的世代间比较[J].生态毒理学报,2007,2(3):297-303[Wang DY,Hu YO,Xu XM.Comparison of multi-biological toxicities between chromium exposed caenorhabditis elegans and their progeny[J].Asian JEcotoxicol,2007,2(3):297-303]
19 Zhao Y,Liu Q,Shakoor S,Gong J R,and Wang D Transgenerational safety of nitrogen-doped graphene quantum dots and the underlying cellular mechanism in Caenorhabditis elegans[J].Toxicol Res,2015,4(2):270-280
20章丛婕,王科洁,刘振亚,黄佳涛,周怀彬,吕建新.C.elegans中clk-1基因对寿命的影响[J].中国细胞生物学学报,2017,39(6):745-755[Zhang CJ,Wang KJ,Liu ZY,Huang JT,Zhou HB,LüJX.Effects of clk-1 on lifespan of Caenorhabditis elegans[J].Chin J Cell Biol,2017,39(6):745-755]
21 Peters CS,Liang X,Li S,Kannan S,Peng Y,Taub R and Diamond RH.ATF-7,a novel bZIP protein,interacts with the PRL-1 protein-tyrosine phosphatase.J Biol Chem,2001,276(17):13718-13726
22 Maekawa T,Kim S,Nakai D,Makino C,Takagi T,Ogura H,Yamada K,Chatton B,Ishii S.Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene[J].EMBO J,2010,29(1):196-208
23 Hall JA,McElwee MK,Freedman JH.Identification of ATF-7 and the insulin signaling pathway in the regulation of metallothionein in C.elegans suggests roles in aging and reactive oxygen species[J].PLoSONE,2017,12(6):e0177432
24 Li WJ,Wang DY,Wang DY.Regulation of there sponse of Caenorhabditis elegans to simulated microgravity by p38 mitogenactivated protein kinase signaling[J].Sci Rep,2018,8(1):857