|本期目录/Table of Contents|

[1]韩佩宇,车 琳,陈圆圆,等.CRISPR/Cas9敲低环氧合酶2表达对黄曲霉毒素B1诱导肝细胞核DNA损伤与脂质蓄积的影响[J].厦门大学学报(自然科学版),2018,57(05):634-642.[doi:10.6043/j.issn.0438-0479.201803016]
 HAN Peiyu,CHE Lin,CHEN Yuanyuan,et al.Effects of Cyclooxygenase 2 Knockdown Using CRISPR/Cas9 on Nuclear DNA Damage and Lipid Accumulation in Aflatoxin B1-induced Hepatocytes[J].Journal of Xiamen University(Natural Science),2018,57(05):634-642.[doi:10.6043/j.issn.0438-0479.201803016]





Effects of Cyclooxygenase 2 Knockdown Using CRISPR/Cas9 on Nuclear DNA Damage and Lipid Accumulation in Aflatoxin B1-induced Hepatocytes
韩佩宇车 琳陈圆圆江 珊段军燕孙宝芳何承勇林育纯林忠宁*
厦门大学 公共卫生学院,分子疫苗学和分子诊断学国家重点实验室,福建 厦门 361102
HAN PeiyuCHE LinCHEN YuanyuanJIANG ShanDUAN JunyanSUN BaofangHE ChengyongLIN YuchunLIN Zhongning*
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics,School of Public Health,Xiamen University,Xiamen 361102,China
CRISPR/Cas9 环氧合酶2 黄曲霉毒素B1 核DNA损伤 脂质蓄积
CRISPR/Cas9 cyclooxygenase 2 aflatoxin B1 nuclear DNA damage lipid accumulation
R 155.3+2
为探讨黄曲霉毒素B1(AFB1)诱导的肝细胞核DNA损伤与脂质蓄积的关系,以慢病毒质粒为载体,采用CRISPR/Cas9技术构建含靶向环氧合酶2(COX-2)编码基因(PTGS2)小向导RNA(sgRNA)的重组质粒,经测序验证成功后,制备假病毒感染HepG2细胞,构建稳定敲低COX-2表达的细胞.通过实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(WB)检测mRNA和蛋白水平,结果显示与HepG2-Cas9-NC对照细胞相比,HepG2-Cas9-PTGS2敲低细胞中PTGS2 mRNA与COX-2蛋白表达水平分别减少至(49.1±1.8)%和(48.1±0.7)%.给予细胞AFB1处理,通过WB和免疫荧光(IF)法检测DNA损伤标志物γH2AX的表达水平,结果显示处理组敲低细胞中,γH2AX蛋白水平和荧光焦点形成数目显著低于对照细胞(p<0.05); 进一步检测脂质合成相关指标,发现敲低细胞中PPARγ蛋白、总胆固醇(TC)、总甘油三脂(TG)水平以及油红O染色阳性脂滴的分布密度,均显著低于对照细胞中(p<0.05).在敲低细胞中回补COX-2后给予AFB1处理,γH2AX蛋白水平和脂质分布密度均显著升高(p<0.05).综上,成功构建了HepG2-Cas9-PTGS2敲低细胞,并发现敲低COX-2表达对AFB1诱导的肝细胞核DNA损伤和脂质蓄积有显著抑制作用,为进一步研究靶向干预COX-2在外源化学物诱导肝细胞毒性中的作用机制提供了细胞模型和依据.
To investigate the relationship between aflatoxin B1(AFB1)-induced nuclear DNA damage and lipid accumulation in he-patocytes, the lentiviral plasmids were used as the vector to construct recombinant plasmids containing the targeting cyclooxygenase 2(COX-2)gene(PTGS2)small guide RNA(sgRNA)using CRISPR/Cas9 technique.The recombinant plasmids were packaged with pseudovirus to infect HepG2 cells for a stable COX-2 knockdown cell line.Quantitative real-time PCR(qRT-PCR)and Wes-tern blot(WB)detection showed that PTGS2 mRNA and COX-2 protein levels in HepG2-Cas9-PTGS2 cells reduced to(49.1&#177;1.8)% and(48.1&#177;0.7)% respectively,compared with HepG2-Cas9-NC control cells.After these cells were treated with AFB1,the expression level of γH2AX,a DNA damage marker,was detected using WB and immunofluorescence(IF).The results showed that the protein level of γH2AX and the fluorescence foci numbers were significantly lower in HepG2-Cas9-PTGS2 cells than in HepG2-Cas9-NC cells(p<0.05).Lipid synthesis related experiments revealed that PPARγ protein expression,total cholesterol(TC),total triglyceride(TG)levels,and Oil Red O staining positive cell density were significantly lower in HepG2-Cas9-PTGS2 cells than in HepG2-Cas9-NC cells(p<0.05).Furthermore,the expression levels of COX-2,γH2AX,and lipid density in AFB1-treated HepG2-Cas9-PTGS2 cells increased after being rescued by wide type COX-2 expression(p<0.05).In summary,COX-2 knockdown HepG2-Cas9-PTGS2 cells were successfully constructed using CRISPR/Cas9 technique,and COX-2 knockdown significantly inhibited AFB1-induced nuclear DNA damage and lipid accumulation in hepatocytes,which provides cell model and basis for studying the mechanism of COX-2 in hepatotoxicity induced by xenobiotics.


[1] KENSLER T W,ROEBUCK B D,WOGAN G N,et al.Aflatoxin:a 50-year odyssey of mechanistic and translational toxicology[J].Toxicol Sci,2011,120(S1):S28-S48.
[2] LIU Y,WANG W.Aflatoxin B1 impairs mitochondrial functions,activates ROS generation,induces apoptosis and involves Nrf2 signal pathway in primary broiler hepatocytes[J].Anim Sci J,2016,87(12):1490-1500.
[3] SMIT E,SOUZA T,JENNEN D G,et al.Identification of essential transcription factors for adequate DNA damage response after benzo(a)pyrene and aflatoxin B1 exposure by combining transcriptomics with functional genomics[J].Toxicology,2017,390:74-82.
[4] ROTIMI O A,ROTIMI S O,DURU C U,et al.Acute aflatoxin B1-induced hepatotoxicity alters gene expression and disrupts lipid and lipoprotein metabolism in rats[J].Toxicol Rep,2017,4:408-414.
[5] COPPOLA M,GLINNI D,MORENO M,et al.Thyroid hormone analogues and derivatives:actions in fatty liver[J].World J Hepatol,2014,6(3):114-129.
[6] MáRTIN-SáNZ P,CáSADO M,BOSCá L.Cyclooxygenase 2 in liver dysfunction and carcinogenesis:facts and perspectives[J].World J Gastroenterol,2017,23(20):3572-3580.
[7] LEE J S,YOO J,KIM H,et al.Tumor stroma with senescence-associated secretory phenotype in steatohepatitic hepatocellular carcinoma[J].PLoS One,2017,12(3):e0171922.
[8] AGHAZADEH-HABASHI A,ASGHAR W,JAMALI F.Drug-disease interaction:effect of inflammation and nonsteroidal anti-inflammatory drugs on cytochrome P450 metabolites of arachidonic acid[J].J Pharm Sci,2018,107(2):756-763.
[9] SHALEM O,SANJANA N E,HARTENIAN E,et al.Genome-scale CRISPR-Cas9 knockout screening in human cells[J].Science,2014,343(6166):84-87.
[10] JINEK M,JIANG F,TAYLOR D W,et al.Structures of Cas9 endonucleases reveal RNA-mediated conformatio-nal activation[J].Science,2014,343(6176):1247997.
[11] ZHOU T J,ZHANG S L,HE C Y,et al.Downregulation of mitochondrial cyclooxygenase-2 inhibits the stemness of nasopharyngeal carcinoma by decreasing the activity of dynamin-related protein 1[J].Theranostics,2017,7(5):1389-1406.
[12] FLYNN R A,ZHANG Q C,SPITALE R C,et al.Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE[J].Nat Protoc,2016,11(2):273-290.
[13] WAN Y,QU K,ZHANG Q C,et al.Landscape and variation of RNA secondary structure across the human transcriptome[J].Nature,2014,505(7485):706-709.
[14] SANDER J D,JOUNG J K.CRISPR-Cas9 systems for editing,regulating and targeting genomes[J].Nat Biotechnol,2014,32(4):347-355.
[15] SANJANA N E,SHALEM O,ZHANG F.Improved vectors and genome-wide libraries for CRISPR screening[J].Nat Methods,2014,11(8):783-784.
[16] HAEUSSLER M,SCHONIG K,ECKERT H,et al.Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPR[J].Genome Biol,2016,17(1):148.
[17] ISHII Y,KURODA K,MATSUSHITA K,et al.Phosphorylation of protein phosphatase 2A facilitated an early stage of chemical carcinogenesis[J].Toxicol Appl Pharmacol,2017,336:75-83.
[18] AROOR A R,LOWERY J R,RICARDO J R,et al.A proteomic analysis of liver after ethanol binge in chronically ethanol treated rats[J].Proteome Sci,2012,10(1):29.
[19] SOLCAN C,GOGU M,FLORISTEAN V,et al.The hepatoprotective effect of sea buckthorn Hippophae rhamnoides berries on induced aflatoxin B1 poisoning in chickens 1[J].Poult Sci,2013,92(4):966-974.
[20] FUJIMORI K,AMANO F.Niacin promotes adipogenesis by reducing production of anti-adipogenic PGF2α through suppression of C/EBPβ-activated COX-2 expression[J].Prostaglandins Other Lipid Mediat,2011,94(3/4):96-103.
[21] GRYGIEL-GRNIAK B.Peroxisome proliferator-activated receptors and their ligands:nutritional and clinical implications-a review[J].Nutr J,2014,13(1):17.
[22] AN Z,YU J R,PARK W Y.T0070907 inhibits repair of radiation-induced DNA damage by targeting RAD51[J].Toxicol In Vitro,2016,37:1-8.
[23] 唐艳艳.EZH2介导ABCA1启动子区域DNA和组蛋白甲基化促进动脉粥样硬化进展[D].衡阳:南华大学,2014:15-16.


收稿日期:2018-03-07 录用日期:2018-05-16
基金项目:国家自然科学基金(81573181,81472997,81773465); 福建省自然科学基金(2015J01344)
Citation:HAN P Y,CHE L,CHEN Y Y,et al.Effects of cyclooxygenase 2 knockdown using CRISPR/Cas9 on nuclear DNA damage and lipid accumulation in aflatoxin B1-induced hepatocytes[J].J Xiamen Univ Nat Sci,2018,57(5):634-642.(in Chinese)
更新日期/Last Update: 1900-01-01