三唑磷对斑马鱼H19和Sox2OT基因表达调控的影响
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摘要
长链非编码RNA (long non-coding RNA, lncRNA)是指长度大于200 nt且不具有编码蛋白质功能的一类RNA分子。当生物体暴露于环境污染物时,其lncRNA通常会异常表达,并与癌症等疾病的发生相关。采用亚致死浓度的三唑磷处理斑马鱼后,利用人源基因芯片初步筛选出了异常表达的斑马鱼lncRNA,并通过生物信息学软件预测了与lncRNA相互作用的微RNA(micro RNA, miRNA)及其靶基因,最后通过实时荧光定量PCR验证了该miRNA及其靶基因在斑马鱼中的表达,从而构建起了长链非编码RNA-微RNA-信使RNA (lncRNA-miRNA-mRNA)的调控网络。荧光定量PCR检测结果表明:与空白对照组相比,经三唑磷处理后,斑马鱼lncRNA Sox2OT基因的表达下调了35%;lncRNA H19基因的表达上调了1.41倍,而与其可能存在相互作用的dre-let-7c基因的表达则下调了78%,同时dre-let-7c的靶基因ddx18的表达上调了2.11倍;差异均达显著水平(P <0.05)。据此推测,在三唑磷的作用下,lncRNA H19可能作为靶基因ddx18的竞争性内源RNA而与dre-let-7c发生相互作用,从而调控靶基因ddx18的表达。因此,lncRNA H19、dre-let-7c及ddx18都有可能作为生物标志物用于监测三唑磷对环境和生物体的影响。
Long non-coding RNAs(lncRNAs) are defined as a class of RNA molecule with a length of over 200 nucleotides and without protein-coding function. On the exposure to environmental pollutants,dysregulated expression of lncRNAs has been observed, which is associated with several diseases such as cancer. A human-derived microarray was used to screen the zebrafish(Danio rerio) lncRNA of zebrafish after sublethal triazophos treatment. The miRNAs and their target genes were predicted by the bioinformatics software. Finally, real-time fluorescent quantitative PCR was used to verify their relative expression in zebrafish, so that a lncRNA-miRNA-mRNA regulatory network was constructed. Results of real-time PCR showed that lncRNA Sox2 OT was reduced by 35% after triazophos treatment compared with that of the control group. The expression of lncRNA H19 was up-regulated by 1.41-fold,while that of dre-let-7 c was reduced by 78%. Furthermore, the expression of ddx18, which may be the target gene of dre-let-7 c, was up-regulated by 2.11-fold. The difference was significant(P < 0.05). It's proposed that lncRNA H19 may interact with dre-let-7 c as a competitive endogenous RNA to regulate the expression of ddx18 after the triazophos treatment. Therefore, lncRNA H19, dre-let-7 c and ddx18 can be potentially used as biomarkers to monitor the effects of triazophos on the environment and organisms.
Long non-coding RNAs(lncRNAs) are defined as a class of RNA molecule with a length of over 200 nucleotides and without protein-coding function. On the exposure to environmental pollutants,dysregulated expression of lncRNAs has been observed, which is associated with several diseases such as cancer. A human-derived microarray was used to screen the zebrafish(Danio rerio) lncRNA of zebrafish after sublethal triazophos treatment. The miRNAs and their target genes were predicted by the bioinformatics software. Finally, real-time fluorescent quantitative PCR was used to verify their relative expression in zebrafish, so that a lncRNA-miRNA-mRNA regulatory network was constructed. Results of real-time PCR showed that lncRNA Sox2 OT was reduced by 35% after triazophos treatment compared with that of the control group. The expression of lncRNA H19 was up-regulated by 1.41-fold,while that of dre-let-7 c was reduced by 78%. Furthermore, the expression of ddx18, which may be the target gene of dre-let-7 c, was up-regulated by 2.11-fold. The difference was significant(P < 0.05). It's proposed that lncRNA H19 may interact with dre-let-7 c as a competitive endogenous RNA to regulate the expression of ddx18 after the triazophos treatment. Therefore, lncRNA H19, dre-let-7 c and ddx18 can be potentially used as biomarkers to monitor the effects of triazophos on the environment and organisms.
引文
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[30]CHANYSHEV M D, KOSOROTIKOV N I, TITOV S E, et al.Expression of microRNAs, CYP1A1 and CYP2B1 in the livers and ovaries of female rats treated with DDT and PAHs[J]. Life Sci, 2014,103(2):95-100.
[31]HALLMANN C A, FOPPEN R P B, VAN TURNHOUT C A M,et al. Declines in insectivorous birds are associated with high neonicotinoid concentrations[J]. Nature, 2014, 511(7509):341-343.
[32]HONG S C, GUO Q, WANG W J, et al. Identification of differentially expressed microRNAs in Culex pipiens and their potential roles in pyrethroid resistance[J]. Insect Biochem Mol Biol,2014, 55:39-50.
[33]HUANG F, CHEN Z F, CHEN H W, et al. Cypermethrin promotes lung cancer metastasis via modulation of macrophage polarization by targeting MicroRNA-155/bcl6[J]. Toxicol Sci, 2018, 163(2):454-465.
[34]HUANG H N, ZHANG K, ZHOU Y Y, et al. MicroRNA-155targetscyb561d2 in zebrafish in response to fipronil exposure[J].Environ Toxicol, 2016, 31(7):877-886.
[35]ZHOU Y, HUANG H, ZHANG K, et al. miRNA-216 and miRNA-499 target cyb561d2 in zebrafish in response to fipronil exposure[J].Environ Toxicol Pharmacol, 2016, 45:98-107.
[2]SáNCHEZ-SANTED F, COLOMINA M T, HERRERO HERNáNDEZ E. Organophosphate pesticide exposure and neurodegeneration[J]. Cortex, 2016, 74:417-426.
[3]SINGH M, RISHI S. Plasma acetylcholinesterase as a biomarker of triazophos neurotoxicity in Young and adult rats[J]. Environ Toxicol Pharmacol, 2005, 19(3):471-476.
[4]SINGH S, TIWARI R K, PANDEY R S. Evaluation of acute toxicity of triazophos and deltamethrin and their inhibitory effect on AChE activity in Channa punctatus[J]. Toxicol Rep, 2018, 5:85-89.
[5]李红艳,张喆,陈海刚,等.三唑磷短期暴露对翡翠贻贝的毒性效应[J].南方水产科学, 2013, 9(5):71-79.LI H Y, ZHANG Z, CHEN H G, et al. Toxic effects of short-term triazophos exposure on mussel Perna viridis[J]. South China Fish Sci,2013, 9(5):71-79.
[6]罗朝晖,黄煜,洪彩熔.三唑磷农药对鲫鱼肝脏过氧化氢酶活性的影响[J].安徽农业科学, 2007, 36:11867-11868.LUO Z H, HUANG Y, HONG C R. Effect of the triazophos on the catalase activity of crucian(Carassus auratus)liver[J]. J Anhui Agric Sci, 2007, 36:11867-11868.
[7]NAVEED A, VENKATESHWARLU P, JANAIAH C. Impact of sublethal concentration of triazophos on regulation of protein metabolism in the fish Channa punctatus(Bloch)[J]. J Neurosci Res,2015, 88(2):360-368.
[8]ZHU B, GONG Y X, LIU L, et al. Toxic effects of triazophos on rare minnow(Gobiocypris rarus)embryos and larvae[J]. Chemosphere,2014, 108:46-54.
[9]JIA L, ZHANG D Y, HUANG H N, et al. Triazophos-induced toxicity in zebrafish:miRNA-217 inhibits nup43[J]. Toxicol Res,2018, 7(5):913-922.
[10]OKAZAKI Y, FURUNO M, KASUKAWA T, et al. Analysis of the mouse transcriptome based on functional annotation of 60, 770 fulllength cDNAs[J]. Nature, 2002, 420(6915):563-573.
[11]乔嘉凯,张丹燕,周胜利,等.斑马鱼长链非编码RNA的研究进展[J].生命科学, 2018, 30(8):822-830.QIAO J K, ZHANG D Y, ZHOU S L, et al. Research progress in long non-coding RNAs of zebrafish[J]. Chin Bull Life Sci, 2018, 30(8):822-830.
[12]DEMPSEY J L, CUI J Y. Long non-coding RNAs:a novel paradigm for toxicology[J]. Toxicol Sci, 2017, 155(1):3-21.
[13]HUANG Q, LIU Y, DONG S. Emerging roles of long non-coding RNAs in the toxicology of environmental chemicals[J]. J Appl Toxicol, 2018, 38(7):934-943.
[14]YE S, YANG L, ZHAO X, et al. Bioinformatics method to predict two regulation mechanism:TF-miRNA-mRNA and lncRNA-miRNAmRNA in pancreatic cancer[J]. Cell Biochem Biophys, 2014, 70(3):1849-1858.
[15]LI H, YU B Q, LI J F, et al. Overexpression of lncRNA H19 enhances carcinogenesis and metastasis of gastric cancer[J]. Oncotarget, 2014,5(8):2318-2329.
[16]YANG S, LEI Y. Transgenerational pancreatic impairment with Igf2/H19 epigenetic alteration induced by p,p’-DDE exposure in early life[J]. Toxicol Lett, 2017, 280:222-231.
[17]LIANG W C, FU W M, WONG C W, et al. The lncRNA H19promotes epithelial to mesenchymal transition by functioning as miRNA sponges in colorectal cancer[J]. Oncotarget, 2015, 6(26):22513-22525.
[18]ZHOU X Y, YE F, YIN C Q, et al. The interaction between MiR-141and lncRNA-H19 in regulating cell proliferation and migration in gastric cancer[J]. Cell Physiol Biochem, 2015, 36(4):1440-1452.
[19]ANDREW T, MANIATIS N, CARBONARO F, et al. Identification and replication of three novel myopia common susceptibility gene loci on chromosome 3q26 using linkage and linkage disequilibrium mapping[J]. PLoS Genet, 2008, 4(10):e1000220.
[20]AMARAL P P, NEYT C, WILKINS S J, et al. Complex architecture and regulated expression of the Sox2ot locus during vertebrate development[J]. RNA, 2009, 15(11):2013-2027.
[21]TOSETTI V, SASSONE J, FERRI A L M, et al. Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis[J]. Plos One, 2017, 12(7):e0180579.
[22]李正龙,徐艺,冷开明,等.长链非编码RNA SOX2OT在肿瘤中的调控作用及对预后的影响[J].现代肿瘤医学, 2018, 26(14):2290-2293.LI Z L, XU Y, LENG K M, et al. Regulatory role of lncRNA SOX2OT in neoplasms and intervention of prognosis[J]. J Mod Oncol, 2018, 26(14):2290-2293.
[23]KERSTIN H, CLARK M D, TORROJA C F, et al. The zebrafish reference genome sequence and its relationship to the human genome[J]. Nature, 2013, 496(7446):498-503.
[24]ZON L I, PETERSON R T. In vivo drug discovery in the zebrafish[J].Nat Rev Drug Discov, 2005, 4(1):35-44.
[25]WANG X X, ZHOU S L, DING X F, et al. Effect of triazophos,fipronil and their mixture on miRNA expression in adult zebrafish[J].J Environ Sci Heal(Part B), 2010, 45(7):648-657.
[26]LIU H, LI J, KOIRALA P, et al. Long non-coding RNAs as prognostic markers in human breast cancer[J]. Oncotarget, 2016,7(15):20584-20596.
[27]PAYNE E M, BOLLI N, RHODES J, et al. Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML[J]. Blood, 2011, 118(4):903-915.
[28]ZHU M J, CHEN Q, LIU X, et al. LncRNA H19/miR-675 axis represses prostate cancer metastasis by targeting TGFBI[J]. FEBS J,2014, 281(16):3766-3775.
[29]张丹燕,贾龙略,郭江峰.长链非编码RNA在毒理学领域的研究进展[J].中国药理学与毒理学杂志, 2017, 31(6):696-700.ZHANG D Y, JIA L, GUO J F. Reseach progress in long non-coding RNA and its toxicology[J]. Chin J Pharmacol Toxicol, 2017, 31(6):696-700.
[30]CHANYSHEV M D, KOSOROTIKOV N I, TITOV S E, et al.Expression of microRNAs, CYP1A1 and CYP2B1 in the livers and ovaries of female rats treated with DDT and PAHs[J]. Life Sci, 2014,103(2):95-100.
[31]HALLMANN C A, FOPPEN R P B, VAN TURNHOUT C A M,et al. Declines in insectivorous birds are associated with high neonicotinoid concentrations[J]. Nature, 2014, 511(7509):341-343.
[32]HONG S C, GUO Q, WANG W J, et al. Identification of differentially expressed microRNAs in Culex pipiens and their potential roles in pyrethroid resistance[J]. Insect Biochem Mol Biol,2014, 55:39-50.
[33]HUANG F, CHEN Z F, CHEN H W, et al. Cypermethrin promotes lung cancer metastasis via modulation of macrophage polarization by targeting MicroRNA-155/bcl6[J]. Toxicol Sci, 2018, 163(2):454-465.
[34]HUANG H N, ZHANG K, ZHOU Y Y, et al. MicroRNA-155targetscyb561d2 in zebrafish in response to fipronil exposure[J].Environ Toxicol, 2016, 31(7):877-886.
[35]ZHOU Y, HUANG H, ZHANG K, et al. miRNA-216 and miRNA-499 target cyb561d2 in zebrafish in response to fipronil exposure[J].Environ Toxicol Pharmacol, 2016, 45:98-107.
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