鲤脾脏高丰度micoRNAlet-7a序列特征、组织表达及功能预测分析
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摘要
先前研究表明let-7a为鲤(Cyprinus carpio)脾脏高丰度mi RNA,但其功能尚不清楚。为全面了解鲤let-7a的潜在生物学功能,研究分析了其成熟序列特征、组织表达谱和靶基因富集情况。结果显示,let-7a在鲤脾脏中存在序列长度变异和碱基替换,长度为18—25 nt,碱基替换类型有9种,种子区序列碱基替换率达2.1%。表达谱分析表明,let-7a为广泛性高丰度mi RNA,具明显的时序表达特征,其在鳃、肠、脾脏和肾脏等免疫相关器官中的表达水平明显高于其他器官。GO富集分析显示,let-7a与转录调控、细胞刺激应答、离子转运、跨膜运输、蛋白质氨基酸磷酸化和细胞迁移等生物学过程密切相关,尤其是靶基因GO term在刺激应答相关的许多生物学过程显著富集。总之,鲤let-7a可广泛参与多种生物学过程调控,尤其是机体各种刺激应答。
Previous studies have shown that the let-7 a was an abundance mi RNA in the spleen of common carp, but its function in the carp spleen is still not clear. Therefore, in this study, the characteristic of let-7 a mature sequence were analyzed, the expression profiles of let-7 a were determined by RT-q PCR in eight tissue samples at two stages of development, and the bioinformatic analysis were performed for the predicted target gene of let-7 a. The results showed that the let-7 a existed sequence length variation and base substitution in the spleen of common carp. The let-7 a length ranged from 18 nt to 25 nt, and the base substitution types of its mature sequence had nine. Especially, the base substitution rate in seed sequence was 2.1%. The expression profile analysis showed that the let-7 a as a generalized high abundance mi RNA exhibited striking temporal expression characteristics. The let-7 a expression levels in the immunity related organs including gill, intestine, spleen and kidney were significantly higher than those in the other organs. GO enrichment analysis suggested that the let-7 a significantly involved in the regulation of transcription, cellular response to stimulus, ion transport, transmembrane transport, protein amino acid phosphorylation, cell motion, and so on. Especially, the predicted target genes of let-7 a were significantly enriched in many biological processes of stimulus response. In a word, let-7 a can widely participate in the regulation of various biological processes, especially in the stimulus response of the body in common carp.
Previous studies have shown that the let-7 a was an abundance mi RNA in the spleen of common carp, but its function in the carp spleen is still not clear. Therefore, in this study, the characteristic of let-7 a mature sequence were analyzed, the expression profiles of let-7 a were determined by RT-q PCR in eight tissue samples at two stages of development, and the bioinformatic analysis were performed for the predicted target gene of let-7 a. The results showed that the let-7 a existed sequence length variation and base substitution in the spleen of common carp. The let-7 a length ranged from 18 nt to 25 nt, and the base substitution types of its mature sequence had nine. Especially, the base substitution rate in seed sequence was 2.1%. The expression profile analysis showed that the let-7 a as a generalized high abundance mi RNA exhibited striking temporal expression characteristics. The let-7 a expression levels in the immunity related organs including gill, intestine, spleen and kidney were significantly higher than those in the other organs. GO enrichment analysis suggested that the let-7 a significantly involved in the regulation of transcription, cellular response to stimulus, ion transport, transmembrane transport, protein amino acid phosphorylation, cell motion, and so on. Especially, the predicted target genes of let-7 a were significantly enriched in many biological processes of stimulus response. In a word, let-7 a can widely participate in the regulation of various biological processes, especially in the stimulus response of the body in common carp.
引文
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[23]Li Z H,Pan X M,Han B W,et al.A let-7 binding site polymorphism rs712 in the KRAS 3′UTR is associated with an increased risk of gastric cancer[J].Tumor Biology,2013,34(5):3159-3163
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[25]Jazdzewski K,Liyanarachchi S,Swierniak M,et al.Polymorphic mature micro RNAs from passenger strand of pre-mi R-146a contribute to thyroid cancer[J].The Proceedings of the National Academy of Sciences of the United States of America,2009,106(5):1502-1505
[26]Brodersen P,Voinnet O.Revisiting the principles of micro RNA target recognition and mode of action[J].Nature Reviews Molecular Cell Biology,2009,10(2):141-148
[27]Borchert G M,Gilmore B L,Spengler R M,et al.Adenosine deamination in human transcripts generates novel micro RNA binding sites[J].Human Molecular Genetics,2009,18(24):4801-4807
[28]Heale B S,Eulalio A,Schulte L,et al.Analysis of A to Iediting of mi RNA in macrophages exposed to Salmonella[J].RNA Biology,2010,7(5):116-122
[29]Kawahara Y,Zinshteyn B,Sethupathy P,et al.Redirection of silencing targets by adenosine-to-inosine editing ofmi RNAs[J].Science,2007,315(5815):1137-1140
[30]Ebhardt H A,Tsang H H,Dai D C,et al.Meta-analysis of small RNA-sequencing errors reveals ubiquitous posttranscriptional RNA modifications[J].Nucleic Acids Research,2009,37(8):2461-2470
[31]Chen X M,Splinter P L,O'Hara S P,et al.A cellular micro-RNA,let-7i,regulates Toll-like receptor 4 expressionand contributes to cholangiocyte immune responses against Cryptosporidium parvum infection[J].The Journal of Biological Chemistry,2007,282(39):28929-28938
[32]He X Y,Chen J X,Zhang Z,et al.The let-7a micro RNAprotects from growth of lung carcinoma by suppression of k-Ras and c-Myc in nude mice[J].Journal of Cancer Research and Clinical Oncology,2010,136(7):1023-1028
[2]Sontheimer E J,Carthew R W.Silence from within:endogenous si RNAs and mi RNAs[J].Cell,2005,122(1):9-12
[3]Koralov S B,Muljo S A,Galler G R,et al.Dicer ablation affects antibody diversity and cell survival in the Blymphocyte lineage[J].Cell,2008,132(5):860-874
[4]Zhu X,Hu Y,Wang K Z,et al.The expressional characterization of mir-222 in mandarin fish(Siniperca chuatsi)[J].Acta Hydrobiologica Sinica,2015,39(2):315-320[朱鑫,胡毅,王开卓,等.翘嘴鳜mi R-222的表达特征.水生生物学报,2015,39(2):315-320]
[5]Raida M K,Buchmann K.Development of adaptive immunity in rainbow trout,Oncorhynchus mykiss(Walbaum)surviving an infection with Yersinia ruckeri[J].Fish&Shellfish Immunology,2008,25(5):533-541
[6]Li G,Zhao Y,Wen L,et al.Identification and characterization of micro RNAs in the spleen of common carp immune organ[J].Journal of Cellular Biochemistry,2014,115(10):1768-1778
[7]Reinhart B J,Slack F J,Basson M,et al.The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J].Nature,2000,403(6772):901-906
[8]Thornton J E,Gregory R I.How does Lin28 let-7 control development and disease[J]?Trends in Cell Biology,2012,22(9):474-482
[9]Viswanathan S R,Daley G Q.Lin28:A micro RNA regulator with a macro role[J].Cell,2010,140(4):445-449
[10]Kumar M,Sahu S K,Kumar R,et al.Micro RNA let-7Modulates the immune response to Mycobacterium tuberculosis infection via control of A20,an inhibitor of the NF-κB pathway[J].Cell Host&Microbe,2015,17(3):345-356
[11]Jiang L,Cheng Z,Qiu S,et al.Altered let-7 expression in Myasthenia gravis and let-7c mediated regulation of IL-10 by directly targeting IL-10 in Jurkat cells[J].International Immunopharmacology,2012,14(2):217-223
[12]Swaminathan S,Suzuki K,Seddiki N,et al.Differential regulation of the Let-7 family of micro RNAs in CD4+Tcells alters IL-10 expression[J].The Journal of Immunology,2012,188(12):6238-6246
[13]Kumar M,Ahmad T,Sharma A,et al.Let-7 micro RNA-mediated regulation of IL-13 and allergic airway inflammation[J].Journal of Allergy and Clinical Immunology,2011,128(5):1077-1085
[14]Wang S J,Geng Q,Zheng X Y,et al.Regulatory role of the let-7/mi R-98 family in IL-6 expression[J].Journal of Shandong University(Health Sciences),2012,50(1):62-65[王树军,耿芹,张秀英,等.Let-7/mi R-98家族对IL-6的调节作用.山东大学学报(医学版),2012,50(1):62-65]
[15]Schulte L N,Eulalio A,Mollenkopf H J,et al.Analysis of the host micro RNA response to Salmonella uncovers the control of major cytokines by the let-7 family[J].The EMBO Journal,2011,30(10):1977-1989
[16]Lehmann S M,Krüger C,Park B,et al.An unconventional role for mi RNA:let-7 activates Toll-like receptor 7and causes neurodegeneration[J].Nature Neuroscience,2012,15(6):827-835
[17]Satoh M,Tabuchi T,Minami Y,et al.Expression of let-7i is associated with Toll-like receptor 4 signal in coronary artery disease:effect of statins on let-7i and Toll-like receptor 4 signal[J].Immunobiology,2012,217(5):533-539
[18]López-Bellido R,Barreto-Valer K,Sánchez-Simón F M,et al.Cocaine modulates the expression of opioid receptors and mi R-let-7d in zebrafish embryos[J].PLo S One,2012,7(11):e50885
[19]Shi Z Y,Wu M L,Fu Y S.Cloning,expression and target gene prediction of let-7 in Paralichthys olivaceus metamorphosis[J].Journal of Fishery Sciences of China,2011,18(6):1211-1218[施志仪,吴明林,付元帅.牙鲆let-7基因的克隆与表达及其靶基因预测.中国水产科学,2011,18(6):1211-1218]
[20]Wu M L,Shi Z Y,Fu Y S,et al.Expression character and target predictions of let-7d during metamorphosis in Japanese flounder(Paralichthys olivaceus)[J].Journal of Shanghai Ocean University,2012,21(3):344-350[吴明林,施志仪,付元帅,等.Let-7d在牙鲆变态发育期间的表达及靶基因预测.上海海洋大学学报,2012,21(3):344-350]
[21]Ambros V.The functions of animal micro RNAs[J].Nature,2004,431(7006):350-355
[22]Zhang J,Zhang L,Fan R,et al.The polymorphism in the let-7 targeted region of the Lin28 gene is associated with increased risk of type 2 diabetes mellitus[J].Molecular and Cellular Endocrinology,2013,375(1-2):53-57
[23]Li Z H,Pan X M,Han B W,et al.A let-7 binding site polymorphism rs712 in the KRAS 3′UTR is associated with an increased risk of gastric cancer[J].Tumor Biology,2013,34(5):3159-3163
[24]Smits K M,Paranjape T,Nallur S,et al.A let-7 micro R-NA SNP in the KRAS 3′UTR is prognostic in early-stage colorectal cancer[J].Clinical Cancer Research,2011,17(24):7723-7731
[25]Jazdzewski K,Liyanarachchi S,Swierniak M,et al.Polymorphic mature micro RNAs from passenger strand of pre-mi R-146a contribute to thyroid cancer[J].The Proceedings of the National Academy of Sciences of the United States of America,2009,106(5):1502-1505
[26]Brodersen P,Voinnet O.Revisiting the principles of micro RNA target recognition and mode of action[J].Nature Reviews Molecular Cell Biology,2009,10(2):141-148
[27]Borchert G M,Gilmore B L,Spengler R M,et al.Adenosine deamination in human transcripts generates novel micro RNA binding sites[J].Human Molecular Genetics,2009,18(24):4801-4807
[28]Heale B S,Eulalio A,Schulte L,et al.Analysis of A to Iediting of mi RNA in macrophages exposed to Salmonella[J].RNA Biology,2010,7(5):116-122
[29]Kawahara Y,Zinshteyn B,Sethupathy P,et al.Redirection of silencing targets by adenosine-to-inosine editing ofmi RNAs[J].Science,2007,315(5815):1137-1140
[30]Ebhardt H A,Tsang H H,Dai D C,et al.Meta-analysis of small RNA-sequencing errors reveals ubiquitous posttranscriptional RNA modifications[J].Nucleic Acids Research,2009,37(8):2461-2470
[31]Chen X M,Splinter P L,O'Hara S P,et al.A cellular micro-RNA,let-7i,regulates Toll-like receptor 4 expressionand contributes to cholangiocyte immune responses against Cryptosporidium parvum infection[J].The Journal of Biological Chemistry,2007,282(39):28929-28938
[32]He X Y,Chen J X,Zhang Z,et al.The let-7a micro RNAprotects from growth of lung carcinoma by suppression of k-Ras and c-Myc in nude mice[J].Journal of Cancer Research and Clinical Oncology,2010,136(7):1023-1028