用Solexa测序技术分析和鉴定猪胚胎microRNA的研究
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摘要
microRNA（miRNA）是一类大小21-23个碱基的单链小分子RNA，由具有发夹结构约70-90个碱基的单链RNA前体经过Dicer酶加工后生成，其家族能够通过与靶mRNA的3′端非翻译区（UTR）完全或不完全配对而降解靶mRNA或抑制mRNA的正常翻译，进而对基因转录后表达进行调控。与小鼠或其他模式生物相比，在生物医学方面猪与人类的亲缘关系更近。
到目前为止，已经在人和小鼠等哺乳动物中鉴定出数百种miRNA，但关于猪的miRNA报道却较少，特别是猪胚胎生长发育中调节基因表达的miRNA研究更加欠缺。显然，系统地鉴定并分析研究猪胚胎的miRNA，阐明miRNA参与胚胎生长发育调控的分子机制十分的必要。
因此，本实验中我们使用Solexa测序以及生物信息学的方法分析猪33天胚胎（E33）中miRNA的序列以及相对表达情况。结果成功获得猪胚胎头部与内脏组织小分子RNA序列，并在胚胎头部组织中鉴定了75种已知miRNA与194种候选的miRNA，而在内脏组织中鉴定了76种已知miRNA与130种候选的miRNA，同时胚胎头部组织与内脏组织中共有50条miRNA的表达具有显著的差异。另外我们使用PicTar和TargetScan对高表达的miRNA进行靶基因预测，同时KEGG功能分析表明在头部与内脏组织中高度表达的miRNA参与神经、大脑、肌肉以及器官的发育。本结果为下一步研究miRNA调节猪胚胎神经系统以及器官生成等的分子调控机制奠定基础。
MicroRNA (miRNA) are a class of small, non-coding RNAs of~22nucleotidesin length that regulate gene expression by completely or incompletely binding to the3′-untranslated regions (UTR) of target mRNAs with degradation mRNA orinhabiting translation. It is now clear that miRNAs are involved in many physiologicaland biochemical processes, including proliferation, differentiation, and regulation ofgene expression during early embryonic development. The miRBase16.0(2010)shows that there are175,673,408, and1,048annotated miRNAs for Caenorhabditiselegans, Mus musculus, Rattus norvegicus, and Homo sapiens, respectively. However,there are only211miRNAs described for Sus scrofa. In particular, the full expressionpatterns of miRNAs regulating gene expression are still poorly understood inembryonic development.
Therefore, based on the construction of head region and organ region small RNAlibraries, we used the Solexa sequencing technology to identify the expression ofembryonic miRNAs by the researching embryo day33of pigs (E33). Meanwhile, wepredicted the target genes of abundant miRNAs in cDNA libraries by using ofbiological information software and preliminary study functions of them, thenanalyzing the different expression of the highly expressed miRNAs in head, organregions and adult tissues. The results show that:(1) After Solexa sequencing, the twolibraries of head and organ were successfully founded including11,617,079and11,458,732raw reads, in which11,400,040and11,256,616were high-quality smallRNA sequences, respectively.(2) Bioinformatics analysis of the distinct miRNAsidentified,75previously known miRNAs and194candidate miRNAs were identifiedin head library,76known miRNAs and130predicted candidate miRNAs wereidentified in organ library. In addition,20miRNAs were verified to express in bothhead and organ regions by Poly(A)-tailed PCR.(3)50known miRNAs were differentially expressed between head and organ libraries, in which31miRNAs inhead library were significantly higher than these in organ library,19miRNAs in organlibrary were significantly higher than these in head library and22miRNAs nodifferences.(4) The expression of10abundant miRNAs in head and organ regionwere analyzed by qRT-PCR, miR-9expression levels was highest in head region andmiR-1expression levels was highest in organ region. Moreover, the expression ofmiR-1, miR-103, and miR-140*were highest in muscle, brain, and lung, respectively.(5) Furthermore, we performed additional investigation for identifying the potentialtarget mRNAs of10highly expressed miRNAs using PicTar and TargetScan, finallyselecting total50target genes. KEGG Pathway analysis showed that the50targetgenes may be involved in the pig embryonic developmental formation of nervoussystem, limbs, anterior-posterior and dorsal-ventral pattern.
In this study, we successfully identified miRNAs of embryo day33of pigs andpredicted target genes of partial miRNAs, analyzing function of target genes. Ourresults provide valuable information for investigators interested in the regulation ofembryonic development in pigs and other animals and lay the foundation for furtherinvestigation the action and molecular mechanisms of miRNAs in pig embryonicdevelopment.
到目前为止，已经在人和小鼠等哺乳动物中鉴定出数百种miRNA，但关于猪的miRNA报道却较少，特别是猪胚胎生长发育中调节基因表达的miRNA研究更加欠缺。显然，系统地鉴定并分析研究猪胚胎的miRNA，阐明miRNA参与胚胎生长发育调控的分子机制十分的必要。
因此，本实验中我们使用Solexa测序以及生物信息学的方法分析猪33天胚胎（E33）中miRNA的序列以及相对表达情况。结果成功获得猪胚胎头部与内脏组织小分子RNA序列，并在胚胎头部组织中鉴定了75种已知miRNA与194种候选的miRNA，而在内脏组织中鉴定了76种已知miRNA与130种候选的miRNA，同时胚胎头部组织与内脏组织中共有50条miRNA的表达具有显著的差异。另外我们使用PicTar和TargetScan对高表达的miRNA进行靶基因预测，同时KEGG功能分析表明在头部与内脏组织中高度表达的miRNA参与神经、大脑、肌肉以及器官的发育。本结果为下一步研究miRNA调节猪胚胎神经系统以及器官生成等的分子调控机制奠定基础。
MicroRNA (miRNA) are a class of small, non-coding RNAs of~22nucleotidesin length that regulate gene expression by completely or incompletely binding to the3′-untranslated regions (UTR) of target mRNAs with degradation mRNA orinhabiting translation. It is now clear that miRNAs are involved in many physiologicaland biochemical processes, including proliferation, differentiation, and regulation ofgene expression during early embryonic development. The miRBase16.0(2010)shows that there are175,673,408, and1,048annotated miRNAs for Caenorhabditiselegans, Mus musculus, Rattus norvegicus, and Homo sapiens, respectively. However,there are only211miRNAs described for Sus scrofa. In particular, the full expressionpatterns of miRNAs regulating gene expression are still poorly understood inembryonic development.
Therefore, based on the construction of head region and organ region small RNAlibraries, we used the Solexa sequencing technology to identify the expression ofembryonic miRNAs by the researching embryo day33of pigs (E33). Meanwhile, wepredicted the target genes of abundant miRNAs in cDNA libraries by using ofbiological information software and preliminary study functions of them, thenanalyzing the different expression of the highly expressed miRNAs in head, organregions and adult tissues. The results show that:(1) After Solexa sequencing, the twolibraries of head and organ were successfully founded including11,617,079and11,458,732raw reads, in which11,400,040and11,256,616were high-quality smallRNA sequences, respectively.(2) Bioinformatics analysis of the distinct miRNAsidentified,75previously known miRNAs and194candidate miRNAs were identifiedin head library,76known miRNAs and130predicted candidate miRNAs wereidentified in organ library. In addition,20miRNAs were verified to express in bothhead and organ regions by Poly(A)-tailed PCR.(3)50known miRNAs were differentially expressed between head and organ libraries, in which31miRNAs inhead library were significantly higher than these in organ library,19miRNAs in organlibrary were significantly higher than these in head library and22miRNAs nodifferences.(4) The expression of10abundant miRNAs in head and organ regionwere analyzed by qRT-PCR, miR-9expression levels was highest in head region andmiR-1expression levels was highest in organ region. Moreover, the expression ofmiR-1, miR-103, and miR-140*were highest in muscle, brain, and lung, respectively.(5) Furthermore, we performed additional investigation for identifying the potentialtarget mRNAs of10highly expressed miRNAs using PicTar and TargetScan, finallyselecting total50target genes. KEGG Pathway analysis showed that the50targetgenes may be involved in the pig embryonic developmental formation of nervoussystem, limbs, anterior-posterior and dorsal-ventral pattern.
In this study, we successfully identified miRNAs of embryo day33of pigs andpredicted target genes of partial miRNAs, analyzing function of target genes. Ourresults provide valuable information for investigators interested in the regulation ofembryonic development in pigs and other animals and lay the foundation for furtherinvestigation the action and molecular mechanisms of miRNAs in pig embryonicdevelopment.
引文
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[3] BENTWICH I. Prediction and validation of microRNAs and their targets.[J] FEBS Lett,2005,579(26):5904-5910.
[4] KIM V N, NAM J W. Genomics of microRNA.[J] Trends Genet,2006,22(3):165-173.
[5] LIM L P, GLASNER M E, YEKTA S, et al. Vertebrate microRNA genes.[J] Science,2003,299(5612):1540.
[6] LEWIS B P, SHIH I H, JONES-RHOADES M W, et al. Prediction of mammalian microRNAtargets.[J] Cell,2003,115(7):787-798.
[7] LEE R C, FEINBAUM R L, AMBROS V. The C. elegans heterochronic gene lin-4encodessmall RNAs with antisense complementarity to lin-14.[J] Cell,1993,75(5):843-854.
[8] REINHART B J, SLACK F J, BASSON M, et al. The21-nucleotide let-7RNA regulatesdevelopmental timing in Caenorhabditis elegans.[J] Nature,2000,403(6772):901-906.
[9] GIRALDEZ A J, CINALLI R M, GLASNER M E, et al. MicroRNAs regulate brainmorphogenesis in zebrafish.[J] Science,2005,308(5723):833-838.
[10] ZHAO Y, SAMAL E, SRIVASTAVA D. Serum response factor regulates a muscle-specificmicroRNA that targets Hand2during cardiogenesis.[J] Nature,2005,436(7048):214-220.
[11] XU P, VERNOOY S Y, GUO M, et al. The Drosophila microRNA Mir-14suppresses celldeath and is required for normal fat metabolism.[J] Curr Biol,2003,13(9):790-795.
[12] ESAU C, KANG X, PERALTA E, et al. MicroRNA-143regulates adipocyte differentiation.[J] J Biol Chem,2004,279(50):52361-52365.
[13] ESAU C, DAVIS S, MURRAY S F, et al. miR-122regulation of lipid metabolism revealed byin vivo antisense targeting.[J] Cell Metab,2006,3(2):87-98.
[14] GLAZOV E A, COTTEE P A, BARRIS W C, et al. A microRNA catalog of the developingchicken embryo identified by a deep sequencing approach.[J] Genome Res,2008,18(6):957-964.
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