长链非编码RNA在不同受孕能力精子中差异表达的初步分析
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摘要
目的:通过对两组不同受孕能力的精子差异长链非编码RNA(lnc RNA)的分析,寻找与受孕能力相关的lnc RNA,探讨其在受精过程中可能的作用。方法:各取5份高、低受孕力的精液标本,提取大RNA,建c DNA库并测序。应用生物信息学方法,组装并预测lnc RNA。采取NOIseq方法筛选两组间有差异表达的lnc RNA,绘制箱图和火山图;运用Cluster软件进行表达模式聚类分析。采用基因本体(GO)及Pathway分析差异表达的lnc RNA功能分布,利用lnc RNA-mRNA互作分析和上下游顺式作用元件的交集分析,来预测lnc RNA的功能。结果:预测到471 615个lnc RNA,其中新lnc RNA 463 596个,已知lnc RNA 8 019个。差异表达分析结果显示,有4 052个lnc RNA存在显著差异,其中上调的985个,下调的3 067个。箱图和火山图显示出两组间存在明显的差异,表达模式聚类分析显示出两组间存在明显的差异。GO功能分类注释得出,差异表达的lnc RNA主要参与新陈代谢、生物调节、细胞膜及细胞器形成、蛋白核酸结合等; Pathway分析显示,差异表达的lnc RNA主要与运输分解代谢、细胞运动、信号分子相互作用、信号转导、以及发育和免疫系统信号通路等有关。5个lnc RNA的预测功能表明,它们可能与受精过程中的精子运动、顶体反应、信号转导相关。结论:差异表达的lnc RNA可能在受精过程中有重要作用,可能成为评估精子质量的生物标志物。
Objective:To search for conception capacity-related long non-coding RNAs(lnc RNA) and explore their possible roles in fertilization.Methods:We obtained 10 semen samples,5 of high and the other 5 of low fertilizing ability,extracted large RNAs,established a c DNA library,and performed RNA sequencing with the Hi Seq 2000 sequencing system.Using the bioinformatics method,we assembled and predicted lnc RNAs,screened differentially expressed genes between the two groups by NOIseq,analyzed the lnc RNAs with the box plot and volcano plot,and determined their expression patterns by hierarchical cluster analysis.We examined the functional classification of differentially expressed lnc RNAs by pathway and gene ontology(GO) enrichment and predicted those of some lnc RNAs by lnc RNA-mRNA interaction analysis and intersection analysis with up-and down-stream cis-acting elements.Results:A total of 147 1615 lnc RNAs were identified in all the semen samples,including 463 596 novel ones and 8 019 known ones,with 4 052 differentially expressed lnc RNAs,985 upregulated and the other 3 067 downregulated.Box plot and volcano plot filtering analyses showed statistically significant differences in the expressions of the lnc RNAs between the two groups,and so did hierarchical cluster analysis.GO functional annotations manifested the involvement of the differentially expressed lnc RNAs in the metabolic process,biological regulation,membrane and organelle formation,and protein-nucleotide binding.Pathway analysis showed that the differentially expressed lnc RNAs were related to transport and catabolism,cell motility,signaling molecular interactions,signaling transduction,and signaling pathways in the development and immune systems.The functions of the 5 lnc RNAs predicted were shown to be associated with sperm motility,acrosomal reaction and signal transduction during fertilization.Conclusion:Differentially expressed lnc RNAs may play an important role in fertilization and become biomarkers for the assessment of sperm quality.
Objective:To search for conception capacity-related long non-coding RNAs(lnc RNA) and explore their possible roles in fertilization.Methods:We obtained 10 semen samples,5 of high and the other 5 of low fertilizing ability,extracted large RNAs,established a c DNA library,and performed RNA sequencing with the Hi Seq 2000 sequencing system.Using the bioinformatics method,we assembled and predicted lnc RNAs,screened differentially expressed genes between the two groups by NOIseq,analyzed the lnc RNAs with the box plot and volcano plot,and determined their expression patterns by hierarchical cluster analysis.We examined the functional classification of differentially expressed lnc RNAs by pathway and gene ontology(GO) enrichment and predicted those of some lnc RNAs by lnc RNA-mRNA interaction analysis and intersection analysis with up-and down-stream cis-acting elements.Results:A total of 147 1615 lnc RNAs were identified in all the semen samples,including 463 596 novel ones and 8 019 known ones,with 4 052 differentially expressed lnc RNAs,985 upregulated and the other 3 067 downregulated.Box plot and volcano plot filtering analyses showed statistically significant differences in the expressions of the lnc RNAs between the two groups,and so did hierarchical cluster analysis.GO functional annotations manifested the involvement of the differentially expressed lnc RNAs in the metabolic process,biological regulation,membrane and organelle formation,and protein-nucleotide binding.Pathway analysis showed that the differentially expressed lnc RNAs were related to transport and catabolism,cell motility,signaling molecular interactions,signaling transduction,and signaling pathways in the development and immune systems.The functions of the 5 lnc RNAs predicted were shown to be associated with sperm motility,acrosomal reaction and signal transduction during fertilization.Conclusion:Differentially expressed lnc RNAs may play an important role in fertilization and become biomarkers for the assessment of sperm quality.
引文
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[2]Rajender S,Avery K,Agarwal A.Epigenetics,spermatogenesis and male infertility.Mutat Res,2012,727(3):62-71.
[3]Dada R,Kumar M,Jesudasan R,et al.Epigenetics and its role in male infertility.J Assist Reprod Genet,2012,29(3):213-223.
[4]张其杰,许洁,秦超.男性不育症的表观遗传学研究进展.中华男科学杂志,2017,23(6):566-569.
[5]Wu Z,Liu X,Liu L,et al.Regulation of lncRNA expression.Cell Mol Biol Lett,2014,19(4):561-575.
[6]徐晨,宋宁.精子发生过程中的表观遗传学调控.中华男科学杂志,2014,20(5):387-391.
[7]夏静,郑英.生精细胞发育相关lncRNA的研究进展.中国男科学杂志,2016,30(9):63-66.
[8]Zhang X,Gao F,Fu J,et al.Systematic identification and characterization of long non-coding RNAs in mouse mature sperm.PLo SOne.2017,12(3):e0173402.
[9]Johnson GD,Lalancette C,Linnemann AK,et al.The sperm nucleus:Chromatin,RNA,and the nuclear matrix.Reproduction,2011,141(1):21-36.
[10]Sendler E,Johnson GD,Mao S,et al.Stability,delivery and functions of sperm RNAs at fertilization.Nucleic Acids Res,2013,41(7):4104-4117.
[11]Wichman L,Somasundaram S,Breindel C,et al.Dynamic expression of long noncoding RNAs reveals their potential roles in spermatogenesis and fertility.Biol Reprod,2017,97(2):313-323.
[12]Wu R,Su Y,Wu H,et al.Characters,functions and clinical perspectives of long non-coding RNAs.Mol Genet Genomics,2016,291(3):1013-1033.
[13]Selvaraju S,Parthipan S,Somashekar L,et al.Current status of sperm functional genomics and its diagnostic potential of fertility in bovine(Bos taurus).Syst Biol Reprod Med,2018,64(6):484-501.
[14]Kataruka S,Akhade VS,Kayyar B,et al.Mrhl long noncoding RNA mediates meiotic commitment of mouse spermatogonial cells by regulating Sox8 expression.Mol Cell Biol,2017,37(14):pii:e00632-16.
[15]Jiang GJ,Zhang T,An T,et al.Differential expression of long noncoding RNAs between sperm samples from diabetic and nondiabetic mice.PLo S One,2016,11(4):e0154028.