空间飞行MG63细胞重力敏感新miRNA HN22的鉴定与功能分析
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摘要
目的筛选空间飞行引起的MG63成骨细胞差异表达新miRNA并进行功能分析。方法对空间飞行后的MG63成骨细胞small RNA(sRNA)进行测序及生物信息学分析,得到空间飞行导致显著变化的新miRNA。利用地面回转模拟微重力效应实验验证其表达变化情况;采用miRNA转染分析其对成骨细胞分化的影响;采用多种miRNA靶基因预测软件综合预测其靶基因并检测其潜在靶基因的表达情况;通过GO注释和Panther pathway进行靶基因功能分析。结果新miRNA HN22在空间飞行和地面回转模拟微重力条件下均发生显著表达变化;HN22转染显著提高成骨分化相关基因表达。靶基因预测结果分析显示HN22可影响成骨细胞Wnt信号通路与钙黏蛋白信号通路。Wnt信号通路中的Wnt5a与HN22的表达趋势相反,是其潜在的靶基因。结论HN22可响应微重力变化,并靶向Wnt信号通路中的Wnt5a与钙黏蛋白信号通路影响成骨细胞分化。
Objective To screen and identify differential expression of novel miRNAs during spaceflight and analyze their functions.Methods Through the small RNA sequencing and bioinformatics analysis of spaceflight osteoblasts(MG63),differentially expressed novel miRNAs were obtained and their expressions were verified under simulated microgravity.Then the influence of novel miRNA on osteoblasts differentiation were studied by miRNA transfection.Finally,functional analysis of the predicted target genes of novel miRNA was conducted by GO annotation and Panther pathway analysis.Results The novel miRNA HN22 was differentially expressed during space flight and simulated microgravity.HN22 transfection significantly enhanced expression of osteogenic differentiation-related genes and Wnt5 a.Bioinformatic functional analysis showed that HN22 affected Wnt signaling pathway and cadherin signaling pathway in osteoblasts.The expression of HN22 and Wnt5 ashowed negative correlation under simulated microgravity condition.Conclusion HN22 can respond to microgravity and regulate osteoblast differentiation through Wnt signaling pathway(Wnt5 a)and cadherin signaling pathway.
Objective To screen and identify differential expression of novel miRNAs during spaceflight and analyze their functions.Methods Through the small RNA sequencing and bioinformatics analysis of spaceflight osteoblasts(MG63),differentially expressed novel miRNAs were obtained and their expressions were verified under simulated microgravity.Then the influence of novel miRNA on osteoblasts differentiation were studied by miRNA transfection.Finally,functional analysis of the predicted target genes of novel miRNA was conducted by GO annotation and Panther pathway analysis.Results The novel miRNA HN22 was differentially expressed during space flight and simulated microgravity.HN22 transfection significantly enhanced expression of osteogenic differentiation-related genes and Wnt5 a.Bioinformatic functional analysis showed that HN22 affected Wnt signaling pathway and cadherin signaling pathway in osteoblasts.The expression of HN22 and Wnt5 ashowed negative correlation under simulated microgravity condition.Conclusion HN22 can respond to microgravity and regulate osteoblast differentiation through Wnt signaling pathway(Wnt5 a)and cadherin signaling pathway.
引文
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[2] Sibonga JD.Spaceflight-induced bone loss:is there an osteoporosis risk[J].Current Osteoporosis Reports,2013,11(2):92-98.
[3] Smith SM,Heer M,Shackelford LC,et al.Bone metabolism and renal stone risk during international space station missions[J].Bone,2015,81:712-720.
[4] Poole KE,Warburton EA,Reeve J.Rapid long-term bone loss following stroke in a man with osteoporosis and atherosclerosis[J].Osteoporos Int,2005,16(3):302-305.
[5] Valenti MT,Dalle Carbonare L,Mottes M.Osteogenic differentiation in healthy and pathological conditions[J].International Journal of Molecular Sciences,2016,18(1):14.
[6] Amiel J,De Pontual L,Henrion-Caude A.MiRNA,Development and disease[J].Advances In Genetics,2012,80:1-36.
[7] Lian JB,Stein GS,Van Wijnen AJ,et al.MicroRNA control of bone formation and homeostasis[J].Nature Reviews Endocrinology,2012,8(4):212-227.
[8] Hassan MQ,Tye CE,Stein GS,et al.Non-coding RNAs:Epigenetic regulators of bone development and homeostasis[J].Bone,2015,81:746-756.
[9] Li B.MicroRNA regulation in osteogenic and adipogenic differentiation of bone mesenchymal stem cells and its application in bone regeneration[J].Current Stem Cell Research&Therapy,2018,13(1):26-30
[10] Chen Z,Zhang Y,Liang C,et al.Mechanosensitive miRNAs and bone formation[J].International Journal of Molecular Sciences,2017,18(8):1684
[11]陈圣菊,王飞,李文炯,等.小鼠运动敏感型血清miRNA的筛选[J].航天医学与医学工程,2018,31(2):224-228.Chen SJ,Wang F,Li WJ,et al.Screening of serum exercise-sensitive miRNA in mice[J].Space Medicine&Medical Engineering,2018,31(2):224-228.
[12] Wang X,Guo B,Li Q,et al.Mir-214targets ATF4to inhibit bone formation[J].Nature Medicine,2013,19(1):93-100.
[13] Wang H,Sun Z,Wang Y,et al.MiR-33-5p,A novel mechano-sensitive microRNA promotes osteoblast differentiation by targeting HMGA2[J].Scientific Reports,2016,6:23170.
[14] Sun Z,Cao X,Hu Z,et al.MiR-103inhibits osteoblast proliferation mainly through suppressing Cav1.2expression in simulated microgravity[J].Bone,2015,76:121-128.
[15] Zuo B,Zhu J,Li J,et al.MicroRNA-103afunctions as a mechanosensitive microRNA to inhibit bone formation through targeting Runx2[J].J Bone Miner Res,2015,30(2):330-345.
[16] Hu Z,Wang Y,Sun Z,et al.MiRNA-132-3p inhibits osteoblast differentiation by targeting Ep300in simulated microgravity[J].Scientific Reports,2015,5:18655.
[17] Liu S,Li D,Li Q,et al.MicroRNAs of bombyx mori identified by solexa sequencing[J].BMC Genomics,2010,11:148.
[18] Lewis BP,Burge CB,Bartel DP.Conserved seed pairing,often flanked by adenosines,indicates that thousands of human genes are microRNA targets[J].Cell,2005,120(1):15-20.
[19] Betel D,Koppal A,Agius P,et al.Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites[J].Genome Biology,2010,11(8):R90.
[20] Betel D,Wilson M,Gabow A,et al.The microRNA.org resource:targets and expression[J].Nucleic Acids Research,2008,36(S1):D149-D153.
[21] Rehmsmeier M,Steffen P,Hochsmann M,et al.Fast and effective prediction of microRNA/target duplexes[J].RNA(New York,Ny),2004,10(10):1507-1517.
[22] Mi H,Thomas P.Panther pathway:an ontology-based pathway database coupled with data analysis tools[J].Methods in Molecular Biology,2009,563:123-140.
[23] Huntley RP,Harris MA,Alam-Faruque Y,et al.A method for increasing expressivity of gene ontology annotations using a compositional approach[J].BMC Bioinformatics,2014,15:155.
[24] Sun Z,Cao X,Zhang Z,et al.Simulated microgravity inhibits l-type calcium channel currents partially by the upregulation of miR-103in MC3T3-E1osteoblasts[J].Scientific Reports,2015,5:8077.
[25] Govoni KE.Insulin-like growth factor-i molecular pathways in osteoblasts:potential targets for pharmacological manipulation[J]. Current Molecular Pharmacology,2012,5(2):143-152.
[26] Williams JN,Kambrath AV,Patel RB,et al.Inhibition of CaMKK2enhances fracture healing by stimulating indian hedgehog signaling and accelerating endochondral ossification[J].Journal of Bone and Mineral Research,2018,33(5):930-944
[27] Renaud J,Chedotal A.Time-lapse analysis of tangential migration in Sema6aand Plexina2knockouts[J].Molecular and Cellular Neurosciences,2014,63:49-59.
[28] Maeda K,Kobayashi Y,Udagawa N,et al.Wnt5a-ror2signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis[J].Nature Medicine,2012,18(3):405-412.
[29] Nemoto E,Ebe Y,Kanaya S,et al.Wnt5asignaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis[J].Biochemical and Biophysical Research Communications,2012,422(4):627-632.