icroRNA介导的IGF1基因沉默对鹿茸软骨细胞增殖的影响
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- 英文论文题名：Effect of microRNA-mediated IGF1 gene silencing on the proliferation of deer antler cartilage cells
- 论文作者：李婷 ; 李沐 ; 孟星宇 ; 刘宁 ; 胡薇
- 英文论文作者：Li Ting ; Li Mu ; Meng Xingyu ; Liu Ning ; Hu Wei ; College of Life Sciences ; Jilin Agriculture University
- 年：2016
- 作者机构：吉林农业大学生命科学学院;
- 论文关键词：MicroRNA ; 胰岛素样生长因子1 ; 基因沉默 ; 鹿茸软骨细胞
- 英文论文关键词：MicroRNA ; insulin-like growth factor 1 ; gene silencing ; deer antler cartilage cells
- 会议召开时间：2016-06-01
- 会议录名称：“十二五” 鹿科学研究进展
- 语种：中文
- 分类号：S825
- 学会代码：ZXMY
- 学会名称：中国畜牧业协会
- 页数：9
- 文件大小：561k
- 原文格式：O
摘要
【目的】构建靶向梅花鹿胰岛素样生长因子1(IGF1)基因的microRNA真核表达载体,研究microRNA介导的IGF1基因沉默对鹿茸软骨细胞增殖的影响。【方法】根据IGF1 mRNA序列设计并合成4对pre-microRNA前体片段,定向克隆于pcDNA6.2-GW/EmGFP-miR真核表达载体中,构建重组质粒pcDNA6.2-Gw/EmGFPIGF1-miR-1,pcDNA6.2-Gw/EmGFP-IGF1-miR-2,pcDNA6.2-Gw/EmGFPIGF1-miR-3和pcDNA6.2-Gw/EmGFP-IGFl-miR-4。测序分析插入序列的完整性;将重组质粒转染鹿茸软骨细胞,利用相对荧光定量PCR技术检测IGF1基因mRNA的表达量;在此基础上选择表达量最低的pcDNA6.2-Gw/EmGFP-IGF1-miR重组质粒转染鹿茸软骨细胞,Western blotting分析IGF1的蛋白表达水平,MTT法和流式细胞仪检测重组质粒对鹿茸软骨细胞体外增殖和细胞周期的影响。【结果】测序结果显示,构建的4组重组质粒插入片段的碱基序列完全正确。转染pcDNA6.2-Gw/EmGFPIGF1-miR重组质粒后,鹿茸软骨细胞中IGF1基因mRNA的表达水平均有所下降,其中转染pcDNA6.2-Gw/EmGFP-IGF1-miR-2组的表达水平最低,筛选出pcDNA6.2-Gw/EmGFP-IGF1-miR-2组为最佳干扰靶点质粒。与对照组比较,IGF1的蛋白表达水平降低;pcDNA6.2-Gw/EmGFP-IGF1-miR-2转染组的鹿茸软骨细胞的增殖受到抑制,细胞周期S期细胞百分比减少,表明鹿茸软骨细胞停滞在G_0/G_1期。【结论】梅花鹿IGF1的表达水平受miRNA的调控,表明在鹿茸快速生长过程中miRNA具有重要的调控作用。
【Objective】To construct Cervus nippon Insulin- like growth factor 1(IGF1) gene microRNA eukaryotic expression vector and research the effect of microRNA-midiated IGF1 gene silenceing on the proliferation of antler cartilage cells.【Method】According to the sequence of IGFl mRNA,four pairs of pre- microRNA were designed and synthesized,then cloned into the GFP reporter pcDNA6.2- GW/EmGFP-miR vector,the recombinant plasmid were pcDNA6.2- Gw/EmGFP- IGFl- miR- 1,pcDNA6.2- Gw/EmGFP-IGFl- miR-2,pcDNA6.2- Gw/Em GFP- IGFl- miR- 3 和 pcDNA6.2- Gw/EmGFP-IGFl- miR- 4.The integrity of the insert fragrents was verified through sequencing analysis.Then transfected into the antler cartilage cells.The IGFl gene expression levels was detected by real- time PCR,and the protein levels was detected by western blotting respectively,then chose the lowest expression level of pcDNA6.2- Gw/EmGFP- IGFl- miR recombinant plasmid and transfected the recombinant into the cartilage cells.The cells proliferation and cell cycle were measured by MTT and FCM.【Result】The sequence analysis showed that the sequences of insert fragments in microRNA expression recombinants completely correct.The expression of IGFl mRNA in antler cartilage cells transfected was down- regulated,the expression level of the pcDNA6.2- Gw/EmGFP- IGF1- miR- 2 group was the lowest,and the pcDNA6.2- Gw/EmGFP- IGFl- miR- 2 was selected the best interference target plasmid.Compared with the negative control group,the protein expression in antler cartilage cells transfected was down- regulated,the cells proliferation of pcDNA6.2- Gw/EmGFP- IGF1-miR- 2 transfected group was inhibitied,and the cells percentage of S phase in the cell cycle were reduced.The result indicated that the deer antler cartilage cells arrested in the G_0/G_1phase.【Conclusion】The expression level of IGFl in Cervus nippon is regulated by the miRNA,and displayed that the miRNA has an important role in the antler growth process.
【Objective】To construct Cervus nippon Insulin- like growth factor 1(IGF1) gene microRNA eukaryotic expression vector and research the effect of microRNA-midiated IGF1 gene silenceing on the proliferation of antler cartilage cells.【Method】According to the sequence of IGFl mRNA,four pairs of pre- microRNA were designed and synthesized,then cloned into the GFP reporter pcDNA6.2- GW/EmGFP-miR vector,the recombinant plasmid were pcDNA6.2- Gw/EmGFP- IGFl- miR- 1,pcDNA6.2- Gw/EmGFP-IGFl- miR-2,pcDNA6.2- Gw/Em GFP- IGFl- miR- 3 和 pcDNA6.2- Gw/EmGFP-IGFl- miR- 4.The integrity of the insert fragrents was verified through sequencing analysis.Then transfected into the antler cartilage cells.The IGFl gene expression levels was detected by real- time PCR,and the protein levels was detected by western blotting respectively,then chose the lowest expression level of pcDNA6.2- Gw/EmGFP- IGFl- miR recombinant plasmid and transfected the recombinant into the cartilage cells.The cells proliferation and cell cycle were measured by MTT and FCM.【Result】The sequence analysis showed that the sequences of insert fragments in microRNA expression recombinants completely correct.The expression of IGFl mRNA in antler cartilage cells transfected was down- regulated,the expression level of the pcDNA6.2- Gw/EmGFP- IGF1- miR- 2 group was the lowest,and the pcDNA6.2- Gw/EmGFP- IGFl- miR- 2 was selected the best interference target plasmid.Compared with the negative control group,the protein expression in antler cartilage cells transfected was down- regulated,the cells proliferation of pcDNA6.2- Gw/EmGFP- IGF1-miR- 2 transfected group was inhibitied,and the cells percentage of S phase in the cell cycle were reduced.The result indicated that the deer antler cartilage cells arrested in the G_0/G_1phase.【Conclusion】The expression level of IGFl in Cervus nippon is regulated by the miRNA,and displayed that the miRNA has an important role in the antler growth process.
引文
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[2]Suttie J M,Fennessy P F,Gluckman P D,et al.Elevated plasma IGF 1 levels in stags prevented from growing antlers[J].Endocrinology,1988,122(6):3005-3007.
[3]Barling P M,Lai A K,Nicholson L F.Distribution of EGF and its receptor in growing red deer antler[J].Cell Biol Int,2005,29(3):229-236.
[4]Price J,Allen S.Exploring the mechanisms regulating regeneration of deer antlers[J].Philos Trans R Soc Lond B Biol Sci,2004,359:809-22.
[5]Bartel D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116:281-297.
[6]Zhang B,Pan X,Anderson T A.MicroRNA:a new player in stem cells[J].J Cell Physiol,2006,209(2):266-269.
[7]Hwang H W,Mendell J T.MicroRNAs cell proliferation,cell death,and tumorigenesis[J].Br J Cancer,2006,94(6):776-780.
[8]Vancheret H.Post-transcriptional small RNA pathways in plants;mechanisms and regulations[J].Genes Dev,2006,20(7):759-771.
[9]Sheng Y,Engstrom P G,Lenhard B.Mammalian microRNA prediction through a support vector machine model of sequence and structure[J].PLoS One,2007,2(9):e946.
[10]Yu X Y,Song Y H,Geng Y J,et al.Glucose induces apoptosis of cardiomyocytes via micro RNA-1 and IGF-1[J].Biochem Biophys Res Commun,2008,376(3):548-552.
[11]Mount J G,Muzylak M,Allen S,et al.Evidence that the canonical Wnt signalling pathway regulates deer antler regeneration[J].Dev Dyn 2006;235(5):1390-1399.
[12]Li C,Suttie J M.Tissue collection methods for antler research[J].Eur J Morphol,2003,41(1):23-30.
[13]Moazed D.Small RNAs in transcriptional gene silencing and genome defence[J].Nature,2009,457(7228):413-420.
[14]Lee Y,Kim M,Han J,et al.MicroRNA genes are transcribed by RNA polymerase 11[J].EMBO J,2004,23(20):4 051-4 060.
[15]Zeng Y.Principles of micro-RNA production and maturation[J].Oncogene,2006,25(46):6156-6162.
[16]Li X,Carthew R W.A microRNA mediates EGF receptor signaling and promotes photoreceptor differentiation in the Drosophila eye[J].Cell,2005,123(7):1267-1277.