microRNA介导的IGF1基因沉默对鹿茸软骨细胞增殖的影响

Effect of microRNA-mediated IGF1 gene silencing on the proliferation of deer antler cartilage cells

【目的】构建靶向梅花鹿胰岛素样生长因子1基因(IGF1)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IGF1mRNA序列设计并合成4对premicroRNA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/EmGFP-IGF1-miR-1、pcDNA6.2-Gw/EmGFP-IGF1-miR-2、pcDNA6.2-Gw/EmGFP-IGF1-miR-3和pcDNA6.2-Gw/EmGFP-IGF1-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IGF1mRNA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蛋白的表达水平,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/EmGFP-IGF1-miR重组质粒后,鹿茸软骨细胞中IGF1mRNA的表达水平均有所下降,其中转染重组质粒pcDNA6.2-Gw/EmGFP-IGF1-miR-2组的表达水平最低,因而筛选重组质粒pcDNA6.2-Gw/EmGFP-IGF1-miR-2为最佳干扰质粒。与对照组相比,IGF1蛋白的表达水平降低;重组质粒pcDNA6.2-Gw/EmGFP-IGF1-miR-2转染组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G0/G1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】This study aimed to construct microRNA eukaryotic expression vector of Cervus nippon insulin-like growth factor 1（IGF1）gene and investigate the effect of microRNA-midiated IGF1 gene silenceing on the proliferation of antler cartilage cells.【Method】According to the sequence of IGF1 mRNA,four pairs of pre-microRNA were designed and synthesized before being cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector to construct the recombinant plasmids：pcDNA6.2-Gw/EmGFP-IGF1-miR-1,pcDNA6.2-Gw/EmGFP-IGF1-miR-2,pcDNA6.2-Gw/EmGFP-IGF1-miR-3,and pcDNA6.2-Gw/EmGFP-IGF1-miR-4.The integrity of the insert fragrents was verified through sequencing analysis before the recombinant plasmids were transfected into the antler cartilage cells.IGF1 gene expression levels were detected by Real-time PCR,and the protein levels were detected by Western blotting.Then,the lowest expression level of pcDNA6.2-Gw/EmGFP-IGF1-miR recombinant plasmid was chosen and transfected 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 were completely correct.The expression of IGF1 mRNA in transfected antler cartilage cells was decreased.The expression level of the pcDNA6.2-Gw/EmGFP-IGF1-miR-2 group was the lowest,and the pcDNA6.2-Gw/EmGFP-IGF1-miR-2 was the best interference plasmid.Compared with the negative control group,the protein expression in transfected antler cartilage cells was down-regulated,the cell proliferation of pcDNA6.2-Gw/EmGFP-IGF1-miR-2 transfected group was inhibitied,and the ratio of cells with S phase in the cell cycle reduced.The result indicated that the deer antler cartilage cells were in the steady state of G0/G1 phase.【Conclusion】The expression level of IGF1 in Cervus nippon is regulated by miRNA, and miRNA plays an important role in antler growth process.