Hu Wei, Li Mu, Hu Rui, Li Ting, Meng Xingyu
1 Department of Biochemistry and Molecular Biology, College of Life Sciences, Jilin Agriculture University , Changchun, Jilin Province, China .
DNA Cell Biol. 2015 Apr;34(4):282-9. doi: 10.1089/dna.2014.2421. Epub 2015 Mar 10.
Insulin-like growth factor-1 (IGF-1) is a multipromoter gene that has complex biological functions and plays an important role in Chinese sika deer antler cell differentiation and proliferation. microRNAs and their roles in deer antler growth have attracted much attention. In the present study, to investigate the effect of microRNAs on the regulation of IGF-1 during the rapid growth of antlers, miRNA GeneChip analysis and TargetScan Human software were used to screen microRNAs that bind to the 3' untranslated region (3'UTR) of IGF-1. The results indicated that a significantly differential expression of miR-18b was observed in cartilage and mesenchymal of antler tip tissue and the presence of miR-18b-binding sites within the IGF-1 3'UTR. A miR-18b mimic was then transfected into antler cartilage cells to overexpress miR-18b and the expression levels were quantified by real-time PCR. Real-time PCR showed that the expression level of miR-18b in transfected cells was significantly increased compared with the control group (p<0.01). Dual luciferase assays revealed that miR-18b decreased the fluorescence value of the luciferase reporter gene in the group transfected with the wild-type vector of IGF-1 3'UTR. In contrast, the relative luciferase activity in the group transfected with the mutant vector of IGF-1 3'UTR did not change obviously. MTT assays and cell cycle analyses confirmed that overexpression of the miR-18b mimic inhibited the proliferation of cartilage cells. In contrast, transfection of a miR-18b inhibitor increased the cell proliferation rate. Furthermore, Western blot analyses revealed that overexpression of miR-18b mimics downregulated the protein levels of IGF-1, while IGF-1 expression increased after transfection of miR-18b inhibitors. Taken together, our findings show that miR-18b is a potentially novel target in deer antler cell proliferation. miR-18b may modulate IGF-1 expression of sika deer antler.
胰岛素样生长因子-1（IGF-1）是一种多启动子基因，具有复杂的生物学功能，在中国梅花鹿鹿茸细胞分化和增殖中发挥重要作用。微小RNA及其在鹿茸生长中的作用已引起广泛关注。在本研究中，为了探究微小RNA在鹿茸快速生长过程中对IGF-1的调控作用，使用miRNA基因芯片分析和TargetScan Human软件筛选与IGF-1的3'非翻译区（3'UTR）结合的微小RNA。结果表明，在鹿茸顶端组织的软骨和间充质中观察到miR-18b的显著差异表达，并且在IGF-1的3'UTR内存在miR-18b结合位点。然后将miR-18b模拟物转染到鹿茸软骨细胞中以过表达miR-18b，并通过实时PCR对表达水平进行定量。实时PCR显示，与对照组相比，转染细胞中miR-18b的表达水平显著增加（p<0.01）。双荧光素酶测定表明，miR-18b降低了用IGF-1 3'UTR野生型载体转染组中荧光素酶报告基因的荧光值。相反，用IGF-1 3'UTR突变载体转染组中的相对荧光素酶活性没有明显变化。MTT测定和细胞周期分析证实，miR-18b模拟物的过表达抑制了软骨细胞的增殖。相反，转染miR-18b抑制剂可提高细胞增殖率。此外，蛋白质印迹分析表明，miR-18b模拟物的过表达下调了IGF-1的蛋白质水平，而转染miR-18b抑制剂后IGF-1表达增加。综上所述，我们的研究结果表明，miR-18b是鹿茸细胞增殖中一个潜在的新靶点。miR-18b可能调节梅花鹿鹿茸的IGF-1表达。
