Department of Biochemistry and Molecular Biology, College of Life Sciences, Jilin Agriculture University, Changchun, Jilin 130118, P.R. China.
Mol Med Rep. 2013 Aug;8(2):523-8. doi: 10.3892/mmr.2013.1515. Epub 2013 Jun 10.
Insulin‑like growth factor‑1 (IGF‑1) is critical in the proliferation and regeneration of Chinese sika deer antler cells. The regulation of IGF‑1 is complex and remains unclear. In the present study, miRNA GeneChip® and TargetScan Human software were used to identify microRNA‑1 (miR‑1), which binds to the 3'-untranslated region (3'UTR) of IGF‑1. An miR‑1 mimic was transfected into antler cartilage cells in order to induce the overexpression of miR‑1. The expression levels of miR‑1 in antler cartilage cells were determined by quantitative polymerase chain reaction (qPCR). A high‑throughput luciferase reporter screen was used to demonstrate the potential regulation of IGF‑1 by miR‑1. miR‑1 suppressed the luciferase activity of the pmiR‑IGF‑1 by ~50% compared with the negative control (NC). An MTT assay and cell cycle analyses confirmed that the overexpression of miR‑1 significantly inhibited the proliferation of cartilage cells (P<0.05). Furthermore, western blot analysis revealed that overexpressed miR‑1 downregulated the protein levels of IGF‑1. The 3'UTR of IGF‑1 was found to have an miR‑1 combining site, which allowed the inhibition of IGF‑1 protein expression, as demonstrated by a luciferase reporter assay, and miR‑1 was shown to be an important and effective means of regulating IGF-1. In conclusion, miR‑1 downregulated the protein expression of IGF‑1 by directly targeting the 3'UTR of IGF‑1. miR‑1 may be crucial for inhibiting the proliferation of deer antler cartilage cells. Our findings provided the evidence for the first time that miR‑1 directly regulates the expression of IGF‑1 by directly targeting its 3'UTR.
胰岛素样生长因子 1（IGF-1）在中华麋鹿鹿角细胞的增殖和再生中起关键作用。IGF-1 的调节非常复杂，目前仍不清楚。本研究使用 miRNA GeneChip®和 TargetScan Human 软件鉴定出 microRNA-1（miR-1），它可以与 IGF-1 的 3'非翻译区（3'UTR）结合。将 miR-1 模拟物转染到鹿角软骨细胞中，以诱导 miR-1 的过表达。通过定量聚合酶链反应（qPCR）测定鹿角软骨细胞中 miR-1 的表达水平。高通量荧光素酶报告基因筛选用于证明 miR-1 对 IGF-1 的潜在调节作用。与阴性对照（NC）相比，miR-1 抑制了 pmiR-IGF-1 的荧光素酶活性约 50%。MTT 测定和细胞周期分析证实，miR-1 的过表达显著抑制了软骨细胞的增殖（P<0.05）。此外，Western blot 分析表明，过表达的 miR-1 下调了 IGF-1 的蛋白水平。IGF-1 的 3'UTR 具有 miR-1 结合位点，允许通过荧光素酶报告基因测定抑制 IGF-1 蛋白表达，并且 miR-1 被证明是调节 IGF-1 的重要有效手段。总之，miR-1 通过直接靶向 IGF-1 的 3'UTR 下调 IGF-1 的蛋白表达。miR-1 可能对抑制鹿鹿角软骨细胞的增殖至关重要。我们的研究结果首次提供了证据表明，miR-1 通过直接靶向其 3'UTR 直接调节 IGF-1 的表达。
