Effects of microRNA-mediated IGF-1 gene silencing on telomerase activity and cell proliferation in deer antler cells

This study aimed to assess deer antler cell proliferation and telomerase activity after transfection with IGF-1microRNA eukaryotic expression recombinants. Four pairs of pre-microRNA were designed and synthesized based on the IGF-1 mRNA sequence, cloned into the pcDNA6.2-GW/EmGFPmiR eukaryotic vector and transfected into deer antler cells. IGF-1 expression after transfection with recombinants was measured by Real-time PCR and Western blotting. Telomerase activity in deer antler cells was assessed by TRAP-PCR-EB. Finally, the effects of IGF-1 silencing on cell proliferation and cell cycle were determined by MTT assay and flow cytometric analysis, respectively. The oligonucleotide sequences for microRNA expression were successfully inserted, and IGF-1 expression levels were significantly decreased in the pIGF1-miR-2 recombinant group in comparison with negative control and normal cells (P < 0.01). The pIGF-1-miR-2 recombinant was selected as the optimal interfering target. We found that IGF-1 silencing by pIGF-1-miR-2 significantly reduced telomerase activity in deer antler cells (P <0.01). In addition, cell proliferation was inhibited, with a decreased percentage of cells in the S phase, indicating cell arrest in the G0/G1 phase. These findings demonstrate that miRNA-mediated IGF-1 gene silencing decreases telomerase activity in deer antler cells, and provide further evidence that IGF-1 gene expression is correlated with telomerase activity during rapid growth of the antlers.