The effect of IGFBP3 gene knockout by the CRISPR/Cas9 system on the IGF-1 pathway in murine cells

The IGF-1 signaling pathway has been deeply involved in the aging mechanism. The insulin-like growth factor binding protein 3 (IGFBP-3) is a protein that binds to IGF-1 that regulates growth, survival, and aging. The purpose of this study was to investigate the impact of the IGFBP3 gene knockout (KO) on the expressions of aging-related proteins and genes using the CRISPR/Cas9 system. The IGFBP3 gene knockout (KO) was performed by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses were used to verify the induction of mutation. First, Sanger DNA sequencing was used to analyze the IGFBP3 gene knockout in murine cells (B16F1). The isolation of three colonies with the mutated DNA sequences in the IGFBP3 gene was validated. In addition, the expression levels of the IGFBP3 gene and protein in the edited B16F1 cells were lower than in those of normal B16F1 cells in western blot analysis as well as RT-PCR and qPCR. Moreover, IGFBP3 gene KO cells enhanced the level of SA-ß-gal staining and short telomere length compared to normal B16F1 cells. In particular, it was found that the expression levels of senescence-related proteins such as PI3K, AKT1, PDK1, and p53 were higher in IGFBP3 gene KO cells than in normal cells in both the absence and presence of IGF-1. Therefore, the above findings could provide a clue that IGFBP3 could play a key role in the aging mechanism.