OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: Therefore, the above findings could provide a clue that IGFBP3 could play a key role in the aging mechanism.
目的:本研究的目的是使用CRISPR/Cas9系统研究IGFBP3基因敲除(KO)对衰老相关蛋白和基因表达的影响。
方法:IGFBP3基因敲除(KO)通过CRISPR/Cas9系统进行。使用SangerDNA测序和Indel分析来验证突变的诱导。
结果:首先,SangerDNA测序用于分析鼠细胞(B16F1)中的IGFBP3基因敲除。验证了在IGFBP3基因中具有突变的DNA序列的三个集落的分离。此外,在蛋白质印迹分析以及RT-PCR和qPCR中,编辑的B16F1细胞中IGFBP3基因和蛋白质的表达水平低于正常B16F1细胞。此外,与正常B16F1细胞相比,IGFBP3基因KO细胞增强了SA-β-gal染色水平和较短的端粒长度。特别是,发现衰老相关蛋白如PI3K的表达水平,在不存在和存在IGF-1的情况下,IGFBP3基因KO细胞中的AKT1,PDK1和p53均高于正常细胞。
结论:因此,上述发现可能为IGFBP3在衰老机制中起关键作用提供了线索。