Gene expression profiles and bioinformatics analysis of insulin-like growth factor-1 promotion of osteogenic differentiation

Abstract

Background: Insulin-like growth factor-1 (IGF-1) promotes osteoblast differentiation and mineralization. The objective of this study was to investigate the effects of IGF-1 on proliferation, mineralization, alkaline phosphatase (ALP) synthesis, and gene expression of osteoblast differentiation in MC3T3-E1 osteoblasts cells, and to explore gene expression profiling differential genes. Methods: MC3T3-E1 osteoblasts cells were cultured in medium with or without IGF-1. The ALP assay was employed to determine the osteoblast mineralization, and Alizarin red S to stain for calcium deposits, which were the indicators of mature osteocytes. The living cell number was assessed by the Cell Counting Kit-8 method. RNA-seq analysis was applied to identify genes that were differentially expressed in with or without IGF-1 as well as genes that varied between these two groups. The expression of osteogenic marker genes was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Result: The cell number of osteoblasts exposed to IGF-1 at 200 μg/L significantly increased compared with the control group. The ALP activity in IGF-1-treated cells was higher than that in the control group. IGF-1 can increase ALP synthesis in osteoblasts in vitro. RNA-seq analysis showed that 677 triggered differentially expressed genes by IGF, of which 383 genes were downregulated and 294 genes were upregulated. Gene ontology (GO) analysis showed that IGF-1 caused a significant change in gene expression patterns. Conclusions: This result suggested that IGF-1 could probably promote the synthesis of organic matrix and mineralize action of bone. Osteogenic-related genes (DMP1, PHEX, SOST, BMP2, RUNX2, OPN, and OCN) were significantly upregulated both in GO analysis and in pathway analysis to perform qRT-PCR. Western blot analysis demonstrated that the Notch pathway was highly upregulated in MC3T3-E1 cells.