Abstract
ETS proto-oncogene 1 (ETS1) has been implicated in osteoporosis (OP), but the exact molecular mechanisms are complex. This work focuses on the impact of ETS1 on the osteogenic differentiation and the molecules involved. A mouse pre-osteoblast cell line MC3T3-E1 was used for in vitro experiments. ETS1 was upregulated during the process of osteogenic differentiation of MC3T3-E1 cells. Overexpression of ETS1 promoted expression of osteogenic markers, alkaline phosphate concentration, and calcareous accumulation in cells. ETS1 was found to specifically bind to miR-128 promoter to suppress its transcription, while miR-128 could target homeobox A13 (HOXA13). Therefore, ETS1 suppressed miR-128 transcription to upregulate HOXA13 expression. Overexpression of HOXA13 promoted the osteogenic differentiation ability of cells and increased the protein level of β-catenin. Either overexpression of miR-128 or downregulation of β-catenin by CWP232228, a β-catenin-specific antagonist, blocked the promoting roles of ETS1 in cells. To conclude, this study provided evidence that ETS1 suppresses miR-128 transcription to activate the following HOXA13/β-catenin axis, therefore promoting osteogenic differentiation ability of MC3T3-E1 cells. This finding may offer novel ideas for OP treatment.
Highlights
Osteoporosis (OP) is the most frequent skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue resulted from an imbalance in bone turnover, namely a greater rate of bone resorption by the osteoclasts than the rate of bone formation by the osteoblasts, leading to enhanced bone fragility and a consequent increase in fracture risk (Nitta et al, 2017; Anastasilakis et al, 2018; Briot et al, 2018)
We first confirmed that the mRNA expression of ETS proto-oncogene 1 (ETS1) was gradually increased in MC3T3-E1 cells during osteogenic differentiation (Figure 1A), and a similar trend was presented with regard to its protein level (Figure 1B)
To further validate the impact of ETS1 on the osteogenic differentiation ability of MC3T3-E1 cells, overexpression of ETS1 was introduced in cells through the transfection of oeETS1, and the successful transfection was confirmed by the Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and western blot results concerning ETS1 expression (Figure 2A)
Summary
Osteoporosis (OP) is the most frequent skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue resulted from an imbalance in bone turnover, namely a greater rate of bone resorption by the osteoclasts than the rate of bone formation by the osteoblasts, leading to enhanced bone fragility and a consequent increase in fracture risk (Nitta et al, 2017; Anastasilakis et al, 2018; Briot et al, 2018). MiRNAs are the mostly studied non-coding RNAs, and thanks to their functions in posttranscriptional regulation of mRNA expression, they are involved in many cellular and molecular activities and play important roles in many pathological processes including bone remodeling (Sun et al, 2016). Our study hypothesized that ETS1 regulates miR-128 transcription to mediate HOXA13 expression, thereby promoting osteogenic differentiation and bone formation. Altered expression of these molecules was introduced in a mouse pre-osteoblast cell line MC3T3-E1 to validate the hypothesis
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