Synonymous Mutations of Porcine Igf1r Extracellular Domain Affect Differentiation and Mineralization in MC3T3-E1 Cells

Abstract

Owing to the wide application of miniature pigs in biomedicine, the formation mechanism of its short stature must be elucidated. The insulin-like growth factor 1 receptor (IGF-1R), which receives signals through the extracellular domain (ECD) binding with ligands, is crucial in regulating cell growth and bone matrix mineralization. In this study, two haplotypes of Igf1r with four synonymous mutations in the coding sequences of IGF-1R ECD between large pigs (LP) and Bama pigs (BM) were stably expressed in the Igf1r-knockout MC3T3-E1 cells and named as MC3T3-LP cells (LP group) and MC3T3-BM cells (BM group), respectively. IGF-1R expression was lower in the BM group than in the LP group both in terms of transcription and translation levels, and IGF-1R expression inhibited cell proliferation. In addition, IGF-1R expression in the BM group promoted early-stage differentiation but delayed late-stage differentiation, which not only suppressed the expression of bone-related factors but also reduced alkaline phosphatase activity and calcium deposition. Moreover, different haplotypes of Igf1r changed the stability and conformation of the protein, further affecting the binding with IGF-1. Our data indicated that the four synonymous mutations of IGF1R ECD encoded by affect gene transcription and translation, thereby further leading to differences in the downstream pathways and functional changes of osteoblasts.

Biological Functions of Let-7e-5p in Promoting the Differentiation of MC3T3-E1 Cells

“…We suspected that let-7e-5p likely plays a vital role in regulating the differentiation of osteoblasts and then participates in bone formation. The activity of ALP and the expression levels of genes related to osteogenic differentiation ( RUNX2 , OPN , OCN , and OSX ) regulate osteoblast differentiation and bone formation in growing bones; the expression levels of the above genes gradually increase during osteoblast differentiation and are important factors for evaluating cell differentiation ( Wang C. et al, 2020 ; Wei et al, 2020 ). Similar to the results of previous works, our results for the transfection of the let-7e-5p mimic confirmed that let-7e-5p enhanced the osteoblast differentiation potential by increasing the ALP activity ( Xi et al, 2016 ) and regulating RUNX2 , OCN , OPN , and OSX expression in MC3T3-E1 cells.…”

“…The MC3T3-E1 cells were cultured in Dulbecco’s minimal essential medium (DMEM, Sigma, United States) supplemented with 10% fetal bovine serum (FBS, Hyclone, United States) and 1% streptomycin/penicillin (Gibco, United States) at 37°C in a humidified 5% CO 2 incubator. The differentiation medium contained 10 mmol/L β-glycerophosphate (Sigma, United States), 50 mg/mL ascorbic acid, and 10 nM dexamethasone (Sigma, United States) ( Wang C. et al, 2020 ; Wei et al, 2020 ). HEK293T cells were cultured in DMEM (Hyclone, China) supplemented with 10% FBS and 1% penicillin/streptomycin.…”

“…A BCA protein assay kit (KeyGEN BioTECH, China) was applied to detect protein concentrations. Western blot assays were performed as previously reported ( Wang C. et al, 2020 ). In this study, antibodies against AKT (1:2000, CST, United States), phosphor-AKT (1:2000, CST, United States), ERK1/2 (1:2000, CST, United States), and phosphor-ERK1/2 (1:2000, CST, United States) were used after 48 h of transfection to investigate the effects of let-7e-5p on MC3T3-E1 cell proliferation.…”

“…Mineralization is the last stage of osteogenic differentiation, and the formation of mineralized nodules is a crucial marker of osteoblast mineralization ( Wang C. et al, 2020 ). The chelation of Alizarin Red (AR) with the calcium ions that form mineralized nodules is used to determine the osteogenic mineralization capability in tissues or cells ( Puchtler et al, 1969 ; Xi et al, 2016 ; Zheng et al, 2018 ).…”

“…Six hours after transfection, the cells were cultured in differentiation media for 21 days. Then, AR staining (ARS) was performed as previously reported ( Wang C. et al, 2020 ). The results were replicated in at least three independent experiments.…”