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Autophagy is involved in neurofibromatosis type I gene‑modulated osteogenic differentiation in human bone mesenchymal stem cells

Yiqiang Li,Mingwei Zhu,Xuemei Lin,Jingchun Li,Zhe Yuan,Yanhan Liu,Hongwen Xu

Neurofibromatosis type I (NF1) is an autosomal dominant genetic disease that is caused by mutations in the NF1 gene. Various studies have previously demonstrated that the mTOR complex 1 signaling pathway is essential for the NF1‑modulated osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Additionally, the mTOR signaling pathway plays a notable role in autophagy. The present study hypothesized that NF1 could modulate the osteogenic differentiation of BMSCs by regulating the autophagic activities of BMSCs. In the present study, human BMSCs were cultured in an osteogenic induction medium. The expression of the NF1 gene was either knocked down or overexpressed by transfection with a specific small interfering RNA (siRNA) targeting NF1 or the pcDNA3.0 NF1‑overexpression plasmid, respectively. Autophagic activities of BMSCs (Beclin‑1, P62, LC3B I, and LC3B II) were determined using western blotting, electron microscopy, acridine orange (AO) staining and autophagic flux/lysosomal detection by fluorescence microscopy. In addition, the autophagy activator rapamycin (RAPA) and inhibitor 3‑methyladenine (3‑MA) were used to investigate the effects of autophagy on NF1‑modulated osteogenic differentiation in BMSCs. Inhibiting NF1 with siRNA significantly decreased the expression levels of autophagy markers Beclin‑1 and LC3B‑II, in addition to osteogenic differentiation markers osterix, runt‑related transcription factor 2 and alkaline phosphatase. By contrast, overexpressing NF1 with pcDNA3.0 significantly increased their levels. Transmission electron microscopy, AO staining and autophagic flux/lysosomal detection assays revealed that the extent of autophagosome formation was significantly decreased in the NF1‑siRNA group but significantly increased in the NF1‑pcDNA3.0 group when compared with the NC‑siRNA and pcDNA3.0 groups, respectively. In addition, the activity of the PI3K/AKT/mTOR pathway [phosphorylated (p)‑PI3K, p‑AKT, p‑mTOR and p‑p70S6 kinase] was significantly upregulated in the NF1‑siRNA group compared with the NC‑siRNA group, and significantly inhibited in the NF1‑pcDNA3.0 group, compared with the pcDNA3.0 group. The knockdown effects of NF1‑siRNA on the autophagy and osteogenic differentiation of BMSCs were reversed by the autophagy activator RAPA, while the overexpression effects of NF1‑pcDNA3.0 on the autophagy and osteogenic differentiation of BMSCs were reversed by the autophagy inhibitor 3‑MA. In conclusion, results from the present study suggest at the involvement of autophagy in the NF1‑modulated osteogenic differentiation of BMSCs. Furthermore, NF1 may partially regulate the autophagic activity of BMSCs through the PI3K/AKT/mTOR signaling pathway.