The Modulation of Autophagy in Bone Marrow Mesenchymal Stem Cells Via Hoxa11-as Activation of the PI3K-AKT-mTOR Pathway

Abstract

Objective: To investigate the regulatory mechanisms by which long non-coding RNA (lncRNA) Hoxa11-as modulates autophagy in bone marrow mesenchymal stem cells (BMSCs) via the PI3K-AKT-mTOR signaling pathway, and to elucidate its specific roles and potential implications in BMSC autophagy. Methods: BMSCs were divided into three groups: a blank control group, an Hoxa11-as overexpression group, and an si-Hoxa11-as knockdown group. Cells were seeded in six-well plates at a density of 3 × 10^5 cells/mL and cultured in complete medium. The blank control group received no treatment and had the complete culture medium replaced every 48 hours. For the Hoxa11-as overexpression group, BMSCs were transfected with the Hoxa11-as plasmid. After transfection, cells were cultured in complete medium fo an additional 48 hours, after which the BMSCs carrying the Hoxa11-as plasmid were harvested. For the si-Hoxa11-as knockdown group, BMSCs were transfected with the si-Hoxa11-as plasmid. After transfection, cells were cultured in complete medium for an additional 48 hours, after which the BMSCs carrying the si-Hoxa11-as plasmid were harvested. Real-time quantitative PCR (qPCR) was performed on the blank control group, the Hoxa11-as overexpression group, and the si-Hoxa11-as knockdown group to quantify the mRNA expression levels of Hoxa11-as, PI3K, AKT, mTOR, Foxo1, LC3, and Beclin1. Immunofluorescence staining was performed on the blank control group, the Hoxa11-as overexpression group, and the si-Hoxa11-as knockdown group to quantify the average fluorescence intensity of Hoxa11-as, PI3K, AKT, mTOR, Foxo1, LC3, and Beclin1 proteins. Results: The qPCR results showed that in the Hoxa11-as overexpression group, the mRNA levels of Hoxa11-as, PI3K, AKT, mTOR, and Foxo1 were significantly increased compared to the blank control group (P > 0.05), while those of LC3 and Beclin1 were significantly decreased (P < 0.05). In the si-Hoxa11-as knockdown group, the mRNA levels of Hoxa11-as, PI3K, AKT, mTOR, and Foxo1 were significantly reduced (P < 0.05), with a more pronounced decrease observed for Hoxa11-as (P < 0.05). The mRNA expression levels of LC3 and Beclin1 in the experimental group were significantly increased compared with the blank control group (P < 0.05). Additionally, there was a significant positive correlation between the mRNA expression levels of LC3/Beclin1 and mTOR (P < 0.05). Immunofluorescence results demonstrated that in the Hoxa11-as group, PI3K, AKT, mTOR, and Foxo1 were significantly upregulated (P < 0.05), while LC3 was significantly downregulated (P < 0.05). Beclin1 expression did not differ significantly (P > 0.05). In contrast, in the si-Hoxa11-as group, PI3K, AKT, mTOR, and Foxo1 were significantly downregulated (P < 0.05), whereas LC3 was significantly upregulated (P < 0.05). Conclusion: Long non-coding RNA Hoxa11-as significantly activates the PI3K-AKT-mTOR signaling pathway, thereby inhibiting the expression of autophagy-related proteins in BMSCs.