A novel ceRNA regulatory network involving the long noncoding NEAT1, miRNA-466f-3p and its mRNA target in osteoblast autophagy and osteoporosis
Osteoporosis (OP) is a systemic metabolic disorder characterized by a reduction in bone tissue volume. LncRNAs have been reported to act as regulators of several human diseases. Specifically, lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in proliferation, differentiation and apoptosis in osteoclasts and bone marrow mesenchymal stem cells and regulates the occurrence and development of OP. However, the relationship between NEAT1 and osteoblast autophagy and its mechanism are still unclear. Western blotting of LC3 and P62 was used to evaluate the effect of fluid shear stress (FSS) on autophagy in MC3T3-E1 osteoblasts. Total transcriptome sequencing and bioinformatics analyses were performed on osteoblasts loaded with and without FSS. qPCR was performed to examine the expression of NEAT1 in OP bone tissues and osteoblasts. The RNA-FISH was performed to study the localization of lncRNA NEAT1 and miR-466f-3p in MC3T3-E1 osteoblasts. In vitro, western blotting, transmission electron microscopy (TEM), immunofluorescence (IF) staining and qPCR were performed to verify the biological functions of NEAT1, miR-466f-3p and HK2. Subsequently, we conducted bioinformatics analysis and dual luciferase reporter assays to identify the relationships among NEAT1, miR-466f-3p and HK2. Additionally, rescue assays were conducted on osteoblasts to clarify the regulatory network of the NEAT1/miR-466f-3p/HK2 signalling pathway. In vivo, the OVX mouse model was used to investigate the effects of si-NEAT1 on autophagy in OP mice. The distal femur and serum were collected for further micro-CT analysis, blood biochemistry, and haematoxylin-eosin and Alizarin red staining (ARS). Immunohistochemistry (IHC) was performed to assess the protein expression of LC3 and HK2. NEAT1 expression was upregulated in OP tissues and osteoblast lines exposed to FSS. Knockdown of NEAT1 inhibited autophagy in vitro and in vivo. Further studies demonstrated that NEAT1 positively regulated HK2 expression via its competing endogenous RNA effects on miR-466f-3p. Moreover, we found the NEAT1/miR-466f-3p/HK2 axis regulated autophagy in osteoblasts. Knocking down NEAT1 inhibited autophagy in osteoblasts via the miR-466f-3p/HK2 signalling pathway, which may provide new ideas for novel molecular therapeutic targets of postmenopausal OP. KEY MESSAGES: • Fluid shear stress (FSS) can promote autophagy of osteoblast and performed transcriptome sequencing. • NEAT1 is overexpressed in osteoporosis and could regulate osteoblast cells autophagy. • Knockdown of lncRNA NEAT1 inhibited osteoblast cells autophagy by sponging miRNA-466f-3p and targeting HK2 in osteoporosis.
Keywords: Autophagy; HK2; LncRNA NEAT1; MiRNA-466f-3p; Osteoblast.