NEAT1 regulates BMSCs aging through disruption of FGF2 nuclear transport

Abstract

Background: The aging of bone marrow mesenchymal stem cells (BMSCs) impairs bone tissue regeneration, contributing to skeletal disorders. LncRNA NEAT1 is considered as a proliferative inhibitory role during cellular senescence, but the relevant mechanisms remain insufficient. This study aims to elucidate how NEAT1 regulates mitotic proteins during BMSCs aging.

Methods: BMSCs were isolated from alveolar bone of human volunteers aged 26-33 (young) and 66-78 (aged). NEAT1 expression and distribution changes during aging process were observed using fluorescence in situ hybridization (FISH) in young (3 months) and aged (18 months) mice or human BMSCs. Subsequent RNA pulldown and proteomic analyses, along with single-cell analysis, immunofluorescence, RNA immunoprecipitation (RIP), and co-immunoprecipitation (Co-IP), were conducted to investigate that NEAT1 impairs the nuclear transport of mitotic FGF2 and contributes to BMSCs aging.

Results: We reveal that NEAT1 undergoes significant upregulated and shifts from nucleus to cytoplasm in bone marrow and BMSCs during aging process. In which, the expression correlates with nuclear DNA content during karyokinesis, suggesting a link to mitogenic factor. Within NEAT1 knockdown, hallmarks of cellular aging, including senescence-associated secretory phenotype (SASP), p16, and p21, were significantly downregulated. RNA pulldown and proteomic analyses further identify NEAT1 involved in osteoblast differentiation, mitotic cell cycle, and ribosome biogenesis, highlighting its role in maintaining BMSCs differentiation and proliferation. Notably, as an essential growth factor of BMSCs, Fibroblast Growth Factor 2 (FGF2) directly abundant binds to NEAT1 and the sites enriched with nuclear localization motifs. Importantly, NEAT1 decreased the interaction between FGF2 and Karyopherin Subunit Beta 1 (KPNB1), influencing the nuclear transport of mitogenic FGF2.

Conclusions: Our findings position NEAT1 as a critical regulator of mitogenic protein networks that govern BMSC aging. Targeting NEAT1 might offer novel therapeutic strategies to rejuvenate aged BMSCs.

Fig. 1

Age-dependent redistribution of NEAT1 from nucleus to cytoplasm. (A) Representative fluorescence images depicting the expression of NEAT1 and Nestin in the bone marrow of young (upper panels) and aged (lower panels) mice. Nuclei are stained with DAPI (blue), immunofluorescence staining of Nestin (green) and FISH staining of NEAT1(red) are visualized in bone marrow. Scale bar, 40 μm. (B) Representative fluorescence images depicting the subcellular localization of NEAT1 in young (upper panels) and aged (lower panels) BMSCs. Nuclei are stained with DAPI (blue), and NEAT1 is visualized using an RNA-FISH probe (red). Merged images reveal partial cytoplasmic distribution of NEAT1 in aged BMSCs. Scale bar, 20 μm. (C) Quantitative analysis the expression of NEAT1 and localization within nuclear and cytoplasmic compartments in young and aged BMSCs. Data represent mean ± s.d. (D) High-magnification images of NEAT1 localization in individual cells during karyomitosis (top) and postkaryomitosis (bottom). Scale bar, 10 μm. (E) Scatter plot correlating nuclear DNA content (determined by DAPI staining) with cellular NEAT1 expression (quantified by RNA-FISH) during BMSCs osteoblasts differentiation. Data are presented with mean ± s.d

Fig. 2

NEAT1 related to BMSCs senescence, osteoblast differentiation, mitotic cell cycle, and ribosome biogenesis. (A) SA-β-gal staining were applied to aged BMSCs within NEAT1 knockdown, and SA-β-gal positive cells were counted. Scale bar, 40 μm. (B) Western blotting against senescence markers in aged BMSCs following transfection with control siRNA, siNEAT1-1 and siNEAT1-2. Full-length blots are presented in Supplementary Material. (C) RT-qPCR analysis of senescence markers in aged BMSCs after transfection with control siRNA, siNEAT1-1 and siNEAT1-2. (D) Venn diagram depicting the overlap of NEAT1-interacting proteins identified by mass spectrometry following NEAT1 pulldown experiments in BMSCs. (E) List of top-ranked NEAT1-bound proteins in BMSCs based on label-free quantification (LFQ) intensity and peptide coverage, with a false discovery rate (FDR) < 0.01. (F) Gene Ontology (GO) term enrichment analysis of NEAT1-associated proteins in BMSCs, categorized by biological processes (left), molecular functions (middle), and cellular component (right)

Fig. 3

FGF2 and KPNB1 involves in BMSCs proliferation and bone regeneration. (A) Uniform Manifold Approximation and Projection (UMAP) plot illustrating distinct clusters of BMSCs, based on single-cell RNA sequencing data. Clusters are numbered and color-coded, reflecting heterogeneity within the BMSC population. (B) UMAP projection of BMSC cell cycle states, displaying cells in G1, S, and G2/M phases. The majority of cells reside in the G1 phase, with smaller subsets in the S and G2/M phases, indicating proliferative diversity within the population. (C) Partition-based graph abstraction (PAGA) was used to analyze and visualize cellular differentiation trajectories. (D-E) Expression density plots visualizing the distribution of FGF2, KPNB1, CCND1, and PCNA transcripts across BMSC populations.(F) RT-qPCR analysis of FGF2 and KPNB1 expression levels in young and aged BMSCs. Expression levels of FGF2 are upregulated in young BMSCs (*P< 0.05). Data represent mean ± s.d. (G) Immunofluorescence staining of FGF2 (green) and KPNB1 (red) in cultured BMSCs. Nuclei are counterstained with DAPI (blue). Merged images reveal colocalization of FGF2 and KPNB1 within the nuclear compartment. Scale bar, 10 μm. (H) Histological analysis of regenerated alveolar bone from mice. Hematoxylin and eosin (H&E) staining highlights bone regeneration morphology, whereas immunofluorescence staining demonstrates the presence of FGF2 (green) and KPNB1 (red) within the newly formed bone matrix. Nuclei are counterstained with DAPI (blue), and merged images show the spatial localization of FGF2 and KPNB1. Scale bar, 50 μm

Fig. 4

The region of FGF2 that binds to NEAT1 is enriched with nuclear localization motifs. (A) The NEAT1–FGF2 interaction matrix identifies specific regions within FGF2, that strongly associate with particular NEAT1 nucleotide regions, suggesting sequence-specific binding between NEAT1 and FGF2. (B) Sequence alignment of the FGF2 nuclear localization signal (NLS) motif. The 18-kDa FGF2 protein sequence (NP_001348594.1) highlights conserved residues critical for nuclear import. Key positively charged and hydrophobic residues are indicated in red, delineating the NLS domain essential for FGF2 nuclear translocation and interaction with NEAT1. (C) NEAT1 sense and antisense pulldown assays demonstrate specific enrichment of the 18-kDa FGF2 isoform and KPNB1 in BMSCs. (D) RNA immunoprecipitation (RIP) assay showing significant enrichment of NEAT1 bound to 18-kDa FGF2 in BMSCs (**P < 0.01). Data are presented as mean ± s.d. Full-length blots are presented in Supplementary Material. (E) Immunofluorescence staining of FGF2 (green) in cultured aged mouse BMSCs after transfection with control siRNA and siNEAT1. Nuclei are counterstained with DAPI (blue). Scale bar, 20 μm

Fig. 5

NEAT1 reduces interaction between FGF2 and KPNB1. (A-B) Immunoblot analysis of FGF2 expression in aged BMSCs following NEAT1 knockdown and young BMSCs following NEAT1 overexpressing, respectively. Full-length blots are presented in Supplementary Material. (C) Co-immunoprecipitation (co-IP) of FGF2 and KPNB1 from BMSCs with or without NEAT1 knockdown. Full-length blots are presented in Supplementary Material. (D) Co-immunoprecipitation of FGF2 and KPNB1 from NEAT1-overexpressing BMSCs. Full-length blots are presented in Supplementary Material. (E) Gene Ontology (GO) (left) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (right) enrichment analyses of FGF2-interacting proteins, highlighting key biological processes and pathways influenced by FGF2