Functional mechanism and clinical implications of lncRNA NNT-AS1 in delayed fracture healing.

Delayed fracture healing impairs patient quality of life, with emerging evidence highlighting long non-coding RNAs (lncRNAs) as pivotal regulators in this process. LncRNA NNT-AS1 was up-regulated in delayed fracture healing, but its mechanism remains unclear. This study analysed 97 controls with normal healing and 106 cases of delayed fracture healing. RT-qPCR was employed to assess the levels of NNT-AS1, microRNA-181a-5p (miR-181a-5p), and osteoblast markers (RUNX-2, ALP, OPG) in serum or MC3T3-E1 cells. Detection of osteoblast markers and downstream target genes (BMP3) at the protein level via Western blot analysis. Evaluation of NNT-AS1's diagnostic potential in identifying delayed fracture healing through ROC curve analysis. The interaction between NNT-AS1 and miR-181a-5p was validated using dual luciferase reporter assays and RNA immunoprecipitation. Cell viability was assessed via CCK-8 proliferation assays. Clinical data indicated that serum NNT-AS1 levels were elevated in patients with delayed fracture healing, with high diagnostic value as determined by ROC curve analysis. In vitro experiments demonstrated that NNT-AS1 expression decreased during osteogenic differentiation, and its overexpression inhibited osteoblast activity and osteogenic differentiation. Mechanistically, NNT-AS1 may act as a competitive messenger RNA directly targeting miR-181a-5p. Serum miR-181a-5p was down-regulated in delayed healing patients and negatively correlated with NNT-AS1. And miR-181a-5p mimic reversed the inhibitory effects of NNT-AS1 overexpression on osteogenic differentiation and cell viability. BMP3 may be a target gene downstream of the NNT-AS1/miR-181a-5p axis. NNT-AS1 sponged miR-181a-5p to inhibit osteogenesis, serving as a delayed fracture healing biomarker and therapeutic target. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-025-00887-4.