Lnc007185570.1–pal-miR-3120-3p–ppp3cb Axis Regulating the Osteogenic Differentiation and Intermuscular Bone Formation in Megalobrama amblycephala via the Wnt Signaling Pathway

IBs are mineralized spicules originating from osteoblasts differentiated from tenocytes that can reduce the commercial value of fish and pose potential health risks to consumers if accidentally ingested. Runx2b exhibited higher expression in osteoblasts associated with IBs. Knockout of the runx2b gene significantly reduced osteoblast numbers and completely inhibited IB formation, confirming its essential role in osteogenic differentiation. However, how non-coding RNAs regulate this process during IB formation remains poorly understood. In this study, whole-transcriptome sequencing was performed to identify ncRNAs and mRNAs in dorsal and tail muscle tissues from six-month-old M. amblycephala with IBs (WT group, n = 3) and without IBs (Mut group, n = 3). A total of 1762 and 1992 differentially expressed (DE) transcripts were identified in dorsal and tail muscle, respectively. Specifically, in dorsal muscle, 408 DE-circular RNAs (circRNAs), 425 DE-long non-coding RNAs (lncRNAs), 171 DE-microRNAs (miRNAs), and 758 DE-messenger RNAs (mRNAs) were detected; in tail muscle, 337 DE-circRNAs, 414 DE-lncRNAs, 222 DE-miRNAs, and 1019 DE-mRNAs were identified. Gene Set Enrichment Analysis (GSEA) revealed that, in the Mut group, key osteogenic signaling pathways, specifically Wnt, MAPK, and TGF-β, were significantly enriched among downregulated genes. Several key IB formation-responsive ceRNAs were identified based on correlation analysis, and the expression and function of the critical regulatory axis, lnc007185570.1–pal-miR-3120-3p–ppp3cb, were experimentally validated. Functional assays subsequently demonstrated that this axis modulates osteoblast differentiation via the Wnt signaling pathway. These findings offer new insights into the molecular mechanisms governing osteoblast differentiation during IB formation from the perspective of ceRNA regulatory networks.