Multi-Omics Analysis Identifies TTYH3 and MPG as Candidate Osteoporosis-Associated Biomarkers in Osteoblasts and Characterizes Spatial Heterogeneity of SPP1 in the Osteoporotic Bone Microenvironment.

Background

Osteoporosis (OP) arises from dysregulated bone metabolism driven by genetic and epigenetic factors. Osteoblasts are central to bone formation, and their functional heterogeneity-shaped by genetic background and receptor expression profiles-may critically influence OP susceptibility. This study aimed to identify osteoblast-specific genes robustly associated with OP and elucidate their underlying pathogenic mechanisms.

Methods

We analyzed single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (bulk RNA-seq), and spatial transcriptomics (ST) datasets of osteoblasts. Differentially expressed genes (DEGs) were identified from scRNA-seq and bulk RNA-seq datasets, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Three machine learning methods and an artificial neural network (ANN)-based weighting analysis, together with weighted gene co-expression network analysis (WGCNA), were used to prioritize candidate OP-associated genes. ST data were integrated with CellChat analysis based on scRNA-seq data to investigate spatial expression patterns and potential cell-cell communication. Candidate genes were validated by immunohistochemical staining in human femoral head samples and quantitative real-time PCR (qRT-PCR) in MC3T3-E1 cells.

Results

The intersecting DEGs from the scRNA-seq and bulk RNA-seq datasets were potentially related to inhibition of ossification. Three machine learning methods identified five osteoporosis-associated candidate genes: TTYH3, NRBP2, MPG, HSPG2, and GPR153, which were subsequently evaluated using ANN-based weighting analysis. Among these, TTYH3 and MPG were identified as OP-related genes by WGCNA. Integration of the scRNA-seq, ST, and CellChat results suggested that SPP1 was highly expressed in osteoblasts from the OP sample and exhibited a spatially heterogeneous expression pattern. Immunohistochemical staining of human femoral head tissues from individuals with normal bone mass and osteoporosis, together with qRT-PCR analysis in MC3T3-E1 cells, further validated the differential expression of TTYH3 and MPG.

Conclusions

This study identified candidate osteoporosis-associated biomarkers and provided insights into potential pathogenic mechanisms, thereby establishing a basis for future mechanistic and clinical validation.