The incidence of OP is strongly associated with advancing age, demonstrating a higher prevalence among individuals in the middle-aged and elderly population, and accounting for 80% of fractures in those aged 50 years or older[11]. Recovery insufficiency following fragility fractures may give rise to chronic pain, disability, and potentially fatal consequences in severe instances[12]. Effective prevention and treatment strategies for OP primarily rely on early screening and identification of high-risk groups. Currently, with the continuous advancement of research methodologies and technologies, transcriptome sequencing and bioinformatics are increasingly being employed in scientific investigations, providing a theoretical foundation for early disease diagnosis, including OP. In this study, we utilized LASSO, SVM, and RF machine learning algorithms to identify two biomarker - PRKACA and CASP6. The ROC curve demonstrated the diagnostic potential of these genes for OP. GSEA results revealed that PRKACA was associated with pathways such as biosynthesis of amino acids, fructose and mannose metabolism, proteasome regulation, rheumatoid arthritis development; while CASP6 was linked to DNA replication processes, mismatch repair mechanisms, Fat digestion and absorption, homologous recombination events as well as hypotaurine metabolism. Furthermore, drug prediction analysis along with disease prediction indicated that these identified biomarkers could serve as potential target genes for therapeutic interventions.
Pyroptosis, also known as inflammatory necrosis of cells, has been extensively implicated in the pathogenesis and progression of infectious diseases, neurodegenerative disorders, atherosclerosis, and cancer. Therefore, it plays a pivotal role in these disease processes[13, 14]. Further investigation into pyroptosis is imperative for comprehending its involvement in the pathogenesis, progression, and prognosis of associated diseases, thereby providing novel insights for clinical prevention and therapeutic interventions. Hsu SK et al. have demonstrated the pivotal role of inflammation-related pyroptosis in cancer treatment[15]. Li Li Y et al. demonstrated that GSDME-mediated pyroptosis contributes to the pathogenesis of inflammation and fibrosis in obstructive nephropathy[16]. Currently, numerous studies have elucidated the underlying mechanism of pyroptosis in osteoporosis. For instance, Ruan H et al. demonstrated that the inhibition of caspase-1-mediated pyroptosis facilitated osteogenic differentiation, thereby offering potential therapeutic targets for OP[17]. Wang S et al. investigated the pivotal role of pyroptosis in pathologies including osteoarthritis, osteoporosis, and gouty arthritis[18]. In this study, employing LASSO, RF, and SVM-RFE techniques, we identified two biomarkers of significant importance in OP - PRKACA and CASP6. CASP6 (Caspase-6), a pivotal member of the Caspase family, plays a crucial role in regulating apoptosis. Furthermore, an increasing number of studies have elucidated the pivotal role of CASP6 in the pathogenesis of the disease. Guo K et al. demonstrated a significant association between CASP6 and the tumor immune microenvironment, thereby establishing its potential as a prognostic indicator for glioma[19]. The LeBlanc AC study reported that CASP6 could serve as a novel therapeutic target for Alzheimer's disease. However, the relationship between CASP6 and OP remains incompletely elucidated. Notably, Chen Y et al. identified CASP6 as a prognostic factor in osteoarthritis[20]. Cui W et al demonstrated that spinal cord CASP6 contributes to postoperative orthopedic pain in mice with tibial fractures through the involvement of netrin-1[21]. The aforementioned studies suggest a significant association between CASP6 and bone diseases, while our study provides preliminary evidence for the correlation between CASP6 and OP. We demonstrated the efficiency of CASP6 in diagnosing OP by drawing ROC curves, and also found that there were significant differences in CASP6 expression between control samples and OP samples. The findings demonstrate the pivotal role of CASP6 in the diagnosis of OP and warrant further investigation.
The PRKACA gene encodes a catalytic subunit of protein kinase A, which plays a pivotal role in various cellular processes, including differentiation, proliferation, and apoptosis. The study conducted by Zilbermint M et al. demonstrated the presence of somatic PRKACA mutations in patients diagnosed with adrenal adenomas[22]. Based on network pharmacology and molecular docking, Wang J et al. found that the key target related to PRKACA in the treatment of OP is associated with the combined application of ABR and MOR[23]. Mlost J et al. revealed increased mRNA expression of PRKACA in bone marrow tissues of rats injected with monoiodoacetate (MIA)[24]. The findings from our analysis further validate the indispensable role of PRKACA in OP progression, thereby offering a novel avenue for clinical diagnosis of patients with this condition. Subsequent GSEA enrichment results showed that PRKACA differential genes were mainly enriched in Biosynthesis of amino acids, Fructose and mannose metabolism, Proteasome, Legionellosis and Rheumatoid arthritis. Studies have demonstrated that essential amino acids serve as sources of bone energy and regulators of molecular pathways, thereby exhibiting the potential to enhance bone mass[25]. Pernow Y et al. demonstrated alterations in the free amino acid profile associated with idiopathic OP in men, highlighting a potential relationship between tryptophan and osteoblast function[26]. Based on previous studies, we speculated that the biosynthesis of amino acids plays a crucial role in OP development and may represent a novel therapeutic target for this disease. Based on bioinformatics analysis, Chu et al. showed that Zishen Jiangtang Pill affects OP through pathways such as fat digestion and absorption[27]. Studies have shown that obesity and OP are common phenomena in the aging process. Previously, there was a prevailing belief that obesity could enhance bone density; however, recent studies have demonstrated that the positive impact of obesity on bone health does not reduce the incidence of OP and fractures. Conversely, other research has indicated that excessive obesity may actually increase the risk of developing OP. Therefore, it is imperative to investigate the pathways involved in fat digestion and absorption in order to mitigate the risk of OP morbidity. According to our hypothesis, CASP6 may exert inhibitory effects on the fat digestion and absorption pathways associated with OP, while PRKACA may modulate OP by activating amino acid pathways. In the drug prediction of CASP6, myricetin was included. Studies have demonstrated that myricetin exhibits potential in promoting osteogenic differentiation and matrix mineralization, thereby mitigating dexamethasone-induced osteoporosis through modulation of the ERK signaling pathway[28]. Additionally, Shuli Fan et al. confirmed the protective effect of myricetin on streptozotocin-induced diabetic osteoporosis in rats[29].
In summary, this study employed bioinformatics analysis to identify significant biomarkers in OP, namely PRKACA and CASP6. Subsequent PPI, GO, KEGG, and GSEA analyses provided insights into their potential biological functions in OP development. The discovery of these biomarkers offers a novel avenue for future OP treatment and sheds light on the underlying biological mechanisms from a new perspective, thereby serving as a reference for clinical studies on OP. However, it should be noted that the validation of PRKACA and CASP6 through in vivo and in vitro experiments is still pending. Further exploration and investigation are warranted to ascertain the pivotal role played by these genes. This limitation should be acknowledged as a shortcoming of our study; nevertheless, our analytical findings provide valuable insights for future research endeavors focused on OP.