Evaluation of the Changes in microRNA Expression Profiles in Rat Primary Osteoblastic Cells Stimulated with Cotinine

Smoking stands as a significant factor contributing to aberrations in bone metabolism, while microRNAs are intricately linked to the regulation of bone metabolism. This study aimed to identify cotinine-responsive microRNAs (miRNAs) and downstream regulatory pathways of their target genes involved in the regulation of osteoblastic cells, providing a foundation for new treatments targeting miRNAs for the bone metabolism imbalance induced by smoking.

Primary osteoblastic cells of Sprague-Dawley rats were cultured through a modified enzymatic digestion method from the cranial bone of neonatal rats and stimulated with a high concentration of cotinine (1000 ng/mL) for 7 days. Then, miRNA gene chip technology was utilized to detect the changes in miRNA expression profiles in cotinine-stimulated osteoblastic cells, and differential expression profiles of cotinine-responsive miRNAs in osteoblastic cells were identified. Real-time polymerase chain reaction was used to detect the levels of significantly differentially expressed miRNAs in rat osteoblastic cells. Gene ontology (GO) and Kyoto encyclopedia of Genes and Genomes (KEGG) pathway analyses were utilized to predict target genes of these miRNAs to reveal the potential biological functions and pathways.

We identified 6 statistically differentially expressed miRNAs in the miRNA microarray analysis, of which 3 were upregulated and 3 were downregulated. We chose bone metabolism-related miRNAs as the miRNAs of interest. Quantitative real-time polymerase chain reaction was used to detect the expression levels of the differentially expressed miRNAs, and only miR-210 was significantly upregulated (3.34-fold), consistent with the microarray data. GO and KEGG pathway analyses of predicted miR-210 target genes revealed that miR-210 might participate in numerous signaling pathways, such as the RAS, Rap, PI3K-Akt, and calcium signaling pathways.

We found that the strongly upregulated miR-210 may play an important regulatory role in osteoblast cells' biological behavior and bone formation function. The GO analysis results showed that miR-210 mainly involved protein binding, transporter activity, growth factor binding, and ion channel activity. According to the results of the KEGG analysis, miR-210 might negatively regulate the PI3K-Akt signaling pathway, thus affecting the proliferation of osteoblastic cells. These findings suggest that miR-210 may be a potential target for regulating the imbalance of bone metabolism caused by smoking, offering a new direction for clinical treatment of patients with bone metabolism-related diseases.