ABSTRACT: BACKGROUND: We previously demonstrated that temperature-related microwave ablation (MWA) can safely regulate vertebral growth plates of piglets. Therefore, the present study was designed to investigate the effects of different temperatures on chondrocyte proliferation and the molecular mechanisms involved in vitro. METHODS: After treatment for 10 min using different temperatures (37°C, 40°C, 42°C, 44°C, 46°C, 48°C, or 50°C), CCK-8 assay examined the activity of ATDC5 cells at 12 hr. RNA-seq identified differentially expressed genes (DEGs) and the hub genes in ATDC5 cells treated at 37°C, 40°C and 44°C. RT-qPCR verified the differential expression of hub genes in ATDC5 cells. RESULTS: Compared with 37°C, stimulation at 40°C significantly enhanced the activity of ATDC5 cells, and 40°C had no significant effect. In addition, stimulation at 44°C, 46°C, 48°C, or 50°C exhibited the opposite pattern, and ATDC5 cells were particularly less than 50% active after treatment at 46°C, 48°C, or 50°C. Differential expression analysis revealed the presence of 179, 374 and 221 DEGs in 40°C vs. 37°C, 44°C vs. 37°C, and 44°C vs. 40°C, respectively. Enrichment analysis indicated that these DEGs predominantly regulate proliferation, differentiation, necrosis, inflammatory and immune responses, and ECM synthesis/degradation, and that they are associated with the Ras, PI3K/AKT, mTOR, cAMP, and MAPK pathways. Furthermore, Agt, Hspa1a, Hspb1, and Nlrc4 were hub genes in DEGs, and RT-qPCR confirmed that the mRNA expression patterns of these hub genes in ATDC5 cells were essentially consistent with the RNA-seq results. CONCLUSION: Temperature-mediated chondrocyte proliferation was associated with Ras, PI3K/AKT, mTOR, cAMP, and MAPK pathways, and Agt, Hspa1a, Hspb1, and Nlrc4 may be the key regulatory genes.
