(PR158) Understanding the Role of SPP1 Polymorphism in Apical Periodontitis

Objectives: Studies have implicated genetic polymorphisms as modulators of apical periodontitis (AP) risk. However, the effects of AP-associated polymorphisms on gene/protein/cell function remain unknown. Recently, we found a significant association between a polymorphism in secreted phosphoprotein 1 (SPP1), a known activator of the NF-κB pathway, with AP. Here, we assessed the effects of SPP1 overexpression on expression of AP- and NF-κB signaling-relevant genes in vitro.

Methods: SPP1 donor and destination vectors were used to construct the plasmid of interest using gateway cloning techniques into E. Coli electrocompetent cells. The resulting vector containing SPP1 was cloned and transiently transfected into HEK293 cells using Opti-MEM medium and Turbofectin. Cells overexpressing SPP1 (HEK293-SPP1+) and wildtype HEK293 were frozen until use. Total RNA extraction and cDNA synthesis followed standard protocols. mRNA expression of SPP1, IL10, COL11A1, STAT3, IL1B, IL2, RANKL, OPG, and MyD88 in wildtype HEK293 and HEK293-SPP1+ cells was assessed via RT-qPCR. Experiments were performed in triplicates and repeated. Data analysis was conducted using the 2−∆∆CT method, considering target gene expression levels normalized to GAPDH as endogenous control. Fold-changes >5 were considered significant.

Results: With the exception of COL11A1, which was downregulated, all genes tested were upregulated ( > 5 fold-change) in HEK293-SPP1+ cells in comparison to wildtype cells. Specifically, STAT3 and OPG were significantly upregulated in HEK293-SPP1+ cells (87- and 20-fold-change differences, respectively), followed by IL1B, and IL10 ( >10 fold-changes).

Conclusion: SPP1 polymorphisms may contribute to altered NF-κB signaling with gain of or loss of function effects that may mediate bone destruction in AP.