MicroRNA Signatures in Dental Pulp Stem Cells Following Nicotine Exposure

Abstract

Background and Objectives: Nicotine is the most well-studied toxic substance in cigarette smoke and e-cigarette vape. However, smoke and vape are composed of other components that have a negative impact on health. The objective of this study is to investigate whether nicotine has a distinctive impact on molecular mechanisms in stem cells. Methods: The cellular impact of nicotine on the regenerative capacity of human dental pulp stem cells (DPSCs) and the microRNA (miRNA) profile was examined. Bioinformatic analysis was performed to identify miRNA-regulated cellular pathways associated with nicotine exposure. These pathways were then compared to those induced by cigarette smoke condensate (CSC). Results: Prolonged exposure to nicotine significantly impaired the regeneration of DPSCs and changed the expression of miRNAs. Nicotine upregulated the expression of hsa-miR-7977, hsa-miR-3178, and hsa-miR-10400-5p compared to vehicle control. Interestingly, nicotine did not change the expression of hsa-miR-29b-3p, hsa-miR-199b-5p, hsa-miR-26b-5p, or hsa-miR-26a-5p compared to the control. However, the expressions of these miRNAs were significantly altered when compared to CSC treatment. Further analysis revealed that nicotine was distinctively associated with certain miRNA-targeted pathways including apoptosis, ErbB, MAPK signaling, PI3K-Akt, TGF-b signaling, and Wnt signaling. Conclusions: Our work provides evidence on the distinctive miRNA signature induced by nicotine. The information will be important for identifying the unique molecular pathways downstream of nicotine from smoking and vaping in different individuals, providing a new direction for personalized disease prevention, prognosis, and treatment.

Keywords: cell signaling; dental pulp stem cells; miRNAs; nicotine.

Figure 1

Prolonged exposure to nicotine impaired DPSC regenerative capacity. (A) DPSCs were exposed to vehicle control or 100 nM to 1 mM of nicotine for 6 weeks. Cells were passaged weekly at 1 × 10⁶/mL. Total cell numbers were compared at each passage. One-way ANOVA, 1 mM, 100 μM, and 10 uM significantly different than control and lower doses, * p < 0.05, ** p < 0.01, n = 3. (B) Following 6-week exposure to nicotine, each set of DPSCs was reseeded and left undisturbed. Surviving cells were stained and (C) quantified. (D) Quantification of wound scratch area at 0, 24, and 48 h. (E) DPSCs were cultured in osteo- or adipo-differentiation induction medium and treated with 1 mM nicotine or vehicle control for up to 4 weeks. Alkaline phosphatase (ALP) activity was measured to determine osteogenic differentiation. (F) qRT-PCR of adipogenic marker PPARγ. (C–F): Student’s t-test compared to control, * p < 0.05, n = 3.

Figure 2

Pathway intersection. The network of signaling pathways associated with miRNAs which were significantly different in nicotine-treated DPSCs compared to controls. The diagram was generated using the PathIN tool and cytoscape. Unweighted edges refer to common genes between two pathways. The pathways showing the most abundant interactions are highlighted in different colors (red, yellow, black, blue, and green). The less abundant interactions are shown with dashed white lines.