Research Paper Volume 15, Issue 14 pp 7124—7145

Figure 6. The LncRNA SPIRE1/miR-181a-5p/PRLR axis regulated the JAK/STAT3 signaling in mouse mandibular BM-MSCs to modulate the Th17/Treg balance. (A) The knockdown efficiency of PRLR-specific shRNA in mandibular BM-MSCs was confirmed by qPCR analysis of BM-MSCs transfected with plasmid expressing the indicated shRNA. (B) Knockdown of LncRNA SPIRE1 or PRLR, or transfection of miR-181a-5p mimics activated the JAK/STAT3 pathway, as evidenced by increased expression of JAK2 and phosphorylated STAT3 (pSTAT3). The protein levels of JAK2 and pSTAT3 in mandibular BM-MSCs after the indicated transfections were quantitated by western blot assays. (C) Mandibular BM-MSCs from periodontitis mice had more activated JAK/STAT3 signaling than that from normal controls, which was inhibited by the STAT3 inhibitor C188-9. The protein levels of JAK2 and pSTAT3 were quantitated as in (B). (D–J) Mandibular BM-MSCs from periodontitis mice were treated with vehicle or STAT3 inhibitor C188-9, while mandibular BM-MSCs from normal control mice were used as controls. After co-culturing of these BM-MSCs with pre-activated CD4⁺ T cells, the percentages of CD4⁺IL-17⁺ Th17 cells (D, E), RORC mRNA levels (F), soluble IL-17 level in culture medium (G), as well as the percentages of CD4⁺FoxP3⁺ Tregs (H, I), FoxP3 mRNA levels and soluble IL-10 levels in culture medium (J) were measured. n = 3 for each group; ^*P < 0.05, ^**P < 0.01; ^***P < 0.001, between the indicated groups.