Fig. 5

Exosomal miR-140-3p inhibits PDLC pyroptosis by regulating DNA methylation via DNMT1/SOCS1/NFκB axis. (A) The qRT-PCR showed that mRNA level of SOCS1 in rat PDL was significantly suppressed under mechanical stimulation. n = 6. (B) Western blot results revealed that force downregulated the protein level of SOCS1 and upregulated the expression of NFκB and pNFκB. Moreover, SOCS1 siRNA increased NFκB level, suggesting that SOCS1 was a negative regulator of NF-κB signaling pathway. Full‑length blots are presented in Fig. S7: Fig. 5B. (C) MSP shows the methylation level of SOCS1 promoter in PDLCs. Force upregulated the DNA methylation level of SOCS1. Full-length gels are presented in Fig. S7: Fig. 5C. (D) The qRT-PCR showed that Decitabine (inhibitor of DNA methyltransferase) and DNMT1 siRNA significantly increased mRNA level of SOCS1 in mechanically stimulated PDLCs. n = 6. (E) Western blot analysis indicated that decitabine downregulated DMNT1, NFκB and pNFκB expression, but upregulated the protein level of SOCS1. Full‑length blots are presented in Fig. S7: Fig. 5E. (F) MSP results showed that DNA methylation of SOCS1 was suppressed in Force + Decitabine group compared with Force + PBS group. Full-length gels are presented in Fig. S7: Fig. 5F. (G) Western blot results revealed that si-DNMT1 increased SOCS1 expression but decreased DMNT1, NFκB and pNFκB levels. Full‑length blots are presented in Fig. S7: Fig. 5G. (H) MSP showed that si-DNMT1 administration decreased the DNA methylation level of SOCS1. Full-length gels are presented in Fig. S7: Fig. 5H. (I) Sequence alignment between miR-140-3p and its putative binding sites (in red letters) in the DNMT1. Mutation of the miR-140-3p target sites (in blue letters) was also shown. Luciferase reporter assay for the relative luciferase activities of WT and Mut DNMT1 indicated that luciferase activity was significantly reduced by miR-140-3p overexpression in DNMT1-3’UTR WT group. n = 4. (J) Western blot analysis demonstrated that overexpression of miR-140-3p suppressed both DMNT1, NFκB and pNFκB levels, but upregulated the protein level of SOCS1. Full‑length blots are presented in Fig. S7: Fig. 5J. (K) The qRT-PCR showed decreased mRNA level of DNMT1 and NFκB, as well as increased SOCS1 level with miR-140-3p overexpressed. n = 6. (L) Western blot results showed that exosomes downregulated DMNT1, NFκB and pNFκB levels, and upregulated SOCS1 expression. Force-Exos exhibited a more significant effect than Ctrl-Exos. Full‑length blots are presented in Fig. S7: Fig. 5L. (M) Western blot analysis revealed that the inhibition of miR-140-3p in Force-Exos significantly increased SOCS1 expression but suppressed DMNT1, NFκB and pNFκB levels. Full‑length blots are presented in Fig. S7: Fig. 5M. (N) Schematic illustration of exosomal miR-140-3p playing crucial roles in the prevention and treatment of RR. PDLCs exhibit elevated expression of DNMT1 in response to mechanical stimulation, and increased DNMT1 reduces the protein level of SOCS1 by upregulating DNA methylation to impact transcription. The inhibition of SOCS1 level, as a negative regulator in the NFκB signaling pathway, facilitates the assembly of the NLRP3 inflammasome which promotes the activation of caspase-1. Subsequently, pro-IL-1, pro-IL-18, and GSDMD are cleaved into mature forms by activated caspase-1. The GSDMD-N terminal region facilitates the formation of cell membrane pores, resulting in cell membrane rupture and the release of contents including IL-1β and IL-18, which leads to inflammatory microenvironment that directly stimulates osteoclast formation or indirectly enhances M1 polarization and inhibits M2 polarization to promote osteoclastogenesis. Furthermore, within exosomes from the supernatant of mechanically forced DFSCs (Force-Exo), miRNA-140-3p is significantly overexpressed. When treating force-induced RR with Force-Exo, DFSC-derived exosomal miRNA-140-3p targets DNMT1 to downregulate DNA methylation and upregulate protein expression of SOCS1, which decreases the levels of NFκB and downregulates NLRP3-mediated PDLC pyroptosis to reduce M1 polarization, thereby suppressing osteoclast formation and RR