In this study, we confirmed that stabilization of HIF-1α and activation of the NF-κB pathway occur during periodontal tissue remodeling in vivo and in vitro. OTM occurs via a variety of mechanisms, including mechanotransduction(7), local hypoxia(28), sterile inflammation(4), angiogenesis(23), osteoclastogenesis(29), and osteogenesis(30). In bone metabolism, HIF-1α is related to angiogenesis and bone remodeling(11, 31), but the mechanism by which HIF-1α mediates periodontal tissue remodeling is unclear. In mice, NF-κB inhibition decreased OTM(14) and enhanced NF-κB activity decreased osteogenesis in mesenchymal stem cells(32), indicating that the activation of NF-κB pathway plays an essential role in OTM. Therefore, investigation of hypoxic and inflammatory responses and their underlying mechanisms in OTM is needed.
In this study, SNHG8 dramatically decreased during OTM, both in vivo and in vitro. SNHG8 is a tumor-associated lncRNA upregulated in various tumor types, increasing the proliferation, migration, invasion, and metastasis of cancer cells(33). In non-tumor diseases, upregulated SNHG8 serves as a competitive endogenous RNA by sponging miR-425‐5p, and inhibits the SIRT1/NF‐κB signaling pathway to attenuate the ischemia-induced microglial inflammatory response(21). However, SNHG8 also affects myocardial infarction by promoting activity in the NF‐κB pathway(22). This opposite phenomenon reflects the high tissue or cell specificity of lncRNAs and their diverse mechanisms of action, including transcriptional regulation in cis or trans, organization of nuclear domains, and regulation of proteins or RNAs(19, 20). In this study, we first explored whether SNHG8 regulates the NF‐κB pathway in PDLCs. To rule out possible interference of other factors in the OTM complex microenvironment, we used the commonly recognized activator TNF-α to activate the NF‐κB pathway(34). The results confirmed that, in PDLCs, SNHG8 inhibits inflammation and negatively regulates the NF‐κB pathway.
To further explore the mechanism by which SNHG8 regulates the NF-κB pathway, we performed RNA sequencing. The results confirmed the anti-inflammatory effect of SNHG8. However, SNHG8 also has a function in the cellular response to hypoxia. Several downstream genes of HIF-1 related to hypoxia were significantly downregulated by SNHG8 overexpression, suggesting the importance of the interaction between SNHG8 and HIF-1α. Most lncRNAs localize to the nucleus and some exert their effects by binding to transcription factors(20, 35). For instance, lincRNA-p21 binds von Hippel-Lindau (VHL) protein and HIF-1α separately, disrupting their interaction and stabilizing HIF-1α to enhance glycolysis in cancer cells(36). HIF-1 is an αβ-heterodimeric transcription factor, the HIF-1α subunit of which is stabilized by hypoxia, whereas the HIF-1β subunit is a constitutive nuclear protein. The two combine in the nucleus to form HIF-1, which binds to a variety of genes whose promoters contain the hypoxic response element and regulates their transcription(9, 37). In this study, SNHG8 was localized to the nucleus, indicating that SNHG8 could binds to HIF-1α, thereby affecting the function of HIF-1 as a transcription factor.
The interaction strength between SNHG8 and HIF-1α predicted by catRAPID(38–40) was computed using a reference set composed of 100 random protein and 100 random RNA sequences having the same lengths as the factors in this study. Strength values above 50% indicated high specificity for the interaction. The DP is a statistical measure of the interaction propensity, and represents the confidence of the catRAPID prediction. DP values above 75% represent high-confidence predictions. In this study, the DP and interaction strength between SNHG8 and HIF-1α were 99% and 95%, respectively, so the binding is highly specific and reliable. Subsequent RIP and pulldown assays confirmed the binding. However, we used the full-length sequence for prediction and validation. The specific binding site warrants further study.
The NF-κB pathway can be activated during OTM by a variety of mechanisms. Mechanical stimulation triggers p65 phosphorylation directly(13). NF‐κB can also be activated by a series of cell-surface receptors, proinflammatory cytokines, and κB kinase(41–43). In addition, NF‐κB is activated by the deformation of blood vessels and recruitment of circulating monocytes and macrophages by the endothelium(2, 3). The effect of the HIF system on NF‐κB varies(18). HIF-1α activates NF‐κB to promote the survival of neutrophils(44), and negatively regulates NF‐κB in T cells(45). However, the effect of HIF-1α on the NF‐κB pathway in PDLCs during OTM has not been demonstrated. In this study, we showed that the activation of NF-κB in PDLCs under compressive force is HIF-1α-dependent, and that SNHG8 interferes with this regulation by binding to HIF-1α directly; these findings improve our knowledge of OTM. However, lncRNAs can play multiple regulatory roles, so other factors may also be involved. Finally, the upstream regulation of SNHG8, and the influence of the mechanism on downstream osteoclastogenesis, require further exploration.