The circular RNA circ_0099630/miR-940/receptor-associated factor 6 regulation cascade modulates the pathogenesis of periodontitis

Abstract

Background/purpose: Periodontitis is one of the highly prevalent chronic inflammatory conditions in adults. The importance of circular RNAs (circRNAs) in the regulation of inflammation has been gradually reported in recent years, but the role of circRNA circ_0099630 in periodontitis has not been reported.

Materials and methods: The contents of circ_0099630, microRNA-940 (miR-940) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. Inflammatory factor secretion, cell proliferation, and apoptosis were analyzed under the application of Enzyme-linked immunosorbent assay (ELISA), Cell Counting Kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU) and flow cytometry, respectively. The Western blot also analyzed the phosphorylation levels of RELA proto-oncogene (P65) and IkappaBalpha (IκBα), key molecules of the nuclear factor kappa-B (NF-κB) pathway. The relationship between miR-940 and circ_0099630 or TRAF6 was verified by luciferase reporter system and RNA immunoprecipitation (RIP) assay.

Results: Higher abundance of circ_0099630 and TRAF6 and lower miR-940 expression were observed in periodontitis, and circ_0099630 knockdown attenuated the damage of human PDL cells (PDLCs) induced by lipopolysaccharides (LPS). The relationship between miR-940 and circ_0099630 or TRAF6 was evidenced, while miR-940 downregulation diminished the repair effect of si-circ_0099630 on overexpression LPS-induced damage in PDLCs. Similarly, TRAF6 upregulation impaired the mitigating effect of miR-940 overexpression on LPS-induced injury in PDLCs. Circ_0099630 silencing evidently curbed the phosphorylation levels of P65 and IκBα and thus attenuating the inflammatory response by acting on the miR-940/TRAF6 axis.

Conclusion: Silencing circ_0099630 alleviates LPS-induced periodontal ligament cell injury via targeting miR-940/TRAF6/NF-κB in periodontitis.

Keywords: 3′UTRs, 3′ untranslated regions; ATG14, autophagy related 14; CCK8, Cell Counting Kit-8; Cdr1as, circRNA CDR1 antisense RNA; Circ_0099630; ELISA, Enzyme-linked immunosorbent assay; EdU, 5-ethynyl-2′-deoxyuridine; FOXM1, forkhead box M1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharides; MiR-940; NF-κB, nuclear factor kappa-B; PDLCs, periodontal ligament cells; Periodontitis; RIP, RNA immunoprecipitation; RIPA, radioimmune precipitation assay; TNF, tumor necrosis factor; TRAF6; TRAF6, tumor necrosis factor receptor-associated factor 6; U6, U6 small nuclear RNA; ceRNA, competing endogenous RNA; circRNAs, circular RNAs; miR-940, microRNA-940; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 1

Circ_0099630 level in periodontitis tissues and LPS-induced PDLCs was analyzed. (A and B) Circ_0099630 level was detected in periodontitis tissues and LPS-induced PDLCs by qRT-PCR. (C) The execution of qRT-PCR analyzed the levels of circ_0099630 and GAPDH after RNase R treatment. (D) The amplification effects of Oligo(dt)18 primers and random primers on circ_0099630 and GAPDH were examined using qRT-PCR. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.001.

Figure 2

Effects of different concentrations of LPS on inflammatory response, viability, proliferation and apoptosis of PDLCs. (A–F) After treatment of PDLCs with 0 μg/mL, 2 μg/mL, 4 μg/mL, and 8 μg/mL of LPS, (A and B) IL-6 and TNF-α levels were measured by ELISA. (C) CCK8 assay measured cell viability. (D) The application of EdU assay assessed cell proliferation. (E) Apoptosis rate was tested by flow cytometry. (F) Cleaved-caspase-3 and Bax levels were measured using Western blot. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.001.

Figure 3

Effects of circ_0099630 downregulation on LPS-induced PDLCs. (A–H) The tests were divided into four groups (Control, LPS, LPS + si-NC, LPS + si-circ_0099630). (A) Circ_0099630 level was tested using qRT-PCR. (B and C) Under the use of ELISA, IL-6 and TNF-α cell levels were analyzed. (D) Cell viability was detected by CCK8. (E) EdU was applied to examine proliferation. (F) PDLCs apoptosis was assayed using flow cytometry. (G and H) Cleaved-caspase-3 and Bax protein levels were assessed by Western blot. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.001.

Figure 4

The relationship between circ_0099630 and miR-940 was evidenced. (A) The complementary sequences of circ_0099630 and miR-940. (B) Overexpression efficiency of miR-940 mimic was confirmed after the performance of qRT-PCR. (C) Targeting between circ_0099630 and miR-940 was validated using dual-luciferase reporter system. (D) Targeting between circ_0099630 and miR-940 was validated by RIP assay. (E) MiR-940 level in periodontitis tissues was analyzed via qRT-PCR. (F) Pearson's analysis revealed the correlation between miR-940 and circ_0099630 levels in periodontitis tissues. (G) QRT-PCR determined the miR-940 level in LPS-induced PDLCs. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.001.

Figure 5

MiR-940 and circ_0099630 jointly regulated the function of LPS-induced PDLCs. (A–H) There were six groups which were Control, LPS, LPS + si-NC, LPS + si-circ_0099630, LPS + si-circ_0099630+anti-miR-NC, LPS + si-circ_0099630+anti-miR-940. (A) The implementation of qRT-PCR analyzed miR-940 level. (B and C) ELISA detected the IL-6 and TNF-α levels. (D) Cell viability was detected using CCK8 assay. (E) EdU assay was performed to detect proliferation. (F and G) The execution of flow cytometry determined apoptosis. (H) Cleaved-caspase-3 and Bax protein levels were measured by Western blot. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.001.

Figure 6

The relationship between TRAF6 and miR-940 was validated. (A) Binding sites of TRAF6 and miR-940. (B) The targeting relationship of TRAF6 and miR-940 was evidenced through dual-luciferase reporter system. (C) RIP assay proved the relationship of TRAF6 and miR-940. (D) TRAF6 content in periodontitis tissues was analyzed by qRT-PCR. (E) Correlation analysis of TRAF6 and miR-940 levels in periodontitis tissues was revealed using Pearson analysis. (F and G) Western blot measured TRAF6 protein level in periodontitis tissues and LPS-induced PDLCs. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.001.

Figure 7

LPS-induced PDLCs function was co-regulated by miR-940/TRAF6 axis. (A–H) There were six groups, Control, LPS, LPS + miR-NC, LPS + miR-940, LPS + miR-940+pcDNA, LPS + miR-940+TRAF6. (A and B) IL-6 and TNF-α levels were measured through ELISA. (C and D) Cell viability and proliferation were detected after the performance of CCK8 and EdU assays. (F and G) Flow cytometry was executed to analyze the apoptosis. (H) Cleaved-caspase-3 and Bax protein levels were revealed in Western blot. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.001.

Figure 8

Circ_0099630 and miR-940 co-regulated the levels of TRAF6 and key molecules in the NF-κB pathway. (A) TRAF6 protein level was checked by Western blot. (B) Under the application of Western blot, the phosphorylation levels of P65 and IkBα were analyzed. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.001.