Jiangxi Provincial Key Laboratory of Oral Biomedicine, Department of Orthodontics, the Affiliated Stomatological Hospital of Nanchang University, Nanchang, 330006, China.
Jiangxi Provincial Key Laboratory of Oral Biomedicine, Department of Oral Prosthodontics, the Affiliated Stomatological Hospital of Nanchang University, Nanchang, 330006, China.
Mol Med. 2021 Oct 20;27(1):132. doi: 10.1186/s10020-021-00382-4.
The FOXP3/miR-146a/NF-κB axis was previously reported to modulate the induction and function of CD4+ Treg cells to alleviate oral lichen planus. Also, other signaling pathways including microRNA-155-IFN-γ loop and FOXP3/miR-146a/TRAF6 pathways were reported to be involved in the pathogenesis of oral lichen planus. In this study, we aimed to investigate the molecular mechanism underlying the pathogenesis of EOLP.
CircRNA microarray was used to observe the expression of candidate circRNAs in CD4+ T-cells collected from different groups. Real-time PCR and Western blot were conducted to observe the changes in the expression of different miRNAs, mRNAs and proteins. Flow cytometry was performed to compare the counts of Treg cells in the HC and EOLP groups, and ELISA was performed to evaluate the changes in the expression of inflammatory cytokines.
No obvious differences were seen between the HC and EOLP groups in terms of age and gender. Among all candidate circRNAs, the expression of circ_003912 was most dramatically elevated in CD4+ T-cells collected from the EOLP group. The levels of miR-1231, miR-31, miR-647, FOXP3 mRNA and miR-146a were decreased while the expression of TRAF6 mRNA was increased in CD4+ T-cells collected from the EOLP group. The count of Treg cells in the EOLP group was dramatically increased. The levels of inflammatory cytokines including IL-4 IFN-γ, IL-10 and IL-2 were influenced by the presence of circ_003912. In CD4+ T-cells in the EOLP group, the levels of IL-4 and IL-10 were decreased while the levels of IFN-γ and IL-2 were increased. The presence of miR-1231, miR-31 and miR-647 all obviously inhibited the expression of circ_003912, which was validated to sponge the expression of above miRNAs. Also, FOXP3 mRNA was proved to be targeted by miR-1231, miR-31 and miR-647. Transfection of circ_003912 up-regulated the expression of circ_003912, miR-146a and FOXP3 mRNA/protein while down-regulating the expression of miR-1231, miR-31, miR-647, and TRAF6 mRNA/protein. The levels of inflammatory cytokines including IL-4 IFN-γ, IL-10 and IL-2 as well as the speed of cell proliferation were influenced by circ_003912.
In this study, we investigated the molecular mechanisms underlying the pathogenesis of EOLP which involved the functioning of circ_003912. We first demonstrated that circ_003912 was up-regulated in CD4+ T-cells of the EOLP group. And miRNAs including miR-1231, miR-31 and miR-647 were sponged by circ_003912 and down-regulated in CD4+ T cells of the EOLP group, which subsequently up-regulated the expression of FOXP3 and miR-146a, and resulted in the inhibition of NF-kB.
FOXP3/miR-146a/NF-κB 轴被报道可以调节 CD4+Treg 细胞的诱导和功能，从而减轻口腔扁平苔藓的症状。此外，其他信号通路，包括 microRNA-155-IFN-γ 环和 FOXP3/miR-146a/TRAF6 通路，也被报道与口腔扁平苔藓的发病机制有关。在本研究中，我们旨在探讨 EOLP 发病机制的分子机制。
使用 circRNA 微阵列观察来自不同组的 CD4+T 细胞中候选 circRNAs 的表达。实时 PCR 和 Western blot 用于观察不同 miRNAs、mRNAs 和蛋白质表达的变化。流式细胞术用于比较 HC 和 EOLP 组中 Treg 细胞的计数，ELISA 用于评估炎症细胞因子表达的变化。
HC 和 EOLP 组在年龄和性别方面没有明显差异。在所有候选 circRNAs 中，circ_003912 在 EOLP 组的 CD4+T 细胞中的表达最为显著升高。在 EOLP 组的 CD4+T 细胞中，miR-1231、miR-31、miR-647、FOXP3 mRNA 和 miR-146a 的水平降低，而 TRAF6 mRNA 的表达增加。EOLP 组 Treg 细胞的数量明显增加。包括 IL-4、IFN-γ、IL-10 和 IL-2 在内的炎症细胞因子的水平受到 circ_003912 的影响。在 EOLP 组的 CD4+T 细胞中，IL-4 和 IL-10 的水平降低，而 IFN-γ 和 IL-2 的水平升高。miR-1231、miR-31 和 miR-647 的存在明显抑制了 circ_003912 的表达，这被证明可以与上述 miRNAs 结合。此外，FOXP3 mRNA 被证明是 miR-1231、miR-31 和 miR-647 的靶标。circ_003912 的转染上调了 circ_003912、miR-146a 和 FOXP3 mRNA/蛋白的表达，同时下调了 miR-1231、miR-31、miR-647 和 TRAF6 mRNA/蛋白的表达。包括 IL-4、IFN-γ、IL-10 和 IL-2 在内的炎症细胞因子水平以及细胞增殖速度受到 circ_003912 的影响。
在本研究中，我们研究了 EOLP 发病机制的分子机制，涉及 circ_003912 的功能。我们首先证明 circ_003912 在 EOLP 组的 CD4+T 细胞中上调。包括 miR-1231、miR-31 和 miR-647 在内的 miRNAs 被 circ_003912 吸收并在 EOLP 组的 CD4+T 细胞中下调，随后上调 FOXP3 和 miR-146a 的表达，从而抑制 NF-κB。
