Medical School of Ophthalmology & Optometry, Shandong University of Traditional Chinese Medicine, Jinan 250002, China.
Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases, Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan 250002, China.
Mediators Inflamm. 2018 Oct 29;2018:2574067. doi: 10.1155/2018/2574067. eCollection 2018.
Uveitis is a serious eye disease that usually damages young adult's health. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate messenger RNA (mRNA) expression. It is predicted that rno-miR-30b-5p can regulate the expressions of interleukin-10 (IL-10) and Toll-like receptor 4 (TLR4). In this study, the regulatory role of rno-miR-30b-5p in IL-10 and TLR4 gene expressions was validated using luciferase activity assay. Further, the inflammatory manifestation of the anterior segment and pathological examination of the eye were explored in experimental autoimmune uveitis (EAU) rats. Meanwhile, the levels of rno-miR-30b-5p in eye tissues, spleen, and lymph nodes were measured using quantitative PCR (Q-PCR). IL-10 and TLR4 in spleen and lymph nodes were further separately determined by using Q-PCR and Enzyme-Linked Immunosorbent Assay (ELISA). Moreover, rno-miR-30b-5p mimic and its inhibitor were separately transfected into purified T cells, and the levels of IL-10 and TLR4 were detected using PCR, flow cytometry, and ELISA techniques. Results indicate that rno-miR-30b-5p was downregulated in spleen, lymph nodes, and eye tissues whereas the expressions of IL-10 and TLR4 at mRNA and protein levels were upregulated. The levels of IL-10 and TLR4 were negatively correlated to rno-miR-30b-5p levels. The result of in vitro cell transfection experiment indicates that IL-10 and TLR4 expressions were inhibited at mRNA and protein levels after T cells incubated with rno-miR-30b-5p mimic. However, the IL-10 and TLR4 mRNA levels were upregulated in purified T cells from spleen and lymph nodes after treatment with miR-30b-5p antagonist. In addition, there was no evident change of IL-10 and TLR4 proteins in spleen and lymph node T cells between EAU control and negative treatment groups. Flow cytometry analysis revealed that rno-miR-30b-5p mimic could reduce the number of both IL-10 and TLR4 positive cells, whereas rno-miR-30b-5p inhibitor could increase the number of IL-10 and TLR4 positive cells. Our study demonstrates that rno-miR-30b-5p influences the development of uveitis by regulating the level of IL-10 and TLR4 positive cells, thereby playing a role in the pathogenesis of uveitis.
葡萄膜炎是一种严重的眼部疾病，通常会损害年轻成年人的健康。MicroRNAs（miRNAs）是一类调节信使 RNA（mRNA）表达的小非编码 RNA。据预测，rno-miR-30b-5p 可以调节白细胞介素 10（IL-10）和 Toll 样受体 4（TLR4）的表达。在这项研究中，使用荧光素酶活性测定法验证了 rno-miR-30b-5p 对 IL-10 和 TLR4 基因表达的调节作用。此外，在实验性自身免疫性葡萄膜炎（EAU）大鼠中，研究了前节的炎症表现和眼部的病理检查。同时，使用定量 PCR（Q-PCR）测量了眼部组织、脾脏和淋巴结中的 rno-miR-30b-5p 水平。进一步使用 Q-PCR 和酶联免疫吸附测定（ELISA）分别测定了脾脏和淋巴结中的 IL-10 和 TLR4。此外，分别将 rno-miR-30b-5p 模拟物及其抑制剂转染入纯化的 T 细胞中，并使用 PCR、流式细胞术和 ELISA 技术检测 IL-10 和 TLR4 的水平。结果表明，rno-miR-30b-5p 在脾脏、淋巴结和眼部组织中的表达下调，而 IL-10 和 TLR4 的表达在 mRNA 和蛋白水平上均上调。IL-10 和 TLR4 的水平与 rno-miR-30b-5p 的水平呈负相关。体外细胞转染实验结果表明，rno-miR-30b-5p 模拟物孵育后，T 细胞的 IL-10 和 TLR4 表达在 mRNA 和蛋白水平上均受到抑制。然而，用 miR-30b-5p 拮抗剂处理后，脾脏和淋巴结中纯化的 T 细胞的 IL-10 和 TLR4 mRNA 水平上调。此外，在 EAU 对照组和阴性对照组的脾脏和淋巴结 T 细胞中，IL-10 和 TLR4 蛋白没有明显变化。流式细胞术分析表明，rno-miR-30b-5p 模拟物可减少 IL-10 和 TLR4 阳性细胞的数量，而 rno-miR-30b-5p 抑制剂可增加 IL-10 和 TLR4 阳性细胞的数量。我们的研究表明，rno-miR-30b-5p 通过调节 IL-10 和 TLR4 阳性细胞的水平来影响葡萄膜炎的发展，从而在葡萄膜炎的发病机制中发挥作用。
