Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, United States.
Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States.
Invest Ophthalmol Vis Sci. 2023 Apr 3;64(4):1. doi: 10.1167/iovs.64.4.1.
To analyze the changes in the lacrimal gland (LG) miRNAome from male nonobese diabetic (NOD) mice with autoimmune dacryoadenitis compared with LG from healthy male BALB/c and dacryoadenitis-free female NOD mice.
LG from these mice were collected for small RNA sequencing to identify dysregulated miRNAs; hits were validated by RT-qPCR in male NOD and BALB/c LG. Dysregulation of validated species within immune cell-enriched cell fractions and epithelial-enriched cell fractions from LG was probed by RT-qPCR. Ingenuity pathway analysis identified putative miRNA targets, which were examined in publicly available mRNA-seq datasets. Western blotting and confocal imaging of immunofluorescence enabled validation of some molecular changes at the protein level.
Male NOD LG exhibited 15 and 13 significantly up- and downregulated miRNAs, respectively. Dysregulated expression of 14 of these miRNAs (9 upregulated, 5 downregulated) was validated in male NOD versus BALB/c LG by RT-qPCR. Seven of the upregulated miRNAs were increased owing to their abundance in immune cell-enriched cell fractions, whereas four downregulated miRNAs were largely expressed in epithelial-enriched cell fractions. Ingenuity pathway analysis predicted the upregulation of IL-6 and IL-6-like pathways as an outcome of miRNA dysregulation. Increased expression of several genes in these pathways was confirmed by mRNA-seq analysis, whereas immunoblotting and immunofluorescence confirmed Ingenuity pathway analysis-predicted changes for IL-6Rα and gp130/IL-6st.
Male NOD mouse LG exhibit multiple dysregulated miRNAs owing to the presence of infiltrating immune cells, and decreased acinar cell content. The observed dysregulation may increase IL-6Rα and gp130/IL-6st on acini and IL-6Rα on specific lymphocytes, enhancing IL-6 and IL-6-like cytokine signaling.
分析与健康雄性 BALB/c 小鼠和无泪腺炎的雌性 NOD 小鼠相比，自身免疫性泪腺炎的雄性 NOD 小鼠泪腺 (LG) miRNAome 的变化。
收集这些小鼠的 LG 进行小 RNA 测序以鉴定失调的 miRNA；在雄性 NOD 和 BALB/c LG 中通过 RT-qPCR 验证命中。通过 RT-qPCR 探测 LG 中免疫细胞富集细胞群和上皮细胞富集细胞群中验证 miRNA 物种的失调。通路分析鉴定了潜在的 miRNA 靶标，并在公共 mRNA-seq 数据集上进行了检查。Western blot 和免疫荧光共聚焦成像使一些分子变化在蛋白质水平上得以验证。
雄性 NOD LG 分别显示出 15 个和 13 个显著上调和下调的 miRNA。通过 RT-qPCR 验证了这些 miRNA 中 14 个（9 个上调，5 个下调）在雄性 NOD 与 BALB/c LG 之间的失调表达。上调的 7 个 miRNA 是由于它们在免疫细胞富集细胞群中的丰度增加，而下调的 4 个 miRNA 主要在上皮细胞富集细胞群中表达。通路分析预测 miRNA 失调会导致 IL-6 和 IL-6 样途径的上调。这些途径中的几个基因的表达增加通过 mRNA-seq 分析得到证实，而免疫印迹和免疫荧光证实了 Ingenuity 通路分析预测的 IL-6Rα 和 gp130/IL-6st 的变化。
雄性 NOD 小鼠 LG 由于浸润免疫细胞和腺泡细胞含量减少而表现出多种失调的 miRNA。观察到的失调可能会增加腺泡上的 IL-6Rα 和 gp130/IL-6st，以及特定淋巴细胞上的 IL-6Rα，增强 IL-6 和 IL-6 样细胞因子信号传导。
