Gene expression of keratinocytes with miR-885-5p inhibited vs control. Gene expression of keratinocytes with miR-885-5p inhibited vs control

As miR-885-5p is a microRNA downregulated in Cutaneous Lupus Erythematosus (CLE) and it is localted in the epidemis and its function is focused in keratinocytes, human Arrays to study the role of miR-885-5p in Cutaneous Lupus Erythematosius will be conducted.Gene expression in keratinocytes transfected with anti-miR-885-5p will be studied. We have included 24 Samples: Non-stimulated control (4), Non-stimulated anti-miR-885-5p (4), UVB-stimulated control (4), UVB-stimulated antimiR-885-5p (4), IFN alpha-stimulated control (4) and IFN alpha-stimulated anti-miR-885-5p Overall design: Microarray gene expression profiling. We will compare the gene expression of keratinocytes with miR-885-5p inhibited vs control. Keraitnocytes were cultured in 24 well plated and transfected with anti-miR-885-5p or anti-miRNA control according to standard protocols. 24h post-transfection, they will be stimulated with different inflammatory treatments: UVB 25 mJ/cm2, IFN alpha 10 ng/mL or PBS (control, non-stimualted). UVB and IFN alpha are known CLE triggers and revelant in the pathogenesis of CLE. Three comparisons will be analysed in order to understand the role of miR-885-5p: 1) Non-stimulated control vs Non-stimulated anti-miR 885-5p 2) UVB control vs UVB anti-miR-885-5p. 3) IFN alpha control vs IFN alpha anti-miR-885-5p.

鉴于miR-885-5p是一种在皮肤红斑狼疮（Cutaneous Lupus Erythematosus, CLE）中表达下调的微小RNA，且其定位于表皮，功能主要聚焦于角质形成细胞，本研究将开展人类基因芯片实验以探究miR-885-5p在皮肤红斑狼疮中的作用。本研究将分析转染抗miR-885-5p寡核苷酸的角质形成细胞中的基因表达情况。
本研究共纳入24例样本，分为6组，每组4例：未刺激对照组（4例）、未刺激抗miR-885-5p组（4例）、中波紫外线（UVB）刺激对照组（4例）、UVB刺激抗miR-885-5p组（4例）、α干扰素（IFN alpha）刺激对照组（4例）以及α干扰素刺激抗miR-885-5p组（4例）。
整体实验设计为基因芯片基因表达谱分析。本研究将对比miR-885-5p被抑制的角质形成细胞与对照组的基因表达差异。实验中，角质形成细胞接种于24孔板，按照标准实验流程转染抗miR-885-5p或抗微小RNA对照序列。转染24小时后，分别采用不同炎症刺激物处理：25 mJ/cm²的UVB、10 ng/mL的IFN alpha，或磷酸盐缓冲液（PBS，作为未刺激的对照组）。已知UVB与IFN alpha均为皮肤红斑狼疮的触发因素，且与皮肤红斑狼疮的发病机制密切相关。为阐明miR-885-5p的作用，本研究将开展3组对比分析：1）未刺激对照组 vs 未刺激抗miR-885-5p组；2）UVB刺激对照组 vs UVB刺激抗miR-885-5p组；3）IFN alpha刺激对照组 vs IFN alpha刺激抗miR-885-5p组。