Department of Dermatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Department of Cardiovascular Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Autoimmunity. 2021 May;54(3):129-137. doi: 10.1080/08916934.2021.1897976. Epub 2021 Mar 24.
Psoriasis is a chronic, inflammatory skin disease. It was reported that lncRNA Non-coding RNA-activated by DNA damage (NORAD) has potential regulatory effects on skin diseases. Our previous studies found that lncRNA NORAD was highly expressed and its potential target miR-26a was down-regulated in psoriasis model mice. Here, we aimed to investigate the role of NORAD in the development of psoriasis.
IL-22/LPS (interleukin-22/lipopolysaccharide)-stimulated HaCaT (human immortalized keratinocytes) cell model and imiquimod-induced mouse model were established. Keratin 6 (K6), Keratin 16 (K16), Keratin 17 (K17), and Cell division cycle 6 (CDC6) levels were detected by western blot. Cell activity was detected by CCK-8, MTT, and EdU assays. Quantitative real-time PCR was performed to examine the levels of NORAD, miR-26a, CDC6, K6, K16, and K17. Haematoxylin-eosin staining was applied to observe the degree of skin thickening and hyperplasia. Fluorescence hybridization detects the location of NORAD. RNA immunoprecipitation, RNA pull-down, and Luciferase test were performed to detect the interaction between NORAD and miR-26a.
In IL-22/LPS-stimulated HaCaT cells, NORAD, CDC6, and keratinocyte proliferation-related proteins (K6, K16, and K17) were up-regulated and miR-26a was down-regulated. Cell survival and proliferation were also increased. However, the results were reversed after interference with NORAD. Also, experiments revealed that NORAD negatively regulated miR-26a. In IL-22/LPS-stimulated HaCaT cells and skin of imiquimod-induced mice, we found that lower NORAD resulted in an increase of miR-26a and a decrease of CDC6, further decreased levels of keratinocyte proliferation-related proteins (K6, K16, and K17).
银屑病是一种慢性炎症性皮肤病。有报道称，长非编码 RNA 激活的 DNA 损伤（NORAD）对皮肤疾病具有潜在的调控作用。我们之前的研究发现，在银屑病模型小鼠中，lncRNA NORAD 表达水平升高，其潜在靶基因 miR-26a 表达下调。在此，我们旨在研究 NORAD 在银屑病发病机制中的作用。
建立白细胞介素 22/脂多糖（IL-22/LPS）刺激 HaCaT（人永生化角质形成细胞）细胞模型和咪喹莫特诱导的小鼠模型。采用 Western blot 检测角蛋白 6（K6）、角蛋白 16（K16）、角蛋白 17（K17）和细胞分裂周期蛋白 6（CDC6）的水平。通过 CCK-8、MTT 和 EdU 实验检测细胞活性。采用实时定量 PCR 检测 NORAD、miR-26a、CDC6、K6、K16 和 K17 的水平。苏木精-伊红（HE）染色观察皮肤增厚和过度增生的程度。荧光原位杂交检测 NORAD 的定位。采用 RNA 免疫沉淀、RNA 下拉和荧光素酶实验检测 NORAD 与 miR-26a 的相互作用。
在 IL-22/LPS 刺激的 HaCaT 细胞中，NORAD、CDC6 和角质形成细胞增殖相关蛋白（K6、K16 和 K17）上调，miR-26a 下调。细胞存活和增殖也增加。然而，干扰 NORAD 后结果逆转。此外，实验还表明 NORAD 负调控 miR-26a。在 IL-22/LPS 刺激的 HaCaT 细胞和咪喹莫特诱导的小鼠皮肤中，我们发现 NORAD 表达下调导致 miR-26a 表达增加，CDC6 表达减少，进一步导致角质形成细胞增殖相关蛋白（K6、K16 和 K17）表达减少。
