牛皮癣是一种慢性,炎症性皮肤病。据报道,DNA损伤激活的lncRNA非编码RNA(NORAD)对皮肤病具有潜在的调节作用。我们之前的研究发现,lncRNANORAD在银屑病模型小鼠中高表达,其潜在靶标miR-26a下调。这里,我们旨在探讨NORAD在银屑病发生发展中的作用。
建立IL-22/LPS(白介素-22/脂多糖)刺激的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。角蛋白6(K6),角蛋白16(K16),角蛋白17(K17),通过蛋白质印迹检测细胞分裂周期6(CDC6)水平。用CCK-8、MTT、和EdU测定。进行定量实时PCR以检查NORAD的水平,miR-26a,CDC6、K6、K16和K17。应用苏木精-伊红染色观察皮肤增厚和增生程度。荧光原位杂交检测NORAD的位置。RNA免疫沉淀,RNA下拉,和荧光素酶试验检测NORAD和miR-26a之间的相互作用。
在IL-22/LPS刺激的HaCaT细胞中,北美防空司令部,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,体外实验显示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,较低的NORAD导致miR-26a增加和CDC6减少,进一步降低角质形成细胞增殖相关蛋白(K6,K16和K17)的水平.
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 in situ 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, in vitro 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).