miR-34a Mediates IL-23/IL-17 Immune Inflammation and Promotes Cell Proliferation in HaCaT Cells by Targeting SIRT1/NLRP3.

Psoriasis is a common immune-mediated skin disorder. miR-34a, as a significant regulatory component, has been involved in the regulation of immune-inflammatory responses. Previous studies have shown that miR-34a is abnormally expressed in psoriasis cell models. However, the role of miR-34a in psoriasis remains unvalidated by any existing studies. We co-stimulated HaCaT cells with IL-17A, IL-22, TNF-α, IL-1α, and M5 to establish an in vitro model of psoriasis. qPCR was used to detect the expression of miR-34a; ELISA was performed to measure the secretion levels of pro-inflammatory cytokines IL-1β, IL-6, IL-17, and IL-23 in HaCaT cells; CCK-8 and EdU staining assays were used to assess cell growth and proliferation; Western blot analysis was employed to assess the expression levels of relevant proteins, including SIRT1, NLRP3, ASC, pro-Caspase-1, and Caspase-1; dual-luciferase reporter gene assays and RNA immunoprecipitation (RIP) were conducted to investigate the interaction between miR-34a and SIRT1. Additionally, a mouse psoriasis model was induced by imiquimod (IMQ) to validate the role of miR-34a in vivo. qPCR results found that miR-34a was upregulated in HaCaT cells. ELISA results indicated that the levels of IL-1β, IL-6, IL-17, and IL-23 were significantly elevated in the M5-treated group. CCK-8 and EdU staining assays revealed that M5 treatment notably increased the proliferation and viability of HaCaT cells. After treatment with the miR-34a inhibitor, the levels of cytokines and cell proliferation were significantly reduced. Western blot analysis showed that M5 treatment resulted in a significant decrease in SIRT1 protein levels, while the expression levels of NLRP3, ASC, pro-Caspase-1, and Caspase-1 were increased. In contrast, miR-34a-inhibitor treatment showed the opposite results. Dual-luciferase reporter and RIP assays indicated a negative feedback interaction between miR-34a and SIRT1. Rescue experiments showed that overexpression of SIRT1 effectively inhibited NLRP3 expression, significantly reduced IL-23/IL-17 immune-inflammatory responses, and suppressed HaCaT cell proliferation, while overexpression of NLRP3 had the opposite effects. In the IMQ-induced mouse psoriasis model, the IMQ group showed severe psoriasis-like phenotypes, obvious skin pathological changes, upregulated miR-34a expression, and increased skin cytokine levels; all these abnormalities were alleviated by miR-34a inhibitor treatment. miR-34a mediates the IL-23/IL-17 immune-inflammatory response and promotes HaCaT cell proliferation by regulating the SIRT1/NLRP3 pathway.