关键词: RNA, 环状, 毛乳头细胞, circ_0005062, RNA结合蛋白质FUS, p21活化激酶类, 半胱氨酸天冬氨酸蛋白酶1, 炎性损伤, 雄激素性脱发

Abstract: 【Abstract】 Objective To investigate the targeting interaction between circ_0005062 and fused in sarcoma （FUS）/p21-activated kinase 1 （PAK1） mRNA, and to explore molecular mechanisms underlying inflammatory injury in human dermal papilla （HDP） cells via modulating the caspase-1 signaling pathway. Methods Hair follicle tissues were collected from 10 patients with androgenetic alopecia （AGA） and 10 healthy volunteers. Quantitative real-time polymerase chain reaction （qPCR） was performed to determine the expression of circ_0005062, enzyme-linked immunosorbent assay （ELISA） to detect levels of inflammatory cytokines interleukin （IL）-6, IL-1β, IL-18, and tumor necrosis factor-α （TNF-α） in the cell culture supernatant, and Western blot analysis to determine the protein expression of PAK1, cleaved caspase-1/caspase-1, and cleaved N-terminal gasdermin D （GSDMD）in these tissues. An in vitro HDP injury model was established by treating HDP cells with 100 μmol/L dihydrotestosterone （DHT） for 24 hours. Based on this model, circ_0005062-knockdown or PAK1-overexpression HDP cells were constructed. The experimental groups were designed as follows: a DHT group was treated with DHT; a sh-circ group and a sh-NC group were transfected with sh-circ_0005062 and control plasmids, respectively; a FUS group and a FUS-NC group were transfected with FUS overexpression plasmids and control plasmids, respectively; a PAK1 group and a PAK1-NC group were transfected with PAK1 overexpression plasmids and control plasmids, respectively; untreated HDP cells served as the control group. Cell viability was assessed using the MTT assay in the control group, DHT group, DHT + sh-NC group, and DHT + sh-circ group, qPCR was performed to determine the expression of circ_0005062, PAK1 mRNA, and caspase-1 mRNA, ELISA to detect the levels of IL-6, IL-1β, IL-18, and TNF-α in the cell culture supernatant, and Western blot analysis to determine the protein expression of PAK1, cleaved caspase-1/caspase-1, and cleaved N-terminal GSDMD. RNA immunoprecipitation （RIP） assay was conducted to assess the enrichment of circ_0005062 and PAK1 mRNA bound to FUS in cells from the control group, sh-circ group, and sh-NC group. In addition, qPCR was performed to determine the expression of circ_0005062, FUS mRNA, and PAK1 mRNA in the control group, sh-NC + FUS-NC group, sh-circ + FUS-NC group, and sh-circ + FUS group. Statistical analysis was performed using the two independent samples t-test or one-way analysis of variance, and multiple comparisons were conducted using the least significant difference-t test. Results In the clinical samples, the expression level of circ_0005062 in hair follicle tissues was significantly higher in patients with AGA than in healthy controls （2.21 ± 0.17 vs. 1.00 ± 0.03, t = 12.01, P < 0.001）. In addition, the levels of IL-6, IL-1β, IL-18, and TNF-α, as well as the protein expression levels of PAK1, cleaved caspase-1/caspase-1, and cleaved N-terminal GSDMD, were all significantly higher in patients with AGA than in healthy controls （all P < 0.05）. In the cell experiments, cell viability was significantly lower in the DHT group than in the control group, but significantly higher in the DHT + sh-circ group than in the DHT + sh-NC group （both P < 0.05）; compared with the control group, the DHT group showed significantly increased expression levels of circ_0005062, PAK1 mRNA, caspase-1 mRNA, the inflammatory cytokines IL-6, IL-1β, IL-18, and TNF-α, as well as the proteins PAK1, cleaved caspase-1/caspase-1, and cleaved N-terminal GSDMD （all P < 0.05）; however, an opposite trend was observed in the DHT + sh-circ group compared with the DHT + sh-NC group （all P < 0.05）. RIP assays showed that FUS could bind to both circ_0005062 and PAK1 mRNA. After treatment with actinomycin D for 4 hours, the mRNA expression level of PAK1 was significantly lower in the sh-circ + FUS-NC group than in the control group and the sh-NC + FUS-NC group, whereas it was significantly higher in the sh-circ + FUS group than in the sh-circ + FUS-NC group （all P < 0.05）. Compared with the DHT group, the DHT + sh-circ + PAK1-NC group showed significantly decreased expression levels of circ_0005062, PAK1 mRNA, caspase-1 mRNA, the inflammatory cytokines IL-6, IL-1β, IL-18, and TNF-α, as well as the proteins PAK1, cleaved caspase-1/caspase-1, and cleaved N-terminal GSDMD （all P < 0.05）; however, an opposite trend was observed in the DHT + sh-circ + PAK1 group compared with the DHT + sh-circ + PAK1-NC group （all P < 0.05）. In addition, the expression level of circ_0005062 was significantly higher in the DHT group than in the control group, but significantly lower in the DHT + sh-circ + PAK1-NC group than in the DHT group （both P < 0.05）, and there was no significant difference between the DHT + sh-circ + PAK1 group and the DHT + sh-circ + PAK1-NC group （P > 0.05）. Conclusion circ_0005062 may aggravate inflammatory injury in HDP cells by targeting the FUS/PAK1 axis and regulating caspase-1 expression, thereby inhibiting cell proliferation and promoting inflammatory responses.

Key words: RNA, circular, Human dermal papilla cells, circ_0005062, RNA-binding protein FUS, p21-Activated kinases, Caspase 1, Inflammatory injury, Androgenetic alopecia