Expression of miR-20b in plasma of patients with psoriasis vulgaris and its effect on keratinocytes / 中国医师杂志

ABSTRACT

Objective:To investigate the expression of microRNA-20b (miR-20b) in peripheral blood plasma of patients with psoriasis vulgis, and to further analyze the effect and potential mechanism of miR-20b on proliferation and apoptosis of human immortals keratinocytes (HaCaT cell line).Methods:The peripheral blood of 36 patients with psoriasis vulgaris and 36 healthy volunteers (as control group) were collected. The relative expression level of miR-20b in plasma was detected by real-time quantitative polymerase chain reaction (qRT-PCR). HaCaT cells were cultured in vitro and randomly divided into control group (untreated), interleukin-22 (IL-22)treatment group (stimulated with 100 ng/ml IL-22 for 24 h), IL-22+ inhibitor control group (after transfection of inhibitor negative control, stimulated with 100 ng/ml IL-22 for 24 h) and IL-22+ miR-20b inhibitor group (treated with 100 ng/ml IL-22 for 24 h after transfection with miR-20b inhibitor). The relative expression of miR-20b in HaCaT cells was detected by qRT-PCR. The proliferation and apoptosis of HaCaT cells were detected by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Bioinformatics software was used to predict the downstream targets of miR-20b. The targeted binding relationship between miR-20b and killin, p53-regulated DNA replication inhibitor (KLLN) 3′UTR was verified by dual luciferase reporter gene assay. The expression level of KLLN protein in HaCaT cells was detected by Western blot. Results:The plasma level of miR-20b in psoriasis patients was significantly higher than that in healthy controls [(1.62±0.53) vs (1.00±0.42), P<0.001]. The results of qRT-PCR showed that the expression of miR-20b in IL-22 treatment group was higher than that in control group ( P<0.05). The expression of miR-20b in IL-22+ miR-20b inhibitor group was lower than that in IL-22+ inhibitor control group ( P<0.05). CCK-8 results showed that the absorbance values of HaCaT cells in IL-22 treatment group were significantly higher than those in control group at 24, 48, 72 and 96 h (all P<0.05). The absorbance values of HaCaT cells in IL-22+ miR-20b inhibitor group at 48, 72 and 96 h were significantly lower than those in IL-22+ inhibitor control group (all P<0.05). The apoptosis rate of IL-22 treatment group was significantly lower than that of control group [(4.12±0.37)% vs (7.06±0.58)%], with statistically significant difference ( P<0.05). The apoptosis rate of HaCaT cells in IL-22+ miR-20b inhibitor group was significantly higher than that in IL-22+ inhibitor control group [(6.59±0.53)% vs (3.94±0.46)%], with statistically significant difference ( P<0.05). Dual luciferase reporter assay was used to verify the interaction between miR-20b and KLLN, and the results showed that the luciferase activity of KLLN wild-type was significantly inhibited by miR-20b mimics. Western blot results showed that the protein expression of KLLN in the IL-22 treatment group was lower than that in the control group ( P<0.05); the protein expression of KLLN in IL-22+ miR-20b inhibitor group was higher than that in IL-22+ inhibitor control group, with statistically significant difference ( P<0.05). Conclusions:MiR-20b is highly expressed in the plasma of patients with psoriasis vulgaris, and miR-20b may promote the proliferation and anti-apoptotic ability of keratinocytes by targeting KLLN.