关键词: 银屑病, 微RNAs, STAT3转录因子, 角蛋白细胞, 细胞增殖, 细胞凋亡, 犀地凉血方, LncRNA NEAT1

Abstract: 【Abstract】 Objective To investigate the effect of Xidi Liangxue recipe on the proliferation and apoptosis of HaCaT cells through the long non-coding RNA （lncRNA） nuclear-enriched abundant transcript 1 （NEAT1）/microRNA （miR）-485-5p/signal transducer and activator of transcription 3 （STAT3） regulatory network. Methods HaCaT cells were induced by interleukin-17 （IL-17）, and the mRNA and protein expression of lncRNA NEAT1, miR-485-5p and STAT3 was detected in IL-17-induced HaCaT cells and normal human epidermal keratinocytes （NHEK） by quantitative PCR （qPCR） and Western blot analysis, respectively. The location of lncRNA NEAT1 and miR-485-5p in IL-17-induced HaCaT cells was observed by fluorescence in situ hybridization （FISH）, and the targeted regulatory relationship among lncRNA NEAT1, miR-485-5p and STAT3 was verified by double-luciferase reporter gene assay. Chinese herbs were decocted according to the Xidi Liangxue recipe, SD rats were divided into two groups to be gavaged with the above decoctions (medicated group) or physiological saline (control group) for 5 days, and then serum samples were collected from the above two groups of rats separately. The IL-17-induced HaCaT cells were divided into 4 groups: control group treated with the control sera, lncRNA-NEAT1 overexpression group transfected with lncRNA-NEAT1 overexpression vectors and treated with the control sera, Xidi Liangxue recipe group treated with the medicated sera, and Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group transfected with lncRNA-NEAT1 overexpression vectors and treated with the medicated sera. qPCR, Western blot analysis, flow cytometry, and cell counting kit （CCK8） assay were performed to determine the mRNA and protein expression of lncRNA NEAT1, miR-485-5p and STAT3, and to evaluate cell proliferation and apoptosis. The two independent samples t-test was used for comparisons between two groups, one-way analysis of variance for comparisons among multiple groups, and least significant difference （LSD） t-test for multiple comparisons. Results The IL-17-induced HaCaT cell group showed significantly increased relative expression levels of lncRNA NEAT1 and STAT3 mRNA（1.84 ± 0.21, 2.20 ± 0.24, respectively） and significantly increased protein expression of STAT3 and p-STAT3 （1.27 ± 0.13, 2.43 ± 0.16, respectively）, but significantly decreased expression level of miR-485-5p （0.32 ± 0.04）compared with the NHEK group （ lncRNA NEAT1 and STAT3 mRNA: 1.00 ± 0.11, 1.00 ± 0.11, respectively, both P < 0.05; STAT3 and p-STAT3 protein: 1.00 ± 0.11, 1.00 ± 0.10, t = 2.54, 3.02, respectively, both P < 0.05; miR-485-5p: 1.00 ± 0.12, t = 2.94, P = 0.015）. FISH demonstrated that miR-485-5p and lncRNA NEAT1 were co-located in the cytoplasm of HaCaT cells. The double-luciferase reporter gene assay showed that the relative activity of luciferase was significantly lower in the miR-485-5p group than in the negative control group （both P < 0.05） after the transfection with wild-type lncRNA NEAT1 or STAT3 recombinant plasmids, while there were no significant differences between the miR-485-5p group and negative control group after the transfection with mutant lncRNA NEAT1 or STAT3 recombinant plasmids （both P > 0.05）. Compared with the control group, the lncRNA-NEAT1 overexpression group showed significantly increased expression of lncRNA NEAT1 and STAT3 （including STAT3 mRNA, STAT3 protein, and p-STAT3 protein） in HaCaT cells （all P < 0.05）, but significantly decreased miR-485-5p expression （P < 0.05）; the Xidi Liangxue recipe group showed significantly decreased expression of lncRNA NEAT1 and STAT3 （all P < 0.05）, but significantly increased miR-485-5p expression compared with the control group （P < 0.05）; significantly decreased expression of lncRNA NEAT1 and STAT3, but significantly increased miR-485-5p expression was observed in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group compared with the lncRNA-NEAT1 overexpression group （all P < 0.05）. After 24-, 48-, and 72-hour intervention, CCK8 assay showed that the proliferative activity of HaCaT cells was significantly higher in the lncRNA-NEAT1 overexpression group than in the control group （all P < 0.05）, as well as in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group than in the Xidi Liangxue recipe group （all P < 0.05）, and the cellular proliferative activity was significantly lower in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group and Xidi Liangxue recipe group than in the control group （all P < 0.05）. The apoptosis rate was significantly lower in the lncRNA-NEAT1 overexpression group （5.84% ± 0.28%） than in the control group （14.75% ± 0.83%, LSD-t = 3.48, P = 0.002）, but significantly higher in the Xidi Liangxue recipe group （35.72% ± 3.62%） than in the control group （LSD-t = 5.34, P = 0.001）; the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group showed significantly increased apoptosis rate （27.64% ± 2.82%） compared with the lncRNA-NEAT1 overexpres-sion group （LSD-t = 9.06, P < 0.001）. Conclusion The Xidi Liangxue recipe could inhibit the proliferation of IL-17-induced HaCaT cells and promote their apoptosis, which may be related to the intervention in the lncRNA NEAT1/miR-485-5p/STAT3 regulatory network.

Key words: Psoriasis, MicroRNAs, STAT3 transcription factor, Keratinocytes, Cell proliferation, Apoptosis, Xidi Liangxue recipe, LncRNA NEAT1