| Psoriasis is a common recurrent inflammatory skin disease.Inflammatory cell infiltration and excessive keratinocyte proliferation are histomathological markers.But the underlying mechanism has not been elucidated.The transcription factor WT1 is overexpressed in many cancers and has the oncogenic potential to promote cell proliferation and tumor growth.However,the role of WT1 in psoriasis and its mechanism have not been studied so far.Objective: To explore the role of WT1 in psoriasis pathogenesis and its mechanism.To provide a new target for the treatment of psoriasis.Methods:1.RT-q PCR was used to detect WT1 m RNA expression in non-lesion tissues of 10 psoriasis patients,20 psoriasis patients and 20 normal skin tissues,and WT1 m RNA expression in peripheral blood mononuclear cells(PBMCs)of psoriasis patients and normal people.To analyze the correlation between WT1 m RNA expression level in skin lesions and PASI score in patients with psoriasis.2.The expression and localization of WT1 in skin tissue were detected by immunohistochemical method.3.BALB/c mice were used to construct a psoriasis-like mousemodel.Skin of the model group and the control group were used for:(1)m RNA or protein levels of target genes were detected;(2)Immunohistochemical assay;(3)HE staining was used to observe pathological changes.4.WT1 overexpression plasmid and WT1 small interfering RNA were used to construct keratinocyte lines with stable over expression or knockout of WT1.Western blot was used to analyze the expression level of WT1 protein in each group;CCK-8 assay was used to detect the proliferation of Ha Ca T cells in each group;Annexin V-FITC/PI was used to detect the apoptosis of Ha Ca T cells in each group.5.WT1 was overexpressed in Ha Ca T cells,and the differentially expressed genes in the experimental group and the control group were screened by RNA-seq technique.The differentially expressed genes were verified by Ch IP-seq and dual-luciferase reporter assay.6.WT1 overexpressed plasmid,WT1 si RNA and its negative control were transfected into Ha Ca T cells,and the expression level of IL-1β after transfection was detected by ELISA.RT-q PCR and Western blotting were used to verify the transfection effect of WT1 si RNA.7.The level of IL-1β m RNA in the skin lesions of 20 patients with psoriasis was detected by RT-q PCR,and the correlation between the level of IL-1β and WT1 in the skin lesions was analyzed.8.The regulation mechanism of WT1 on IL-1β was studied by Ch IP-q PCR.Result:1.The m RNA and protein expression levels of WT1 in non-lesion and lesion tissues of patients with psoriasis were significantly higher than those in normal control group.WT1 m RNA and protein expression levels were slightly increased in psoriatic lesions compared with non-lesion tissues,but there was no statistical significance.The expression level of WT1 m RNA in psoriatic lesions was positively correlated with PASI score.2.The expression of WT1 was significantly enhanced in the epidermis of psoriatic lesions.The WT1 m RNA and protein expression levels in PBMCs of psoriasis patients were not significantly different from those of normal controls.3.Compared with the control group,the expression of Ki67,a marker associated with keratinocyte proliferation,was significantly increased in IMQ-treated mice,suggesting that IMQ induced keratinocyte overproliferation.Consistent with the results of human samples,the levels of WT1 m RNA and protein in non-lesion and lesion of psoriatic mice induced by IMQ were significantly higher than those in normal skin of control mice.4.Western blot analysis showed that WT1 protein was highly expressed in Ha Ca T cells transfected with WT1 plasmid.CCK-8 results showed that WT1 overexpression promoted the proliferation of Ha Ca T cells,especially on the 4th and 5th day after transfection.Apoptosis analysis by flow cytometry showed that Annexin positive cells and propidium iodide negative cells(representing early apoptotic cells)were significantly reduced in the WT1 plasmid transfection group compared to the control group.5.Western blot analysis showed that the expression of WT1 in Ha Ca T cells transfected with WT1 si RNA was significantly lower than that of negative control.Transfected Ha Ca T cells were collected at different time points for CCK-8 analysis.The results showed that the proliferation ability of Ha Ca T cells in WT1 si RNA transfection group was significantly lower than that in control group.Flow cytometry was used to detect the effect of WT1 gene knockout on Ha Ca T cell apoptosis,and it was found that the percentage of early apoptosis of Ha Ca T cells in the WT1 si RNA transfection group was significantly higher than that in the control group.6.RNA-seq results showed that there were 181 differentially expressed genes between the WT1 overexpression group and the blank control group,among which 101 genes were up-regulated and 80 genes were down-regulated.Thirteen genes that may be associated with psoriasis were screened out and further verified by RT-q PCR.IL-1β was significantly different between the two groups,and the data were consistent with the RNA-seq results.Therefore,IL-1β was included in subsequent studies.7.Ch IP-seq was used to verify whether WT1 interacts with IL-1β.Among 181 genes screened by RNA-seq,72 genes had WT1 binding sites.IL-1β sequencing signals were visualized by UCSC.Compared with the INPUT group,IL-1β had a significant enrichment peak.Therefore,WT1 plays a direct role in transcriptional activation through binding with IL-1β.In luciferase reporter gene assay,the mean fluorescence intensity of the mutant plasmid decreased significantly.In wild-type plasmid transfected cells,luciferase reporter gene expression was significantly increased compared with empty vector co-transfected with WT1 over expressing plasmid.8.After transfection with WT1 overexpressing plasmid,the IL-1βprotein levels in the supernatant of Ha Ca T cells increased gradually.The expression of IL-1β protein and m RNA decreased after WT1 si RNA transfection,and the difference was statistically significant.9.There was a positive correlation between WT1 m RNA expression and IL-1β m RNA expression in psoriatic lesions.10.Ch IP-q PCR showed that the histone acetylation level of IL-1βpromoter region in the overexpressed WT1 group was higher than that in the control group.In addition,WT1-knockdown group showed a lower level of histone acetylation in the IL-1β promoter region than the control group.The expression of the histone acetyltransferase p300 in the IL-1βpromoter region was consistent with the level of histone acetylation.Conclusions:In this study,WT1 expression was increased in psoriatic lesions,promoting keratinocytes proliferation and inhibiting their apoptosis.WT1 m RNA expression was positively correlated with PASI score.Further studies suggested that WT1 may be involved in the occurrence and development of psoriasis by regulating the expression of its downstream target gene IL-1β,and this regulation may be related to the level of histone acetylation in the promoter region of IL-1β.This study confirmed the important role of WT1 in the pathogenesis of psoriasis and its potential as a new therapeutic target for psoriasis. |
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