CRY2 Promotes Psoriasis-associated Keratinocyte Proliferation Through ERK1/2 Signaling Pathway E

Objective:This study is to determine the expression of Cryptochrome2（CRY2）in psoriasis and its role and underlying mechanism in psoriatic keratinocytes.This research will demonstrate the important regulatory role of CRY2 in the pathogenesis of psoriasis.Methods:We first analyzed the expression of CRY2 in psoriatic patients through the data from high-throughput RNA sequencing（RNA-Seq）,GEO database and single-cell RNA sequencing（scRNA-Seq）,and then analyzed the expression of CRY2 in imiquimod（IMQ）-induced psoriasis-like dermatitis in mice.The expression of CRY2 in peripheral blood mononuclear cells（PBMC）of patients with psoriasis was detected by qRT-PCR.CRY2 was silenced or overexpressed in human keratinocyte cell line HaCaT cells by small interfering RNA（siRNA）or overexpressed plasmid transfection,and the effects on cell proliferation and chemokine expression were determined.Western Blot was used to detect the effect of CRY2 on ERK1/2 signal in keratinocytes.We used ERK1/2 inhibitor combined CRY2 silencing to examine the effect on cell proliferation.To further investigate the upstream of CRY2 in psoriasis,we used the IL-17A and TNF-α-induced mouse models and determined the expression of CRY2 by quantitative PCR（qRT-PCR）and immunofluorescence staining.Moreover,we determined the expression of FADS2 in the skin tissues of psoriasis patients before and after Infliximab or Secukinumab treatment by qRT-PCR and immunofluorescence staining.Finally,we silenced CRY2 in HaCaT cells combined with TNF-α stimulation,and used qRT-PCR to detect the expression of chemokines.Results:RNA-seq and scRNA-seq analysis revealed that the expression of CRY2 was markedly decreased in skin lesions of psoriasis patients,especially in keratinocytes.Compared with normal controls,and in peripheral blood mononuclear cells（PBMC）of psoriatic patients,the expression levels of CRY2 and PER3 were reduced,while the expression levels of CLOCK,NRIDI and BMAL1 mRNA did not change.In psoriatic mouse model,CRY2 expression level in was significantly reuded.Silencing CRY2 in HaCaT cells promoted keratinocyte cell proliferation,while overexpression of CRY2 inhibited cell proliferation.Moreover,silencing CRY2 upregulates the expression of chemokines,such as CXCL1 and CXCL8.Further studies showed that CRY2 silencing significantly upregulated ERK1/2 phosphorylation,and inhibition of ERK1/2 phosphorylation suppressed cell proliferation induced by CRY2 silencing.The expression of CRY2 in epidermal keratinocytes was significantly inhibited by subcutaneous injection of TNF-α into the ears of C57BL/6 mice,but IL-17A injection had no effect on the expression of CRY2.The expression of CRY2 in psoriasis patients was significantly increaseafter 10 weeks of TNF-α antibody treatment.However,there was no difference in CRY2 expression before and after IL-17A antibody treatment in psoriatic patients.Silencing CRY2 can further enhanced the expression of CXCL1,CXCL8 and CCL20 induced by TNF-α stimulation.Conclusion:In psoriasis,CRY2 expression was significantly reduced in keratinocytes.Reduced CRY2 expression promoted keratinocyte proliferation and the expression of chemokines,such CXCL1,CXCL8 and CCL20,which may be related to the activation of ERK1/2 signaling pathway.The downregulation of CRY2 expression in psoriasis may be regulated by TNF-α.