| Background:Psoriasis is a multifactorial,immune-mediated,recurrent chronic inflammatory skin disease,the typical clinical manifestations are skin erythema and scaling,and its specific pathogenesis has not been fully elucidated.It is generally believed that the interaction of keratinocytes with immune cells is the key to the formation of psoriasis lesions.Studies have confirmed that keratinocytes in psoriasis skin lesions secrete a large number of chemokines to promote the circulation of activated T cell skin homing,and T lymphocyte activation and migration to the site of onset is a key step in the onset of psoriasis.It can be seen that the regulation of the chemotaxis function of keratinocytes plays a crucial role in the development mechanism of psoriasis.Micro RNA-210(miR-210)is a signature hypoxia-induced micro RNA that regulates the expression of target genes involved in the regulation of biological processes such as cell proliferation,apoptosis,differentiation,migration,mitochondrial metabolism,and angiogenesis.The previous research of our research group shows that miR-210 plays a vital role in psoriasis CD4~+T cell dysfunction and lesion formation.However,whether miR-210 also promotes T cell activation and migration by regulating the chemotaxis of keratinocytes,aggravating the formation of psoriasis lesions has not been studied.Objective:Investigate the regulatory effect of miR-210 on the chemotaxis function of keratinocytes and its specific molecular mechanism,to further elucidate the mechanism of psoriasis skin lesion formation,and to provide a new theoretical basis for the targeted treatment of psoriasis with miR-210.Methods:1.To investigate the expression of chemokines and their receptors in epidermis and CD4~+T cells of psoriatic skin lesions:(1)Real-time PCR was used to detect the m RNA expression levels of chemokines CCL5,CCL20,and CXCL10 in the epidermis of psoriatic skin lesions and normal skin tissues.(2)CD4~+T cells infiltrated in psoriatic skin lesions and CD4~+T cells in psoriatic peripheral blood were sorted,and m RNA expression levels of CCL5 receptor CCR5,CCL20receptor CCR6 and CXCL10 receptor CXCR3 of CD4~+T cells in skin lesions and peripheral blood were detected by real-time PCR.(3)Immunofluorescence was used to detect the expression of chemokine receptors CCR5,CCR6 and CXCR3 in CD4~+T cells of psoriatic skin lesions and normal skin tissues.2.To explore the effect of miR-210 on the secretion of chemokines by keratinocytes and the migration of CD4~+T lymphocytes:(1)Human immortalized keratinocyte lines(Ha Ca T cells)were transfected with Agomir-210,Agomir-NC and Antagomir-210,Antagomir-NC respectively,and the m RNA expression levels of CCL5,CCL20 and CXCL10 in the cell supernatant were detected by real-time PCR.(2)Ha Ca T cells were transfected with Agomir-210,Agomir-NC and Antagomir-210,Antagomir-NC respectively,and the protein expression levels of CCL5,CCL20 and CXCL10 in the cell supernatant were detected by ELISA.(3)The supernatant of Ha Ca T cells transfected with Agomir-210,Agomir-NC and Antagomir-210,Antagomir-NC was collected,and the effect of overexpression and inhibition of miR-210 in keratinocytes on the migration of CD4~+T cells was detected by transwell assay.3.To explore the effect of miR-210 on the secretion of chemokines in mice:(1)Apply imiquimod ointment on the back of wild-type C57BL/6N mice for 7 consecutive days to establish a psoriasis-like mouse model,and at the same time Agomir-210 and Agomir-NC were injected intradermally on days 1,2,3 and 4,respectively;(2)imiquimod ointment was applied to the back of miR-210 knockout(miR-210 KO)mice and wild-type(wild type,WT)mice was used to establish a psoriasis-like mouse model.The Psoriasis area and severity index(PASI)score was performed on the skin lesion phenotype of the above two psoriasis-like mice on the 7th day,the histopathological changes in the skin lesion area of??the mice were analyzed by HE staining,and the m RNA expression levels of chemokines CCL5,CCL20 and CXCL10 in the epidermis of skin lesions were detected by real-time PCR,the protein expression levels of CCL5,CCL20 and CXCL10 in serum were detected by ELISA.4.To explore the molecular mechanism of miR-210 regulating chemotaxis function of keratinocytes:(1)Bioinformatics software was used to predict target genes associated with chemotaxis function of miR-210 and further validated it by using the dual-luciferase reporter gene assay.(2)The expression of miR-210 target genes in the epidermis of psoriatic skin lesions and normal skin tissues was detected by western blot and real-time PCR.(3)The m RNA and protein expression levels of miR-210 target genes in the epidermis of psoriatic skin lesions of WT mice and miR-210 KO mice were detected by Real-time PCR and western blot.(4)Agomir-210,Agomir-NC and Antagomir-210,Antagomir-NC were transfected into Ha Ca T cells,and the m RNA and protein expression levels of miR-210 target genes were detected by real-time PCR and western blot.(5)Ha Ca T cells were transfected with target gene si RNA and control si RNA,and the m RNA expression levels of target gene and CCL5,CCL20 and CXCL10 were detected by real-time PCR.(6)Ha Ca T cells were transfected with Antagomir-210 and Antagomir-NC for 24 hours and then transfected with target gene si RNA and control si RNA for 48 h,and the m RNA expression levels of miR-210target genes and CCL5,CCL20 and CXCL10 were detected by real-time PCR.(7)The supernatant of Ha Ca T cells after transfection of Antagomir-210 and Antagomir-NC for 24 h and then transfection of target gene si RNA and control si RNA for 48h was collected,and the effect of target gene-mediated miR-210 on CD4~+T cell migration in Ha Ca T cells was detected by transwell experiment.Results:1.Compared with normal epidermis,the m RNA expression levels of chemokines CCL5,CCL20 and CXCL10 in the epidermis of psoriatic skin lesions were significantly increased(P<0.05).The m RNA expression levels of chemokine receptors CCR5,CCR6 and CXCR3 in CD4~+T cells infiltrated in psoriatic skin lesions were significantly higher than those infiltrated in peripheral blood(P<0.05.The results of immunofluorescence showed that compared with normal skin tissues,the expression of chemokine receptors CCR5,CCR6 and CXCR3 in CD4~+T cells in psoriatic skin lesions was significantly increased.2.Compared with the control group,overexpression of miR-210 can promote the m RNA expression levels of chemokines CCL5 and CCL20in Ha Ca T cells,and inhibition of miR-210 can reduce the m RNA expression levels of chemokines CCL5,CCL20 and CXCL10 in Ha Ca T cells(P<0.05).Overexpression of miR-210 can significantly promote the secretion of chemokines CCL5,CCL20 and CXCL10 in Ha Ca T cells(P<0.05),and inhibition of miR-210 can significantly inhibit the secretion of chemokines CCL20 and CXCL10 in Ha Ca T cells(P<0.001).Compared with the control group,overexpression of miR-210 in Ha Ca T cells can significantly promote CD4~+T cells migration while inhibition of miR-210 can significantly reduce the migration of CD4~+T cells(P<0.001).3.Compared with mice in Agomir-NC group,the psoriatic skin lesions and pathological changes of mice injected with Agomir-210 were significantly aggravated,the PASI score,hypertrophy of the spinous layer and infiltration of dermal inflammatory cells was significantly increased(P<0.01),the m RNA expression of CCL5 and CCL20 in the epidermis of skin lesions was significantly increased(P<0.05),and the secretion of CCL5,CCL20 and CXCL10 in serum was significantly increased(P<0.05).Compared with WT mice,miR-210 KO mice had obvious psoriatic skin lesions and pathological changes,the PASI score,hypertrophy of the spinous layer and infiltration of dermal inflammatory cells was significantly reduced(P<0.05),the m RNA expressions of CCL5,CCL20 and CXCL10 in the epidermis of skin lesions were significantly reduced(P<0.05),and the secretion of CCL5,CCL20 and CXCL10 in serum was significantly reduced(P<0.01).4.Mi R-210 was predicted to bind with the target gene FoxO3through biological information websites..Compared with HEK293T cells co-transfected with mimic-NC and FoxO3 WT plasmid,the luciferase activity was significantly reduced in cells co-transfected with mimic-210and FoxO3 WT plasmid,while the luciferase activity was significantly increased in cells co-transfected with mimic-210 and FoxO3 Mut plasmid(P<0.05).Compared with normal skin tissues,the m RNA and protein expression levels of FoxO3 were significantly decreased in the epidermis of psoriatic skin tissues(P<0.05).Compared with WT mice,the m RNA and protein expression levels of FoxO3 were significantly increased in the psoriatic skin lesions of miR-210 KO mice(P<0.0001).Compared with the control group,overexpression of miR-210 significantly decreased the m RNA and protein expression of FoxO3,while inhibition of miR-210 significantly increased the m RNA and protein expression of FoxO3 in Ha Ca T cells(P<0.05).Compared with the si-NC group,transfection of si-FoxO3 inhibited the expression of FoxO3 and significantly increased m RNA expression levels of chemokines CCL5,CCL20 and CXCL10 in Ha Ca T cells(P<0.05).Transfection with si-FoxO3 partially reversed the effect of inhibition of miR-210 on the expression of chemokines in Ha Ca T cells and on the migration of CD4~+T lymphocytes(P<0.05).Conclusion:Mi R-210 may promote the secretion of CCL5,CCL20and CXCL10 by keratinocytes by inhibiting the expression of its target gene FoxO3,and then participate in regulating the migration of CD4~+T cells and promote the formation of psoriatic skin lesions.Figures 27 Tables 40 References 126... |
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