| Background: Psoriasis is a chronic inflammatory recurrent disease with erythema and scales as its basic manifestations,which is related to heredity and immunity.In recent years,the study on the relationship between mi RNA and psoriasis has become a hot topic.Increasing researches are performed on the feedback pathway between mi RNA and psoriatic susceptible genes or inflammatory factors.In the past 10 years,our research group has been working on the mi RNA,susceptible genes,and cytokines related to the pathogenesis of psoriasis.We used the Zhuhuang(ZH)granule,a self-made traditional Chinese medicine in our hospital,to intervene psoriasis and explore the mechanism of psoriasis occurrence.In our previous studies,we found that mi R-192,mi R-99 a,mi R-3175,and mi R-124 were differentially expressed in psoriasis and had a two-way regulatory effect;the levels of mi RNAs,susceptible genes,and cytokines were altered in psoriasis lesions,and there might be an interaction network among them.Furthermore,the research team constructed a preliminary network consists of psoriasis-related mi RNAs,susceptibility genes,and cytokines.Although some achievements have been made,the specific mechanism underlying the functions of the network is complex and diversified.Under the intervention of ZH granules,further research on the regulation of mi RNAs and psoriasis susceptibility genes needs to be conducted by the cell model in vitro and animal experiments.A large number of studies on the pathogenesis of psoriasis have shown that the abnormal proliferation of keratinocytes is the core event of psoriasis pathogenesis and the up-regulated expression of fibroblast growth factor receptor 2(FGFR2)in the skin lesions of psoriasis patients plays a vital role in this process.However,the underlying mechanism is not precise.Mi RNA is dysregulated in psoriatic tissues and can negatively regulate target m RNA translation.Our team used bioinformatics software to analyze mi RNAs that may target FGFR2.Based on the prediction by four pieces of software,we took the intersection and found that mi R-124-3p is most likely to regulate theexpression of FGFR2.Objective: 1.To verify the function of mi R-124 in the pathogenesis of psoriasis and verify the role of mi R-124 in mediating the expression of FGFR2 and regulating keratinocyte proliferation;2.To clarify that mir-124-3p/FGFR2 axis can mediate the effect of ZH granule containing serum on the model cells.Methods: The first part,1.Collect the psoriatic skin lesions and normal skin tissues and examine the expression levels of FGFR2 by Immunohistochemical(IHC)staining,QPCR,and Western blot(WB).2.Ha Ca T cells were stimulated with IL-22 and the protein levels of FGFR2 were detected by WB assay;FGFR2 was silenced in IL-22-stimulated Ha Ca T cells,the proliferation and migration of Ha Ca T cells were detected by Ed U and scratch assays;The expression of IL-17 A and TNF? was detected by q PCR assay;The protein levels of FGFR2,keratin6,keratin16,MMP1,MMP9,PI3 K,Akt,and ERK were detected by WB assay.The content of MMP1 and MMP9 was detected by ELISA.3.Bioinformatics analysis was performed to predict and screen for mi RNAs targeting FGFR2 3'UTR;QPCR assay was performed to detect the expression of mi R-124-3p in psoriatic skin lesions and normal skin tissues.Ha Ca T cells were stimulated with IL-22 and QPCR assay was performed to detect the expression of mi R-124-3p;mi R-124-3p overexpression and inhibition was achieved in Ha Ca T cells and WB assay was performed to detect the expression of FGFR2;Dual-Luciferase Report assay was performed to verify the target binding relationship between mi R-124-3p and FGFR2 3'UTR.4.Under the stimulation of IL-22,Ed U and scratch assays were used to detect the proliferation and migration of Ha Ca T cells when the mi R-124-3p was overexpressed;QPCR assay was used to detect the expression of IL-17 A and TNF?;WB assay was used to detect the protein levels of FGFR2,keratin6,keratin16,MMP1,MMP9,PI3 K,Akt,and ERK;ELISA assay was used to detect MMP1 and MMP9 content.5.Under the stimulation of IL-22,mi R-124-3p and/or FGFR2 was overexpressed,and the expression of FGFR2,IL-17 A,and TNF?was detected by QPCR assay;the proliferation and migration of Ha Ca T cells were detected by Ed U and scratch assays;the protein levels of FGFR2,keratin6,keratin16,MMP1,MMP9,PI3 K,Akt,and ERK were detected by WB assay.The content of MMP1 and MMP9 was detected by ELISA.The second part,1.ZH granule serum was prepared and Ha Ca T cells were treated with ZH granule serum or PBS(control).The cell proliferation and migration were detected by MTT and scratch assays,respectively;the expression of IL-17 A and TNF? was detected by QPCR assay;the protein levels of FGFR2,keratin6,keratin16,MMP1,and MMP9 were detected by WB assay.2.Ha Ca T cells were treated with ZH granule serum and PBS(control).The expression of FGFR2 and mi R-124-3p was detected by QPCR and FGFR2 protein by WB.3.Ha Ca T cells were transfected with mi R-124-3p inhibitor and treated with ZH granule serum,MTT and scratch assays were used to detect cell proliferation and migration activity;QPCR assay was used to detect the expression of IL-17 A and TNF?;WB assay was used to detect the protein levels of FGFR2,keratin6,keratin16,MMP1,and MMP9.4.Imiquimod(IMQ)-induced psoriasis model was established in mice.ZH granule was infused into the stomach of mice.Methotrexate was used as positive control.Mice were randomly divided into four groups(PBS,IMQ,IMQ + ZH granule,IMQ + MTX)and photos of representative skin tissues were taken;IHC was used to detect the expression of PCNA and FGFR2 in skin tissues of each group;QPCR was used to detect the expression of FGFR2 and mi R-124-3p in skin tissues.Results: The first part,1.Compared with normal skin,the hyperkeratosis of the epidermis in psoriatic lesions resulted in high expression of FGFR2 m RNA and protein.2.Compared with the PBS group,the expression level of FGFR2 in the RCAT cells in the r IL-22 stimulation group was significantly up-regulated;compared with the si-NC group,the proliferation,and migrationof the cells in the si-FGFR2 interference group was significantly down-regulated,the m RNA expression levels of IL-17 A and TNF? were significantly down-regulated,the protein levels of FGFR2,keratin6,keratin16,MMP1,and MMP9 were significantly down-regulated,the protein contents of MMP1 and MMP9 in the cells were significantly down-regulated,and the PI3K/Akt and ERK pathways were significantly inhibited.3.The results of the bioinformatics analysis showed that mi R-124-3p was the upstream regulatory factor of FGFR2.Compared with normal skin,mi R-124-3p was lower in psoriatic lesions,and mi R-124-3p expression was negatively correlated with FGFR2 expression.Compared with the PBS group,the expression level of mi R-124-3p was significantly down-regulated in the r IL-22 stimulation group;compared with the NC mimics group,the expression level of mi R-124-3p was significantly up-regulated and FGFR2 was significantly down-regulated in the mi R-124-3p mimics group;compared with the anti-NC group,the expression level of mi R-124-3p was significantly down-regulated,and FGFR2 was significantly up-regulated in the anti-mi R-124-3p mimics group.The results of the Dual-Luciferase Report showed that there was a targeted binding site between mi R-124-3p and FGFR2 3'UTR.4.Under the stimulation of r IL-22,compared with NC mimics group,the proliferation and migration of Ha Ca T cells in mi R-124-3p mimics group were significantly down-regulated,the m RNA expression of IL-17 A and TNF? was significantly down-regulated,the protein expression levels of FGFR2,keratin 6,keratin 16,MMP1,and MMP9 were significantly down-regulated,the protein levels of MMP1 and MMP9 were significantly down-regulated,and the protein levels of PI3K/Akt and ERK pathway were significantly inhibited.5.Compared with mi R-124-3p +vector group,the proliferation and migration of Ha Ca T cells in mi R-124-3p +FGFR2 group was restored,the m RNA expression of IL-17 A and TNF? was decreased,the protein expression levels of FGFR2,keratin 6,and keratin 16,MMP1,and MMP9 were decreased,PI3K/Akt and ERK pathways were restored.The second part,compared with PBS group,the proliferation,andmigration of Ha Ca T cells in ZH granule serum group were significantly inhibited,the m RNA expression of IL-17 A and TNF? was significantly down-regulated,and the protein expression levels of keratin 6,keratin 16,MMP1,and MMP9 were significantly down-regulated.2.Compared with the PBS group,the expression of FGFR2 and mi R-124-3p in Ha Ca T cells of the ZH granule serum group was significantly down-regulated.3.Compared with ZH granule + NC inhibitor group,the proliferation and migration activity of Ha Ca T cells in ZH granule + mi R-124-3p inhibitor group was restored,the m RNA expression of IL-17 A and TNF? was down-regulated,and the protein expression levels of FGFR2,keratin 6,keratin 16,MMP1 and MMP9 were down-regulated.4.Compared with the PBS group,the skin of IMQ group was significantly keratinized,the expression of PCNA and FGFR2 was significantly up-regulated,and mi R-124-3p was significantly down-regulated;compared with the IMQ group,the skin of IMQ + ZH granule group and IMQ +MTX group was significantly reduced in keratinization and the corresponding scale,the expression of PCNA and FGFR2 was significantly down-regulated,and mi R-124-3p was significantly up-regulated.Conclusion: 1.The mi R-124-3p/FGFR2 axis can inhibit the proliferation and migration of human keratinocytes and improve the inflammatory microenvironment of psoriasis.The axis might be a potential target for the treatment of psoriasis.2.ZH granule exerts its functions through the mi R-124-3p/FGFR2 axis. |
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