MiR-145-5p Regulates Proliferation Of Keratinocytes And Skin Inflammation In Psoriasis By Targeting MLK3

Background:Psoriasis is a chronic,T cell mediated disease that affects the skin or joints or both and is characterized by red,scaly skin plaques.Psoriasis also has a strong genetic component,and a growing number of psoriasis susceptibility genes which involved in immunity and keratinocyte functions have been identified.In recent years,the study of immunological mechanism has found that the immune microenvironment disorder caused by Treg/Th17(regulatory T cell/T helper cell 17)imbalance is the main cause of psoriasis.Psoriasis is closely related to the factors of environment,infection and mental,and so on.However,the trigger and molecular mechanism of psoriasis have not been fully clarified until now.Therefore,it is of great theoretical significance to explore the specific molecular mechanisms of psoriasis and to find molecular targets that related to psoriasis for guiding its clinical treatment.The regulatory role of microRNA-145-5p(miR-145-5p)in immune-related diseases has attracted increasing attention.However,whether miR-145-5p participates in the regulation of the inflammatory response in psoriasis skin lesion remains unclear.More and more studies have proved that abnormal expression of microRNAs(miRNAs)are involved in the occurrence and development of psoriasis.MiR-145-5p,as an important member of the miRNA family,is closely related to the occurrence and development of a variety of immune-related diseases,including rheumatoid arthritis(RA),chronic glomerulonephritis(CGN)and neuroinflammation.In recent years,it has been found that miR-145-5p can regulate cell proliferation and the expression of many inflammatory factors through regulating mitogen-activated protein kinases(MAPK),nuclear factor-kappa B(NF-?B)signaling pathways and so on,including cytokines IL-1?,IL-1?,IL-2,IL-6,IL-8,TNF-? and CXCL16,which play an important role in the occurrence and development of immune-related diseases.However,it is not clear whether miRNA-145-5p is involved in the pathogenesis of psoriasis.Our previous microarray results showed that the expression of miR-145-5p was significantly down-regulated in psoriatic skin lesions,which suggesting that down-regulation of miR-145-5p in psoriasis skin lesions may be involved in the occurrence and development of psoriasis.Using bioinformatics analysis,we found that Mixed-Lineage Kinase 3(MLK3)was one of the potential target genes of miR-145-5p.MLK3 was first found in human thymus tissues,and it is also widely expressed in human tissues.MLK3 is a member of the serine protein kinase family,including kinase domain,central leucine zipper domain and Src homology 3 domain.As a kind of MAP3K(Mitogen-activated protein Kinase Kinase Kinase)protein kinase,MLK3 can activate downstream protein molecules such as MAP2K 3/4/6/7(mitogen-activated protein Kinase Kinase 3/4/6/7,MAP2K 3/4/6/7,MKKK3/4/6/7)and participate in regulating signalling pathways such as MAPK and NF-kappa B,regulating cell proliferation,apoptosis,differentiation and immune response.MLK3 plays an important role in the regulation of MAPK and NF-kappa B signaling pathway,and is involved in the regulation of inflammatory response,which is closely related to the pathogenesis of many immune-related diseases.However,whether MLK3 is involved in the regulation of skin inflammatory response in psoriasis remains unclear.Objectives:1.To clarify the expression of miR-145-5p and MLK3 in psoriatic lesions and to verify whether there is a targeted regulatory relationship between miR-145-5p and MLK3.2.To study the effects of miR-145-5p and MLK3 on the proliferation and the inflammatory factors secretion of keratinocytes and investigate their molecular mechanisms.3.To clarify the regulatory effects and mechanisms of miR-145-5p on epidermal hyperplasia and skin inflammation in psoriasis.Part I Screening and validation of differentially expressed microRNAs and targeting regulation of MLK3 expression by microRNA145-5pMethods:(1)Microarray gene chip Exiqon MicroCURY LNA microRNA array,7th generation(MicroBase v18,condensed Probe ID version,Exiqon,Vedbaek,Denmark)was used to screen differentially expressed microRNAs in psoriasis lesions and normal control(n=4);qRT-PCR was used to verify the expression levels of the eight most significant down-regulated microRNAs and the three previously verified microRNAs(n=10);5.8S rRNA was used as internal control.The results were calculated by 2-??CT.(2)Fluorescence in situ hybridization(FISH)was used to further verify the expression of miR-145-5p in psoriatic lesions.(3)Using bioinformatics prediction methods(TargetScan,miRmap,miRanda and miRTarbase),we found that MLK3 might be a target of miR-145-5p,which was further verified by double luciferase reporter assay.(4)The expression of MLK3 in the skin lesions of psoriasis patients was analyzed by qRT-PCR and Immunohistochemistry(IHC).Results(1)MiRNA chip results and verification:compared with healthy control tissues,there were 116 microRNAs differently expressed(in fold change>2,P<0.05)in psoriatic lesional skin.We used qRT-PCR to validate eight down-regulated microRNAs and three previously validated microRNAs.The results showed that miR-10b-5p(P<0.05),miR-143-3p(P<0.001),miR-145-5p(P<0.001),miR-196-5p and mir-338-3p(P<0.05)were significantly down-regulated in psoriasis,and the results were consistent with those of microarray.The expression level of miR-21-5p(P<0.001),miR-31-5p(P<0.001)and miR-146a-5p(P<0.01)were significantly increased in psoriasis,which were consistent with previous reports.There was no significant difference in the expression levels of miR-214-5p(P>0.05),miR-3681-5p(P>0.05),and miR-4326(P>0.05).(2)The expression of MiR-145-5p in psoriatic lesions was decreased:FISH results showed that the expression of miR-145-5p in the epidermis of psoriasis patients was lower than that of healthy controls.(3)MiR-145-5p directly targets MLK3:using TargetScan,micromap,microanda and microTarbase databases,we predicted that MLK3 might be a target of miR-145-5p.Using double luciferase reporter gene method,we further verified that miR-145-5p directly regulates the expression level of MLK3.(4)MLK3 was highly expressed in psoriatic lesions:using the methods of qRT-PCR and Immunohistochemistry,we found that the expression of MLK3 in the lesions of psoriasi was significantly higher than that of healthy controls(P<0.01)at both mRNA and protein levels.ConclusionsOur microarray results showed that 116 miRNAs were differentially expressed in psoriasis(fold change>2,P<0.05).Compared with the skin tissues of the normal control group,we found that the expression level of miR-145-5p in psoriatic lesions was decreased.In contrast,the expression level of MLK3 was increased,and their expression levels were negatively correlated.The expression level of MLK3 was directly regulated by miR-145-5p.Part II:miR-145-5p regulates proliferation and chemokine secretion of NHEKs by targeting MLK3 and the related mechanismsMethods:(1)Isolation and culture of normal human epidermal keratinocytes(NHEKs).(2)MiRNA and small interfering RNA(siRNA)transfection.MiR-145-5p mimic/Ctrl(50nM)and miR-145-5p inhibitor/Ctrl(100nM)were transfected with lipo2000.Then,the overexpression and inhibition of miR-145-5p were analyzed by qRT-PCR 24h later.The expression of MLK3 was analyzed by qRT-PCR(24h)and Western blot(48h)after transfection of miR-145-5p mimic/Ctrl(50nM),miR-145-5p inhibitor/Ctrl(100nM)and siMLK3/Ctrl(60nM).Co-transfection of miR-145-5p inhibitor/Ctrl(100nM)and siMLK3/Ctrl(60nM)was carried out to analyze whether siMLK3 could antagonize the biological effects of miR-145-5p inhibitor.(3)CCK-8 proliferation assay.CCK-8 proliferation assay was used to measure the proliferation of NHEKs after different treatments.The effects of miR-145-5p mimic/Ctrl(50nM),miR-145-5p inhibitor/Ctrl(100nM)and siMLK3/Ctrl(60nM)on the proliferation of NHEKs were determined by CCK-8 after transfection of miR-145-5p mimic/Ctrl(50nM),miR-145-5p inhibitor/Ctrl(100nM)and siMLK3/Ctrl(60nM).Co-transfection of miR-145-5p inhibitor/Ctrl(100nM)and siMLK3/Ctrl(60nM)was performed to determine whether the effect of miR-145-5p on NHEKs proliferation was mediated by MLK3.(4)The secretion levels of chemokines(CCL2,CCL7,CCL20,CXCL1,CXCL2,CXCL5,CXCL8 and CXCL-10)secreted by NHEKs were detected by Magnetic Luminex(?)Assays after different treatments.MiR-145-5p mimic/Ctrl(50nM),miR-145-5p inhibitor/Ctrl(100nM)and siMLK3/Ctrl(60nM)were transfected with lipo2000 to determine the effects of miR-145-5p and MLK3 on the secretion of chemokines of NHEKs.Co-transfection of miR-145-5p inhibitor/Ctrl(100nM)and siMLK3/Ctrl(60nM)was performed to determine whether the effect of miR-145-5p on chemokines secretion of NHEKs was mediated by MLK3.(5)Western blot was used to detect the effects of miR145-5p and MLK3 on the expression of the key proteins in NF-kappa B and STAT3 signalling pathways,and explore the mechanisms of the role of microRNA145-5p and MLK3 in psoriasis.Results(1)MiR-145-5p is involved in regulating proliferation of NHEKs.Inhibiting the expression of miR-145-5p in NHEKs can promote the proliferation of NHEKs.Conversely,overexpression of miR-145-5p can inhibit the proliferation of NHEKs.(2)MLK3 is involved in regulating proliferation of NHEKs.Interference the expression of MLK3 in NHEKs can inhibit the proliferation of NHEKs.(3)MiR-145-5p participates in the regulation chemokine secretion of NHEKs after stimulation with IL-17A.The results of liquid multifactorial analysis showed that inhibiting the expression of miR-145-5p in NHEKs can promote the secretion of chemokines induced by IL-17A.In contrast,overexpression miR-145-5p can inhibit the secretion of chemokines induced by IL-17A.(4)MLK3 participates in the regulation chemokine secretion of NHEKs after stimulation with IL-17A.The results of liquid multifactorial analysis showed that inhibiting the expression of MLK3 in NHEKs can inhibit the secretion of chemokines induced by IL-17A.(5)MiR-145-5p regulates the proliferation NHEKs by targeting MLK3.The proliferation of NHEKs induced by miR-145-5p inhibitor can be antagonized by siMLK3,suggesting that the proliferation effect of miR-145-5p inhibitor functions through targeting MLK3.(6)MiR-145-5p regulates the chemokine secretion of NHEKs by targeting MLK3.MiR-145-5p inhibitor promotes IL-17A-induced chemokine secretion and the effect can be antagonized by siMLK3,suggesting that miR-145-5p inhibitor promotes NHEKs chemokine secretion through targeting MLK3.(7)MiR-145-5p regulates NF-kappa B and STAT3 signaling pathways by targeting MLK3,In NHEKs,over-expression of miR-145-5p inhibits the expression of MLK3,inhibits the activation of NF-kappa B and STAT3 signaling pathways.However,interference the expression of miR-145-5p can promote the expression of MLK3 and activate the signaling pathways of NF-kappa B and STAT3.These results suggest that miR-145-5p can regulate NF-kappa B and STAT3 signaling pathways by targeting MLK3.ConclusionsMiR-145-5p is involved in regulating the proliferation and the secretion of chemokines of NHEKs.The mechanism of the effects is closely related to targeting MLK3.MiR-145-5p regulates the signalling pathways of NF-kappa B and STAT3 through targeting MLK3.Part ? MiR-145-5p regulates epidermal hyperplasia and skin inflammation in psoriasis by targeting MLK3Methods:(1)C57 female mice were purchased at age of 8-10 weeks and used to induce psoriasis mice model by topical application of IMQ(62.5 mg/d)*5 days.PASI score and hematoxylin-eosin staining(HE staining)were used to determine whether the mice models were successfully constructed.(2)The expression of miR-145-5p in the epidermis of psoriasis models was manipulated by intraepidermal inj ection of agomiR-145-5p and antagomiR-145-5p.(3)In situ fluorescence hybridization(FISH)was used to analyze the expression of miR-145-5p after local transfection with agomiR-145-5p/Ctrl(1 nom/d × 3d)and antagomiR-145-5p/Ctrl(1 nom/da×3d).(4)The expression levels of MLK3,CCL2,CCL20 and IL-1 7A were detected by qRT-PCR,Western blot and IHC after local transfection of agomiR-145-5p/Ctrl(1 nom/d X 3d)and antagomiR-145-5p/Ctrl(I nom/d×3d).(4)HE staining was used to analyze the effects of agomiR-145-5p(1 nom/d X 3d)and antagomiR-145-5p(1 nom/d X 3d)on epidermal proliferation and inflammatory cell infiltration of psoriasis mouse models.(5)Immunohistochemical technique was used to analyze the effects of local transfection of agomiR-145-5p/Ctrl(1 nom/d × 3d)and antagomiR-145-5p/Ctrl(1 nom/d X 3d)on the expression of MLK3 protein in mouse back skin,the number of Ki-67+ cells,and the chemotactic recruitment of CD3 + T cells and IL-17 +Th17 cells.Results(1)The mouse model of psoriasis was successfully induced by IMQ(62.5 mg/d)*5d.There were typical scaly erythema and plaques on the back of mice,and the severe cases were impregnated and exuded.There were also typical pathological changes of psoriasis:incomplete keratosis,thinning of granular layer,hypertrophy of spinous layer,papillary capillary dilatation,lymphocyte infiltration around blood vessels.(2)qRT-PCR and FISH techniques confirmed that locally transfected agomiR-145-5p overexpressed miR-145-5p,whereas locally transfected antagomiR-145-5p interfered with the expression of miR-145-5p.(3)In vivo,the expression level of miR-145-5p was negatively correlated with MLK3,CCL2,CCL20 and IL-17A.(4)In vivo,the expression of miR-145-5p was negatively correlated with the degree of epidermal hyperplasia.(5)Overexpression of miR-145-5p can reduce the number of Ki-67+ NHEKs in epidermis,decrease the number of CD3+T cells and IL-17+Th17 cells,and alleviate the skin inflammation induced by IMQ in psornatic mice.On the contrary,inhibition of the expression of miR-145-5p,increase the number of Ki-67+ NHEKs in epidermis,increase the number of CD3+T cells and IL-17 Th17 cells,and can aggravate the skin inflammation induced by IMQ in mice models of psoriasis.ConclusionsAnimal experiments results showed that miR-145-5p was involved in regulating the epidermal hyperplasia and the skin inflammation in psoriasis.Down-regulation of miR-145-5p contributes to epidermal hyperplasia and skin inflammation in psoriasis.