The Role Of LncRNA-MSX2P1 In The Regulation Of S100A7 Through MiR-6731-5p In The Pathogenesis Of Psoriasis

Research BackgroundPsoriasis is a chronic recurrent inflammatory skin disease characterized by abnormal proliferation and keratinization of keratinocyte in the genetic background of multiple genes.The prevalence of psoriasis in China is generally around 0.123%-0.47%,which has been increasing year by year in recent years.The disease is a systemic disease,which has the characteristics of long course,easy to relapse and not easy to cure,and it is often seen in young and middle-aged diseases,which seriously affects the quality of life and physical and mental health of the patients.The exact etiology and pathogenesis of psoriasis have not been fully elucidated so far.Therefore,it has become a focus and an urgent problem to be solved in the world to study the pathogenesis and new therapeutic targets of psoriasis.At present,it has been proved that the pathophysiological process of psoriasis involves many factors such as inflammation,immune disorde,abnormal cell proliferation and apoptosis,activation of intracellular signal transduction pathway.The immune imbalance is an important part of the pathogenesis in psoriasis.T cells(including CD4+ T cells and CD8+ T cells),innate immune cells(including KC,γδT cells and plasmacytoid dendritic cells,neutrophils,natural killer T cells and macrophages)and other cellular components by secreting a series of cytokines and chemokines mediated the activation of intracellular signal transduction pathway,and a cascade amplification effect is generated by interacting between KCs,innate immune cells and T lymphocytes.It synergies to local KC in skin lesions,induces excessive proliferation and abnormal differentiation of KC,and lead to inflammatory reaction in psoriatic lesions.This is the basis of the histopathological phenotype of psoriasis.The clinical efficacy of the treatment of psoriasis is achieved through controlling inflammation and suppressing immune response.IL-22 is one of the members of the type II cytokine family,is also known as the IL-10 cytokine family in addition to INF.IL-22 is mainly secreted by the activated Th22,Th17,Th1,NK cells and other immune cells;The IL-22 receptor is a heterodimeric structure consisting of two chains,one of which is the IL-22R1 chain(also referred to as CRF2-9),and the other is the IL-10R2 chain(also referred to as CRF2-4).IL-22 plays biological activity by binding to the corresponding two strands simultaneously.In the skin tissue,KC is a specific target cell for the action of IL-22.IL-22 can bind with the corresponding receptors on KC to bridge the interaction between immune cells and KC,and participate in local immune dysfunction of psoriatic lesions.It has been proven that in psoriatic skin and serum,the expression level of IL-22 is significantly increased,and it is positively correlated with the severity of disease.After effective anti-inflammatory treatment,its expression level can be significantly reduced.This suggests that IL-22,a cytokine associated with inflammation,plays an important role in the immune imbalance of psoriatic lesions.S100A7,it is one of the members of the S100 protein family.In 1991,Madsen and others were originally isolated from the keratinocytes of patients with psoriasis,and are also known as psoriasin.S100A7 is a differentiation related protein,which plays an important role in the process of cell proliferation,cell differentiation,cell apoptosis,oxidative stress,chemotaxis of inflammatory cells,and regulation of gene expression through its ligand binding.S100A7 has strong chemotaxis.It has been shown that S100A7 is highly expressed in psoriatic KC and can be used as chemokines to recruit neutrophils,monocytes and CD4+T lymphocytes to the site of infection,secreting inflammatory factors and causing local inflammatory response in skin lesions.In recent years,with the development of gene detection technology,more and more non-coding RNA sequences have been found.In particular,the study on the role and regulatory mechanism of long non-coding RNAs(long non-coding RNAs lncRNAs)and microRNAs(miRNAs)has become a hot research topic in the field of life science.It has been confirmed that multiple micro RNAs molecules,such as miR-203,miR-99a,miR-31,miR-125b,miR-146a and miR-424,are involved in the inflammatory reaction of psoriasis and the regulation of KC proliferation and differentiation through downstream target genes.However,the role of IncRNA in the pathogenesis of psoriasis is not well understood,and the mechanism of regulation is less studied.IncRNA and miRNA have their own regulatory pathways,but since the hypothesis of competitive endogenous RNA(CeRNA),the interaction of IncRNA and miRNA in disease has been confirmed by more and more experiments.CeRNA is not a newly discovered RNA molecule,but a newly discovered regulatory mechanism.The mechanism suggests that IncRNA and mRNA share miRNA binding sites,which are regulated by miRNA and have a positive correlation with each other.lncRNA and mRNA are called each other ceRNA.There is a complex and fine regulatory network among lncRNA,miRNA,mRNA and other RNA.ceRNA and mRNA,which is called ceRNA..The result is that ceRNA and miRNA are in a stable state.When the expression of ceRNA is abnormal,the expression level of ceRNA cooperatively changes,and the imbalance of stable state is destroyed,which will lead to the occurrence of related diseases.Up to now,the role and mechanism of CeRNA regulatory network in psoriasis have not been reported.So,for the first time,from the perspective of CeRNA,our group exploring the possible role of lncRNA molecules in the pathogenesis of psoriasis,it can help us to understand the genesis and development of psoriasis from the genetic level and provide a new direction for the targeted treatment of psoriasis.Research ObjectiveIn this study,from the CeRNA point of view,using LncRNA and mRNA microarray,differential expression of lncRNAs in IL-22-stimulated keratinocytes was screened.Bio informatics analysis and molecular biology experiments were performed to investigate the expression of long non-coding RNA-MSX2P1(LncRNA-MSX2P1,MSX2P1)and S100A7 in psoriasis,and to analyze the effect of MSX2P1 on the proliferation,cell cycle,apoptosis and expression of related inflammatory factors in keratinocytes,to elucidate the role of MSX2P1 as a CeRNA in the regulation of S100A7 by competitively binding miR-6731-5p in the pathogenesis of psoriasis.Part 1 Screening and verification of LncRNA differential genes and construction of LncRNA-MSX2P1 related CeRNA networkResearch Methods(1)The experiment was performed on human immortalized keratinocytes(HaCaT cell line).The experimental group received IL-22(100 ng/ml)for 24 hours and the control group was not stimulated.It was conducted by two independent experiments.Total RNA was extracted using RNeasy Mini Kit,differentially expressed LncRNA and mRNA were analyzed using the Affymetrix microarray GeneChip Human Gene 2.0 ST Array.We verified those significant differentially expresstion LncRNAs in the other 10 cases independent samples by real-time quantitative PCR(RT-qPCR),with GAPDH as reference,the data was indicated by2-△△Ct.(2)We using bioinformatics method to compare the sequence of chip source lncRNA to obtain the differential expression of IncRNA sequence for the prediction of the target site of lncRNA-miRNA.Combined with the results of RT-qPCR verification and literature review,we selected MSX2P1 as the research object,and used continuous probability distribution method(CUPID)to construct the CeRNA network and predict the corresponding target genes.Result(1)We performed IncRNA array to detect lncRNAs that were differentially expressed among the two groups,Of the 1,438 differentially expressed IncRN As,we found that 154 IncRNA expression differences(fold change>1.5,P<0.05).of those,103(66.9%)were up-regulated and 51(33.1%)were down-regulated.(2)We used RT-qPCR to validate the expression levels of 10 IncRNAs,compared to the control group.Our data showed that MSX2P1,IGKV1D-27,LOC100216479,LOC100287834,and IGLV2-23 were up-regulated in the experimental group(P<0.05,P<0.01).LOC440895,IGKV3D-15,LOC100505942,FLJ45340,and LOC284632 were down-regulated in the experimental group(P<0.05,P<0.01).The expression trend is consistent with the result of the chip.Each experiment was repeated for three times,and the differences were statistically significant.(3)Bioinformatics methods identify the IncRNA related ceRNA network of differential expression of IL-22-stimulated keratinocytes.In combination with the results of RT-qPCR verification,we selected MSX2P1-miR-6731-5p-S100A7 for further functional experiments and molecular mechanism studies.Part 2 Expression of LncRNA-MSX2P1 and S100A7 in psoriatic lesions and keratinocytesResearch Methods(1)H&E staining and immunohisto chemical staining(IHC)were performed on 10 cases of psoriatic lesions and 10 cases of normal skin tissues,and typical images of S1 00A7 immunohistochemical staining were obtained.(2)In the clinical level,RT-qPCR and Western blotting were used to detect the expression difference of MSX2P1 and S100A7 in psoriatic lesions and normal skin tissues,and the correlation between the expression level of MSX2P1 and S100A7 was analyzed.(3)In vitro,the expression levels of MSX2P1 and S100A7 in IL-22(100 ng/ml)mediated keratinocyte(HaCaT cell line)and primary human keratinocyte line(HNEK)were detected by RT-qPCR and Western blotting.Nuclear separation of keratinocytes was carried out and the expression of MSX2P1 in the nucleus and cytoplasm was detected by RT-qPCR.Result(1)The results of H&E staining showed that 10 clinical samples from the skin of patients were all consistent with histopathological changes of psoriasis vulgaris.The results of immunohistochemical staining showed that S100A7 was highly expressed in epidermal keratinocytes of psoriasis,which was higher than that of normal skin tissues.(2)RT-qPCR results showed that the expression levels of MSX2P1 and S100A7 in 10 psoriatic lesions were higher than those in normal skin tissues,and the difference was statistically significant(P<0.001).The results of western blotting showed that the expression of S100A7 protein was strongly positive in psoriasis lesions with high expression of MSX2P1,but in the normal skin tissues with low expression of MSX2P1,the expression of S100A7 protein was weakly positive.There was a positive correlation between the expression level of S100A7 and MSX2P1(R = 0.635,P=0.0487).(3)In vitro,results of RT-qPCR showed that the expression level of MSX2P1 and S100A7 in IL-22-stimulated keratinocytes were all up-regulated.MSX2P1 is mainly expressed in the cytoplasm.The difference was statistically significant(P<0.01).In addition,the expression level of S100A7 protein was increased in keratinocytes treated by IL-22 stimulated.Part 3 The Regulation and Mechanism of LncRNA-MSX2P1through competitive combination of miR-6731-5p to S100A7Research Methods(1)Knockdown of MSX2P1 expression by lenti-shMSX2P1 and verify the expression level of MSX2P1,miR-6731-5p and S100A7 using RT-qPCR and Western blotting.We knocked down the expression of MSX2P1 and investigated the cell proliferation,cell cycle,and apoptosis of IL-22-stimulated keratinocytes by the CCK-8 assay,flow cytometry and Annexin V/PI staining.ELISA method was used to detect the expression level of cytokines IL-23、TNF-α、IL-12β,and Western blotting was used to detect the protein expression levels of IL-23、NF-κB、TNF-α、IL-12β、HLA-C and CCHCR1.(2)RT-qPCR and Western blotting were used to detect the expression level of miR-6731-5p and S100A7 in IL-22-stimulated keratinocytes transfected with miR-6731-5p mimics and inhibitor.We also detected the effect of miR-6731-5p on the cell proliferation,cell cycle,and apoptosis of IL-22-stimulated keratinocytes by the CCK-8 assay,flow cytometry and Annexin V/PI staining.(3)Through dual luciferase reporter gene assay,we detected the combination of miR-6731-5p and MSX2P1,miR-6731-5p and S100A7 mRNA,respectively,and the target gene was verified.(4)Knockdown of S100A7 expression by siS100A7,and verify the expression level of S100A7 by using RT-qPCR and Western blotting.We transfected with siS100A7 and miR-6731-5p inhibitor,and investigated the cell proliferation,cell cycle,and apoptosis of IL-22-stimulated keratinocytes by the CCK-8 assay,flow cytometry and Annexin V/PI staining.ELISA method was used to detect the expression level of cytokinesIL-23、TNF-α、IL-12β,and Western blotting was used to detect the protein expression levels of IL-23、NF-κB、TNF-α、IL-12β、HLA-C 和CCHCR1.Result(1)We used lentiviral-vector shRNA targeting MSX2P1 and found that the expression levels of MSX2P1 significantly decreased(P<0.01),which indicated that the interference was successful.Similarly,the decrease in MSX2P1 reduced the expression levels of mRNA and protein of S100A7 while the expression levels of miR-6731-5p significantly increased.Furthermore,cell proliferation was significantly down-regulated in IL-22-stimulated keratinocytes after knocked down the expression of MSX2P1 compared with cells in the control group by the CCK-8 assay(p<0.01).IL-22 mediated keratinocytes infected with lenti-shMSX2P1,we observed that the cycle transition from G1 to S phase was arrested,which inhibited the growth by flow cytometry.The number of apoptotic cells in IL-22-stimulated keratinocytes infected with lenti-shMSX2P1 significantly increased compared with cells in the control group,as evaluated by Annexin V/PI staining.Knockdown of MSX2P1 expression by lenti-shMSX2Pl,the expression level of IL-23、TNF-α、IL-12βdecreased using ELISA,and the protein expression level of IL-23、NF-κB、TNF-α、IL-12β、HLA-C and CCHCR1 decreased.(2)Transfected with miR-6731-5p inhibitor group,the expression levels of miR-6731-5p was significantly reduced(P<0.01),which showed that miR-6731-5p inhibitor was successfully transfected.Similarly,We found that the cells treated with the miR-6731-5p inhibitor increased the mRNA levels of S100A7(p<0.01).Consistent with this result,the suppression of miR-6731-5p also elevated the protein expression of S100A7.We found that cell proliferation significantly increased in IL-22-stimulated keratinocytes transfected with miR-6731-5p inhibitor compared with cells in the control group by the CCK-8 assay(p<0.01).MiR-6731-5p mimics induced cell cycle arrest,the cycle transition from G1 to S phase was arrested,which inhibited the growth by flow cytometry(P<0.01).In addition,the number of cells undergoing apoptosis in IL-22-stimulated keratinocytes transfected with miR-6731-5p mimics significantly increased,while IL-22-stimulated keratinocytes were statistically decreased transfected with miR-6731-5p inhibitor,compared with cells in the control group.(3)The results of double luciferase reporter assay showed that miR-6731-5p could inhibit the fluorescence intensity of firefly(P<0.05)carrying wild type MSX2P1(WT)3’UTR vector,However,there was no significant difference in the fluorescence intensity of the MSX2P1 3’UTR vector carrying the mutant(MUT)seed sequence(P>0.05),which confirmed that MSX2P1 can be combined with miR-6731-5p.At the same time,dual luciferase reporter assay also confirmed that miR-6731-5p can directly target S100A7,miR-6731-5p plays a negative regulatory role of S100A7(P<0.01).(4)Knockdown of S100A7 expression downregulated the S100A7 expression of mRNA(P<0.01)and S100A7 protein,which showed that the interference of S100A7 is successful.MiR-6731-5p inhibitor significantly promoted siS100A7-mediated proliferation by the CCK-8 assay(P<0.01).MiR-6731-5p inhibitor significantly suppressed siS100A7-mediated cell cycle arrest assessed by flow cytometry.Cell apoptosis(assessed by Annexin V/PI staining)significantly decreased in IL-22-stimulated keratinocytes transfected with S100A7 siRNAs and miR-6731-5p inhibitor,compared with IL-22-stimulated keratinocytes transfected with S100A7 siRNAs alone(P<0.05).In addition,co-transfected siS100A7 and miR-6731-5p inhibitor cells,the expression level of IL-23、TNF-α、IL-12β was increased,and the protein expression level of IL-23、NF-κB、TNF-α、IL-12β、HLA-C and CCHCR1 was also increased.Conclusion(1)MSX2P1 and S100A7 were highly expressed in psoriatic lesions.(2)IL-22 could up-regulate the expression of MSX2P1 and S100A7 in HaCaT and HNEK.(3)S100A7 is identified as the direct target of miR-6731-5p.(4)MiR-6731-5p negatively regulates S100A7,and inhibites proliferation and promotes apoptosis of IL-22-stimulated keratinocytes.(5)MSX2P1 can directly bind miR-6731-5p as an endogenous "miRNA sponge" and negatively regulate the expression of miR-6731-5p.(6)MSX2P1 indirectly activates the expression of S100A7 and the secretion of other inflammatory factors through inhibiting miR-6731-5p,play a role in the progression of psoriasis by promoting the proliferation of keratinocytes,inhibiting the role of apoptosis.In conclusion,our findings revealed that MSX2P1,as a competitive endogenous RNA molecule,indirectly regulates the function and molecular mechanisms of S100A7 in keratinocytes by binding to miR-6731-5p.The lncRNA-mediated gene regulation mechanism provides a new perspective for further understanding the mechanism of ceRNA mediated gene regulation in psoriasis,and provide a new idea for targeted therapy of psoriasis.