Establishment Of The Regulation Network System Among The Susceptibility Genes-miRNA-cytokines In Psoriasis And MiR-99a Pathway's Evaluation

ObjectivesPsoriasis(Psoriasis,PS)is a both genetic and environmental induced disorder,characterized by excessive proliferation of keratinocytes,immune-mediated chronic inflammatory skin disease,in the clinical scale of erythema as the main performance.Immune signal cytokines and inflammatory factors stimulate cell changes,chromosomal susceptibility genes carry mutations caused by gene expression and functional variation,gene expression regulation microRNA(miRNA)in psoriasis pathology plays an important role.Thus the previous study only focused on a single angle and was not able to understand its complete pathology.System biology(System biology)to check the overall level of multi-level regulation,based on the data of pre-researchers and the integration of different levels of information,the establishment and analysis of a comprehensive understanding of psoriasis pathology,psoriasis susceptibility genes,miRNAs and Inflammatory cytokines such as the existence of the association may be in the pathological process of interaction and regulation.For the pathogenesis of psoriasis to provide a new system of biological perspective and the latest relevant theory.After the establishment of the network,we also selected the miR-99 a pathway found by the network for the existence of the verification,and prove that the miR-99 a pathway(miR-99a/FZD/Cyclin D1)effect in patients with psoriasis and regulation.After being shown in the effect of the miR-99 a pathway,this study has exact clinical efficacy and considerable research based on particle Zhuhuang Granule on miR-99 a pathway(miR-99a/FZD/Cyclin D1)of the control effect,prove the existence of the miR-99 a pathway from the other side,and also to find a way for the treatment of Zhuhuang Granule on psoriasis.Methods(I)Preliminary networks construction of miRNA related genes and cytokines in psoriasis:The psoriasis susceptibility gene miRNA and its related search,psoriasis target search,psoriasis related cytokines and cytokine miRNA search,target identification,speculated that the cytokine related gene network.(1)Search for psoriasis susceptibility genes: We carefully read the relevant literature,screening related susceptibility genes.In addition,we were further screened by thoroughly reading the 3,474 psoriasis-related literature returned from the search.In addition,we also collected psoriasis-related genes from the current public database,including psoriasis-related gene database psoriasis-related gene database(psoriasis-db),genetic association database genetic association database(GAD),DisGeNET,Human online Mendelian inheritance online Mendelian inheritance(OMIM)and functional disease ontology annotation functional disease ontology annotation(FunDO)database to improve the relevant psoriasis susceptibility gene data.(2)Psoriasis-associated miRNAs and their targets: Similar to the collection of psoriasis-related genes,we also manually collect psoriasis-associated miRNAs from different sources,including miRTarbase,mir2 disease,human microRNA lesions database(HMDD v2.0),PhenomiR.The operation is as follows,using the keywords "miRNA" and "psoriasis" or "microRNA" and "psoriasis" or "mIR" and "psoriasis".The database(including miRWalk,miRTarBase,miR2 Disease,miRecord)and miRSel)downloaded the associated miRNAs that may be regulated by these psoriasis-related target genes.In addition,the target gene of psoriasis may be regulated by targetscan,miRwalk,miRanda,RNA22 and other databases.(3)Search for psoriasis-associated cytokines: Similar to the collection of psoriasis-related genes,we also manually collect psoriasis-associated miRNAs from different sources,search for "cytokine" and "psoriasis" for keywords The At the same time from the current public database of psoriasis-related genes,including psoriasis-related gene database psoriasis-related gene database(psoriasis db),genetic association database genetic association database(GAD),DisGeNET,human online Mendelian genetic online Mendelian inheritance in human(OMIM)and functional disease ontology annotation functional disease ontology annotation(FunDO)(20)database.We received a total of 60 psoriasis-related cytokines.At the same time,in order to further obtain the relevant cytokine regulatory target genes,we analyzed the downstream factors related to the cytokine transcription factors.(4)Identification of miRNAs and cytokine targets: We combined the predictive and experimental validation of target miRNA targets.First,we passed miRNA targets,miRTarBase,miRecords(miRecar)by using TargetScan(version 6.2)and miRanda(version 2010.9)and miR2 Disease,(2013.4.27)and TarBase(TarBase_V6,version 2012.1)were predicted and the results were compared with the cytokines and related susceptibility genes.To predict the regulatory interaction between cytokines and susceptible genes / miRNAs,we obtained predicted transcription factor binding site(TFBS)data from the UCSC genome browser and screened those conserved transcription factor binding sites that were conserved in vertebrates The In order to reduce the rate of false positive prediction,we set the Z-value to 2.33 high score as the transcription factor binding site with better confidence.In addition,we have integrated data from ChIP-Seq and ChIP chips from the ENCODE project(http: // genome.Ucsc.edu/ENCODE/)57).Proactively verified transcription factor targets were extracted from the TRANSFAC database(version 2013.4).Cytokine-related control gene networks are deduced by transcription factors that affect the downstream effects of cytokines,as well as the key genes regulated downstream of transcription factors.(5)Network establishment: After improving the relationship between miRNA/susceptibility gene/cytokines,we constructed a psoriasis-associated miRNA-cytokine-susceptibility gene co-regulatory network using python scripting,including miRNA-cells Factor-susceptibility gene feedback loop and intermodulation loop of cytokines and miRNAs in two different forms.(II)Study on the existence and effect of miR-99a/FZD/Cyclin D1 pathway in psoriasis:We selected the miR-99a/FZD/Cyclin D1 pathway in the susceptible gene,miRNA,cytokine interaction network established in the first part of this paper to verify in psoriasis patients and in vitro.First of all,the collection of skin lesions in 23 patients with psoriasis vulgaris,with normal skin tissue for comparison,quantitative real-time PCR(qPCR)miR-99 a,mRNA,FZD5 to detect the expression of FZD8 mRNA in tissue damage in psoriasis skin,parallel FZD5/FZD8 expression correlation and regression analysis of the expression of miR-99 a.Secondly,cultured HaCaT cells transfected with miR-99 a mimics and miR-99 a inhibitor,the determination of quantitative real-time PCR miR-99 a expression after transfection,Western blot assay the expression changes of HaCaT cells detected by FZD5/FZD8 miR-99 a mimics and miR-99 a inhibitor after transfection.Again,the use of miR-99 a mimics and protein expression of FZD gene transfection vector,measured the activity of HaCaT cells were transfected by MTT,BrdU determination of effect of miR-99 a on the proliferation of HaCaT incorporation method was detected on HaCaT cells miR-99 a FZD5/FZD8 beta-catenin,CyclinD1 expression of downstream protein blot determination.Then,by detecting luciferase activity,to determine whether miR-99 a can inhibit the expression of FZD5 / FZD8 by directly binding to FZD5 / FZD8 of 3'UTR.(III)Study on the effect of Zhuhuang granule containing plasma intervention on HaCaT cells miR-99a/ FZD/Cyclin D1 pathway:We selected the miR-99a/ FZD/CyclinD1 pathway in the susceptible gene,miRNA,cytokine interaction network established in the first part of this paper to verify in psoriasis patients and in vitro.It is found that the miR-99a/ FZD/CyclinD1 pathway is clearly present in patients with psoriasis.On this basis,the psoriasis drug has Shiraia particles as considerable research foundation,we also investigated the effects of the drug intervention this pathway.We were preparing tabasheer medicated plasma particles,concentration calculation of optimal HaCaT cell medicated plasma with MTT method,the cultured cells were divided into the inner granule group(A group),miR-99 a mimic transfected control group(B group)and blank plasma group(group C),miR-99 a inhibitor transfection combined with granule group(D shiraiabambusicola group),was treated with medicated plasma,the expression of miR-99 a was measured by real-time fluorescence quantitative PCR,Western blotting was used to detect the expression of HaCaT in each group were measured by FZD5/FZD8 cells and HaCaT cells were FZD5/FZD8 beta-catenin,CyclinD1 expression of the downstream effects of changes in determination of plasma containing intervention HaCaT cell activity by MTT.Results(I)Preliminary networks construction of miRNA related genes and cytokines in psoriasis:The 478 genes of psoriasis susceptibility were obtained successfully,and the clustering of gene function was obtained by bioinformatics genomic annotation of Kyoto University.We found that 94 of the 478 genes were involved in the interaction between cytokines and cytokine receptors,while the other one was related to the Jak/STAT signaling pathway,and the 51 was related to the differentiation of Th17 lymphocytes.Therefore,these results suggest that psoriasis is an autoimmune disease is highly dependent on cell factor;received a total of 60 psoriasis related cytokines,95 expression changes in human psoriasis,and there are potential regulatory function of miRNA,based on the literature data obtained information of susceptibility genes and miRNA 314 cell factor the regulation and the establishment of a regulatory network,including 103 for psoriasis cuticle cells induced gene expression or miRNA.(II)Study on the existence and effect of miR-99a/FZD/Cyclin D1 pathway in psoriasis:We found that the down-regulation of specific expression of miR-99 a in psoriatic skin lesions,and can inhibit the proliferation of HaCaT cells;found to directly by the target,miR-99 a can also regulate the curl protein 5(FZD5)/-8(FZD8)coil protein expression.In addition,we found that FZD5/FZD8 signal transduction downstream factor downstream target gene CyclinD1 beta-catenin and beta-catenin can be inhibited by miR-99a;the inhibition effect of miR-99 a on beta-catenin and CyclinD1 can through forced overexpression of FZD5/FZD8 to eliminate the.To sum up,for the first time,we have targeted FZD5/FZD8 by downstream factor beta-catenin and CyclinD1,which can inhibit the proliferation of HaCaT cells by miR-99 a,and provide diagnostic markers and new targets for the treatment of psoriasis.(III)Study on the effect of Zhuhuang granule containing plasma intervention on HaCaT cells miR-99a/FZD/Cyclin D1 pathway:It is found that the upregulation of miR-99 a expression,the expression of FZD5 and FZD8 protein inhibited the expression of its downstream beta-catenin,CyclinD1 protein was also inhibited,so that the activity of HaCaT cells decreased,proving once again that miR-99a/FZD/Cyclin D1 pathway exists in psoriasis.Zhuhuang granule containing plasma can produce the effect of the same miR-99 a and mimic,FZD5,FZD8 protein expression also inhibited the expression of its downstream,while suppressing the beta-catenin,CyclinD1,HaCaT cell activity decreased;so it is concluded that the mechanism of Zhuhuang Granule on treatment of psoriasis may be related to the effect of simulated miR-99 a.The use of miR-99 a inhibitor to block the expression of miR-99 a,expression of Shiraia granule containing no inhibition of FZD5 plasma,FZD8,beta-catenin,CyclinD1 protein,can decrease the activity of HaCaT cells,to verify the therapeutic effect of Zhuhuang granule containing plasma is through the effect model of miR-99 a mediated.ConclusionsWe predicted and verified the relationship between cytokines and miRNA and susceptible genes,and established the regulatory network of miRNA-cytokine gene.The Internet helps people from different levels and angles in understanding the pathogenesis of psoriasis.In this study,we predicted the existence of the miR-99a/FZD/Cyclin D1 pathway in psoriasis,and this pathway has been verified in clinical and experimental;we also verify the therapeutic effect of Zhuhuang granule containing plasma is likely through the effect model of miR-99 a mediated.