The Primary Study Of LncRNA Expression Profile And Genomic Methylation Abnormality In Keloid

BackgroundKeloid disease（KD）is a type of benign tumor unique to human skin.Long non-coding RNA（lncRNA）regulating gene transcription and DNA methylation modification participate in a wide-spread biological processes and diseases,especially in numerous cancers.However,studies reported that IncRNA and DNA methylation are involved in keloid,the specific mechanism is unknown.ObjectiveThe aim of this study is to primarily explore the expression of lncRNA in keloid and potential biological roles of lncRNA involved in the pathogenesis of keloid;and the regulation of DNA methylation in the promoter region in keloid formation.Methods1.The IncRNA/mRNA microarray,IncRNA subgroup analysis,pathway analysis and coding-non-coding RNA co-expression analysis were used to screen differentially expressed IncRNA/mRNA（DE-lncRNA/mRNA）and find the relationship between DE-lncRNA and DE-mRNA in 5 cases of keloid tissue（KT）（experimental group）and 5 cases of normal tissue（NT）（control group）.2.Isolation of keloid-derived fibroblasts（KFBs）was from intralesional tissues of keloid,and normal-fibroblasts（NFBs）after healthy men’s circumcision.3 cases of KFBs and 3 cases of NFBs were utilized for IncRNA/mRNA microarray and pathway analysis screening the overlapped DE-lncRNA/mRNA expressed both in KT and KFBs;and the relative expression level of target IncRNA was detected by quantitative Real-time PCR（qRT-PCR）in 79 KTs and 21 NTs.3.The methylated DNA immunoprecipitation-sequencing（MeDIP-Seq）and differentially methylated in the promoter regions analysis were used to find out the relationship between overlapped DE-mRNA and differentially methylated in the promoter regions in 3 cases of KFBs and 3 cases of NFBs.And the relative expression level of target DE-mRNA was detected both in tissue samples and cell samples,such as 5 KTs and 5NTs,3 KFBs and 3NFBs,via qRT-PCR.Then,the target DE-mRNA was subjected to qRT-PCR validation in larged samples,such as 49 KTs and 24 NTs.Results1.There were 3680 DE-lncRNAs and 5448 DE-mRNAs identified in the KT compared to NT.Among the DE-lncRNAs,2238 were up-regulated and 1442 were down-regulated.Furthermore,2526 up-regulated and 2922 down-regulated DE-mRNAs were confirmed in the KT compared to NT（fold change≥2 and p<0.05）.2.The up-regulated transcripts were mainly involved in extracellular structure organization and extracellular matrix organization,the most active pathway related with these molecules was ECM-receptor interaction.And other pathways,such as focal adhesion,Wnt signaling pathway and PI3K/Akt signaling pathway were involved.Whereas,the down-regulated transcripts were primarily involved in metabolic process and oxidation-reduction process.The most active pathway related with these molecules was valine,leucine and isoleucine degradation,and carbon metabolism pathway was involved.3.In the DE-lncRNAs subgroup analysis,72.5%of genic lncRNAs were regulated synergistically with their associated mRNAs,and the rest was in the opposite direction.There were 393 enhancer-like lncRNAs and 864 long intergenic non-coding RNAs（lincRNAs）with their nearby genes.4.In the most active pathway in up-regulation,there were 5 DE-mRNAs including COL5A2,COL6A2,ITGA1,ITGB1and THBS3 had relative DE-genic lncRNAs including ENST00000419029,uc010gqe.2,uc003jox.1,ENST00000414157 and ENST00000447623,respectively.Whereas,only DE-mRNA-ACAT2 had relative DE-genic lncRNA-NR₀37166 in the most active pathway in down regulation.Similarly,8 kinds of DE-mRNAs in Wnt pathway were changed along with their relative DE-genic lncRNAs,DE-mRNAs-PRKACB had one relative DE-enhancer-like lncRNA-ENST00000439186,and 3 kinds of DE-mRNAs including CTBP1,FZD1 and PRKACB had 4 DE-lincRNAs,including TCONS₀0007951,TCONS₀013884,TCONS₀0014352 and TCONS₀0001546.In PI3K/Akt pathway,9 kinds of DE-mRNAs had the change of their relative DE-genic lncRNAs,and 2 kinds of DE-mRNAs,such as COL5A1 and THBS2,had relative DE-enhancer-like lncRNAs,including ENST00000423455,ENST00000439730 and ENST00000413324;5 kinds of DE-mRNAs had their relative DE-lincRNAs.5.From the mRNAs-lncRNAs co-expression analysis,ITGB1 and COL5A2 were identified to have the most amounts of associated IncRNAs.Moreover,one mRNA was related to many lncRNAs,and one IncRNA was related to many mRNAs.6.There were 1231 DE-lncRNAs and 1744 DE-mRNAs in KFBs compared with NFBs.Among the DE-lncRNAs,738 were up-regulated and 493 were down-regulated.Moreover,1165 DE-mRNAs were up-regulated and 579 DE-mRNAs were down-regulated（fold change≥2 and p<0.05）.7.There were 71 overlapped DE-lncRNAs and 271 overlapped DE-mRNAs expressed both in KT and KFBs.Among them,59 DE-lncRNAs were up-regualted,12 DE-lncRNAs were down-regualted and,up-and down-regulated DE-mRNAs were 183 and 88,respectively.8.The up-regulated DE-lncRNAs,such as uc003jox.1,ENST00000414157 and ENST00000439703,were tightly associated with PI3K-Akt pathway.Furthermore,the relative expression level of lncRNA-uc003jox.1 and lncRNA-ENST00000439703 were high in KT compared with NT.9.Compared with NFBs,there were 758 mRNA-associated differentially methylated regions（DMRs）in promoters in KFBs,of which 380 were differentially hypermethylated regions associated genes and 378 were differentially hypomethylated regions associated genes.10.There were 4 overlapped up-regulated DE-mRNAs（STEAP1,SLC38A5,CRISPLD2 and IL32）with differentially hypomethylated regions in promoters and 1 overlapped down-regulated DE-mRNAs-ALX1 with differentially hypermethylated regions in promoters.11.The targeted DE-mRNAs（STEAP1,SLC38A5 and IL32）were expressed high in KFBs compared with NFBs,and targeted DE-mRNAs（STEAP1,SLC38A5 and CRISPLD2）had increased expression in KT compared with NT.Conclusion1.The interplay of numerous lncRNAs and mRNAs are involved in the pathogenesis and development of keloid.2.The PI3K/Akt signaling pathway is activated and the associated IncRNAs,including uc003jox.1 and ENST00000439703 have increased expression in keloid.3.The hypomethylated regulation in promoters of up-regulated mRNAs,such as STEAP1,SLC38A5 and CRISPLD2,plays a role in the pathogenesis of keloid.