The Expression Of HedgeHog Signaling Pathway And Epithelial-Mesenchymal Transformation Associated With Lncrnas In Keloid

Objective: Current studies have shown that Hedgehog（HH）signaling pathways and epithelial mesenchymal transition（EMT）play an important role in the development of embryonic development,cell proliferation and differentiation,and the development of tumor and fibrotic diseases.Recent studies have found that they also have abnormal changes in pathological scars,but their specific roles and molecular mechanisms remain to be elucidated.Our previous study showed that the expression of long chain non coded RNA（lncRNAs）,which is closely related to the signal pathway,has changed significantly in the pathological scar.On this basis,we use a chip to search for differential expression of lncRNAs and mRNAs related to Hedgehog signaling pathway and EMT in keloids and normal skin tissues,and conduct a series of bioinformatics analysis.The molecular mechanism of keloid formation is discussed from a new perspective.It will provide new ideas and theoretical basis for further searching for new targets in clinical treatment.Methods:（1）The epidermis of the keloid and adjacent normal skin were removed respectively.The total RNA was extracted by Trizol and tested for quality.After the quality examination was qualified,the fluorescent labeling,Arraystar human Lnc PathTM Hedgehog chip（8x15K）were hybridized,and the cross images were scanned,and the Agilent Feature Extraction v11.0.1.1 software was used.The data were analyzed by AgilentGeneSpring GX V12 software and the differential expression profiles of lncRNAs and mRNAs related to Hedgehog signaling pathway were detected.Select the up regulation of lncRNALOC100271722 related to the HH signaling pathway,the down lncRNA HNF1A-AS1,And the up regulation of mRNA,including SFRP2,WNT7 B,HNF1A,the down regulation of mRNA WIF1,GAPDH as the internal reference,and fluorescence quantitative PCR verification.Different-ially expressed mRNAs were subjected to Gene Ontology（GO）and Pathway functional annotation cluster analysis using DAVID Bioinformatics Resources 6.7 to obtain functional enrichment of differential m RNA-related functions.（2）After passing the quality test,it is purified and subjected to fluorescencelabeling and hybridization with Arraystar human LncPathTM EMT chip（8x15K）.The hybridization image is then scanned and processed using Agilent Feature Extraction v11.0.1.1 software.Data analysis was performed using Agilent’s GeneSpring GX v12 software to detect differential expression profiles of lncRNAs and mRNAs associated with EMT signaling pathways.Select the up regulation of lncRNAsAK055628,NR₀33321 related to EMT.The down lncRNAENST00000399188,the up regulation of mRNA ENST00000304636,NM₀06475,the down regulation of m RNA NM₀53056,GAPDH was used as an internal reference,and fluorescence quantitative PCR was used to verify the reliability of the chip results.Combined with previous reports and databases,we analyzed subclasses and target genes of differentially expressed lncRNAs.Results:（1）Compared with normal skin epidermis,30 lncRNAs and 33 m RNAs related to Hedgehog signaling pathway were screened for differential expression.16 lncRNAs were significantly up-regulated in keloid epidermis,including AK055628,MIAT,MIR31 HG,RP11-264F23.3,and AC073257.2.Significant down-regulation of14 lncRNAs significantly down-regulated in keloid epidermis,including: RP11-12M9.3,XLOC₀07437,XLOC₀09485,RP5-1042I8.7,HNF1A-AS1,etc.Thirteen mRNAs were significantly up-regulated,including SFRP2,ANGPT2,APC2,IVL,and GLI2;20 mRNAs were significantly down-regulated,including WIF1,KRT1,CCND1,BCL2,and NOTCH2.The qPCR verification results are consistent with the chip detection results.GO analysis and KEGG pathway analysis showed that the up-regulated mRNAs involved in cell proliferation,growth and tissue remodeling,and down-regulated mRNAs involved in biological processes such as cell death and apoptosis.Both lncRNA AC073257.2 and its target gene Gli2 up-regulated in keloid,and Arraystar lncRNA database predicted that lncRNA AC073257.2 might have the functional characteristics of enhancer and could positively regulate the expression of its upriver target gene Gli2 at the transcriptional level and thus effect cell growth and proliferation.In the keloid HNF1A-AS1 down-regulated while its target gene HNF1 A up-regulated,and lncRNA HNF1A-AS1 might negatively control the expression of its neighboring target gene HNF1 A horizontally during or after the transcription and thereby impact on the growth of keloid.（2）In the keloid we found that 26 lncRNAs and 39 mRNAs were differential expression relating to EMT,among those 9 lncRNAs raised obviously,17 lncRNAs lowered obviously,17 mRNA raised obviously,22 mRNA lowered obviously,and the qPCR verification result was in accordance with the chip test result.The differential expression of lncRNA NR₀33321 may play an important role in the process of keloid fibrosis.The neighboring target gene lncRNA ENST00000379816 may regulate the target gene MT1 B by the base pairing or cis acting,which may play the role in the epithelial proliferation and differentiation.lncRNA ENST00000399188 and ENST00000454001 can act as enhancers of EP300 and LATS2 respectively,which may influence the transcriptional level of adjacent protein-coding gene promoter and regulate cell proliferation,migration and the process of keloid EMT.lncRNA ENST00000573934 can bind to miRNA by competitive endogenous mechanism and regulate the expression of corresponding protein-coding genes,so participate in the process of epidermal EMT.These differential expression of lncRNAs can affect the expression of the nearby protein-coding gene by acting as an enhancer or competitive endogenous mechanisms,which may regulate the EMT process and the development of keloid.All these results may be useful to research the function and mechanism of one or two lncRNA step by step,which can more clearly clarify the regulatory mechanism of lncRNAs in the process of epidermal EMT,and seek novel therapeutic method for the keloid.The prediction of differential expression lncRNAs show the target genes involved in regulating the EMT process,suggesting that lncRNAs in keloid skin play a regulatory role in the process of EMT.Conclusion:（1）Compared with normal skin tissue,the lncRNAs and m RNAs related to Hedgehog signaling pathway and the lncRNAs and mRNAs related to EMT were significantly different from those of human keloid tissue,which may be closely related to the development and prognosis of keloid.（2）The up regulation of lncRNA AC073257.2 in keloid tissue has the functional characteristics of the enhancer,which may regulate the expression of the upstream target gene Gli2 at the transcriptional level,and the down regulated HNF1A-AS1 may negatively regulate the expression of its adjacent target gene HNF1 A at the transcriptional or post transcriptional level.（3）Up-regulation of lncRNA ENST00000379816 in keloid tissue may regulate its adjacent target gene MT1 B through base complementary pairing or cis-acting,thereby participating in epithelial proliferation and differentiation;Down-regulated lncRNA ENST00000399188 and ENST00000454001 may act as enhancers of EP300 and LATS2,respectively.Interfere with transcription of promoters of adjacent protein encoding genes,thereby affecting cell proliferation,migration,and epithelial mesenchymal transition.Up-regulation of lncRNAs ENST00000573934 may bind miRNA through a competitive endogenous mechanism,regulating its target genes ETS1,HDAC3,PCBP1,and EPB41L5 expression.（4）Differentially expressed lncRNAs may participate in the keloid Hedgehog signaling pathway and EMT changes by affecting the expression of adjacent protein-coding genes,acting as enhancers or competing endogenous mechanisms,resulting in the formation and evolution of keloids.Its detailed role and its molecular mechanism remain to be further explored and clarified.