Analysis Of Differentially Expressed LncRNAs And CircRNAs In Hypertrophic Scar And The Study On Autophagy And Exosome Intervention

ObjectiveHypertrophic scar（HS）is a major problem that disturbs clinical practice.Finding the gene target of HS is the main direction of the treatment.Non-coding RNAs and autophagy play important roles in gene regulation.But their biological effects in HS are unclear.Studies found that microRNAs（miRNAs）were involved in the regulation of autophagy and affected the formation of HS.As Competitive endogenous RNA（ceRNA）,long non-cording RNAs（lncRNAs）and circular RNAs（circRNAs）can be combined with miRNAs to participate in biological regulation.However,their relationship with autophagy and the biological function in HS are still unknown.In addition,exosomes have been shown to play important roles in the diagnosis and treatment of diseases through information exchange and material transfer between cells.Up to now,there are no relevant studies on the signal transfer of exosomes about lncRNAs or circRNAs in HS.Therefore,this study proposed to analysis of the differences of lncRNAs and circRNAs expression in HS,and changes of lncRNAs and circRNAs after autophagy regulation,to further explore the biological functions of lncRNAs or circRNAs in HS,and the relationship with autophagy.Meanwhile,the effects of exosomes transport were also analyzed.As an ideal carrier,exosomes might be implanted autophagy-related lncRNAs or circRNAs for gene therapy of HS.Methods1.Normal skin and HS specimen collection of clinical patients were adopted;then extracted the total RNA and got the quality control;LncRNA,circRNA and mRNA in the tissues were examed by RNA high-throughput sequencing;and qRT-PCR was performed for the verification of RNA-seq.2.Bioinformatics analysis software was used to study the expression profile characteristics of lncRNA,circRNA and mRNA,and to conduct clustering and enrichment analysis.The related CNC and ceRNA networks were constructed to analyze the co-expression of lncRNA-circRNA-mi RNA-mRNA,and determine themajor biochemical metabolism and signal transduction pathways involved in the gene expression of differentially expressed genes.And to analyze the possible biological effects.3.In vitro cultured and transmitted human HS fibroblasts.After starvation culture,rapamycin was used to induce autophagy;and then cell RNA extraction and quality control were processed.LncRNA and circRNA in the two groups were detected by RNA sequencing.The differences between lncRNA and circRNA in fibroblasts after autophagy intervention were analyzed.Through network construction,the lncRNA and circRNA associated with autophagy were further searched,and its possible biological function was analyzed.4.On human amniotic membrane in vitro cultivation for mesenchymal stem cells were processed.After hungry training,overspeed centrifugal was used for extraction the exosomes from the supeRNAtant fluid.And joined it to fibroblasts,then the cells RNA extraction and quality inspection were conducted.LncRNA and circRNA in the two groups were detected by RNA sequencing,and the difference expression of lncRNA and circRNA in fibroblasts after exocrine intervention was analyzed.Through network construction,the transshipment and regulation effects of stem cells derived exosomes on related lncRNA and circRNA were further explored.Result1.536 differentially expressed mRNA,3469 lncRNA and 11 circRNA were identified between the hyperplastic scar and normal skin controls.Among them,mRNA 345,lncRNA 2479 and cicRNA 10 were up-regulated,and the mRNA 191,lncRNA 990 and cicRNA were down-regulated.2.Enrichment analysis of differentially expressed genes showed that they mainly attended extracellular matrix metabolism,DNA transcription regulation and regulation of gene expression and biological function.All of them mainly participated in the regulation of TGF beta signal pathways,PI3K/Akt signal pathways,ECM-receptor signal pathways,etc.3.CNC network analysis results indicated that: the H19,INHBA AS1,TGFB3AS1,LINC00299,AC048380.1 and AC037198.1;circ-Chr17:50187014₅0195976_-,circ-Chr17:50189167₅0194626_-,circ-Chr17:50189167₅0198002_-,circ-Chr17:50189858₅0195330_-were increased significantly.And CASC11,circ-chr9:125337017₁25337591₊ and circ-chr12:120782654₁20784593_were significantly reduced.All these genes related to INHBA,SMAD7,COL1A1,TGFB3,ID2,MYC,etc.4.CeRNA network analysis hinted that: INHBA-AS1 and circ-Chr17:₅0195976_-50187014,circ-Chr17:₅0194626_-50189167,circ-Chr17:₅0198002_-50189167,circ-Chr17:50189858₅0195330_-could competitive combinate with mi R-145-5 p,miR-125-a-5 p,miR-129-5 p,mi R-7-5 p and targeted the expression of HTR2 A,GLIS3,LOX,SERPINE1,EYA4,CDH2,ADAMTS2,etc.5.In the fibroblasts with autophagy intervention group,186 differentially expressed lncRNA,56 circRNA and 190 mRNA were identified.Among them,84 lncRNA,41 cicRNA and 70 mRNA were up-regulated,and 102 lncRNA,15 cicRNA and 70 mRNA were down-regulated.6.The results of enrichment and network analysis showed that the difference genes were mainly involved in the biological processes of cell growth and metabolism,extracellular matrix construction,vascular growth,and transformation of growth factor receptor.And most of them participated in autophagy related signaling pathways,such as MAPK signaling pathway,signaling pathway,m TOR signaling pathway,etc.7.The results of CNC network analysis indicated that XLOC₀00035,XLOC₀00038,XLOC₀00292,circ-chr3:47037665₄7046621_-,circ-chr8:130358016₁30361771_-were positively correlated with TGFB3 and INHBE,which were negatively correlated with SMURF2,INHBA and BMP6.And XLOC₀00146was negatively correlated with TGFB3.8.The ceRNA network analysis suggested that XLOC₀00038,circ-chr8:130358016₁30361771_-could competed combine to the binding sites of miR-195-5p,miR-497-5p,mi R-15a-5p,miR-15b-5p,and targeted to SMURF2.At the same time,circ-chr3:47037665₄7046621_-,XLOC₀00038 can be competitive with miR-130a-5p,miR-23a-3p,miR-23b-3p and mir-23 c which targeting at SMURF2.9.In the autophagy intervention group,lncRNA SNHG1 expression wasincreased,circ-chr8:130358016₁30361771_-,circ-chr3:47037665₄7046621_-expression were decreased.All of them might be combined with miR-195 to reduce the expression of SMURF2.10.In the exosomes intervention group,172 lncRNA,45 circRNA and 262 mRNA showed significant differences expression.In which 138 lncRNA,32 cicRNA and 205 mRNA were up-regulated,and 34 lncRNA,13 cicRNA and 57 mRNA were down-regulated.11.The results of enrichment and network analysis showed that the difference genes were mainly related to cell differentiation,cell migration,cell matrix construction,nucleotide synthesis and metabolism,transcription factor regulation and other life activities.All of them mainly were involved in the regulation of cell adhesion,TGF-beta,HIF-1,PI3K-Akt,ECM-receptor signaling pathway.12.CNC network analysis results indicated that: XLOC₀00038,XLOC₀00026,circ-chr1:176555406₁76557241₊,circ-chr1:1815755₁825499_-,circ-chr1:1815755₁839238_-,circ-chr19:3660965₃662001_-,circ-chr8:130152735₁30180880_-,circ-chr8:140879470₁40890769_-were positively associated with SPP1,HGF and NR4A1 and negatively correlated with COL1A1,PIK3 CD,FGF18,ITGA2,CREBBP,EP300 and EGFR.13.The ceRNA network analysis suggested that XLOC₀00026,circ-chr1 :176555406₁76557241₊ could be jointly competitive with miR-27a-3p binding site and targeting at ITGA2 and HGF;XLOC₀00038,circ-chr8: 130152735₁30180880_,circ-chr1:176555406₁76557241₊,circ-chr8:140879470₁40890769_-could be competitive with mi R-497-5p,miR-195-5p,miR-15a-5p,miR-15b-5p,miR-424-5p and miR-6838-5p,and target at ITGA2,FGF18,etc.14.In the exosomes intervention group,the expression of circ-chr8:130152735₁30180880_-,circ-chr1:176555406₁76557241₊,circ-chr8:140879470₁40890769_-were decreased.We speculated that exosome might be involved in the out transport of these circRNA and reduce the competitive combination of miRNAs,and increase ITGA2 expression.Conclusion1.There were different expressions of lncRNAs and circRNAs between humanhypertrophic scar and normal skin tissues.The differences RNAs participate in the TGF beta,PI3K/Akt,ECM-receptor signal pathway and closely associated with hyperplastic scar formation.2.Different expressions of lncRNAs and circRNAs in the cultured fibroblasts with autophagy were confirmed.They were involved in the various signal pathways of autophagy.Autophagy could interact with differential lncRNAs or cirRNAs to regulate development of hypertrophic scar.3.There were different expressions of lncRNAs and circRNAs in the cultured fibroblasts with exosomes.The reduction of related genes might be related to the out transport by exosomes of stem cell.Exosomes implanted autophagy-related lncRNAs or circRNAs might be the effective means of hypertrophic scar gene therapy.