Mitomycin C Inhibits Post-ESD Esophageal Stricture By Regulating LncRNA-ATB And MiR-200b

Part 1 Integrative analysis of miRNA-mRNA expression profiles in esophageal fibrosis after ESDBackground:The incidence of esophageal fibrosis and benign esophageal stricture(BES)has increased in recent years due to the curative therapy for early-stage esophageal carcinoma,including partial esophagectomy and esophageal endoscopic submucosal dissection(ESD).However,the prevention or treatment of benign esophageal stricture is still a clinical tough.The aim of the present study was to identify key miRNAs and associated pathways of esophageal fibrosis after the ESD procedure and to find the potential targets for preventing or treating the benign esophageal stricture.Methods:During the esophageal ESD procedure,the mucosal and partial submucosal layer was directed.After the ESD procedure,the esophageal tissue in the remaining submucosal layer located in the inner side of the wound edge,referred to as normal esophageal(NE)tissue,was collected.And 1 week thereafter,post-operative esophageal(PE)tissue in the similar site was also obtained.Five cases in each group were enrolled.Microarray chip analysis was used to identify dysregulated microRNAs(miRNAs/miRs)between NE and PE tissues.According to the differentially expressed(DE)miRNAs,putative target genes were predicted according the two databases(miRDB and miRWalk).Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis and DEmiRNA interaction network analysis were performed.Reverse transcription-quantitative PCR(RT-qPCR)was performed to validate the RNA microarray results.Results:A total of 199 miRNAs were determined to be dysregualted between NE and PE tissues.Compared with the expression in the NE group,83 miRNAs were significantly upregulated,while 116 miRNAs were significantly downregulated.According to the dysregualted miRNAs and their function analysis,5 pivotal miRNAs were confirmed,including miR-223-3p,miR-142-5p,miR-582-5p,miR-21-3p and miR-218-5p.And then,forkhead box O1(FOXO1),paired box 6(PAX6),phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3CA)and adrenoceptor β1(ADRB1)were predicted as DE genes according to the DE miRNAs and the two databases(miRDB and miRWalk).The results suggested that certain pathways were markedly dysregulated,including FOXO,MAPK,AMP-activated protein kinase and signaling pathways regulating the pluripotency of stem cells and proteoglycans in cancer.According to the RT-qPCR results,the expression levels of FOXO1,PAX6,ADRB1,miR-223-3p,miR-582-5p,miR-21-3p and miR-218-5p were consistent with the integrated analysis.Conclusion:In conclusion,FOXO1,PAX6,PIK3CA and ADRB1 may have a role in esophageal fibrosis,regulated by miR-223-3p,miR-142-5p,miR-582-5p,miR-21-3p and miR-218-5p.The present results provided an improved understanding of the changes in the microenvironment during the process of esophageal fibrosis,as well as novel potential targets for the treatment of esophageal fibrosis and BES.Part 2 Microarray of lncRNA/mRNA and interaction network analysis after post-ESD esophageal fibrosisBackground:The incidence of esophageal fibrosis and benign esophageal stricture(BES)has increased in recent years due to the curative therapy for early-stage esophageal carcinoma,including partial esophagectomy and esophageal endoscopic submucosal dissection(ESD).However,the prevention or treatment of benign esophageal stricture is still a clinical tough.According to the published studies,lncRNAs regulate the process of fibrosis in many organs via various mechanisms.The aim of the present study was to identify key lncRNA and their target genes of esophageal fibrosis after the ESD procedure and to find the potential targets for preventing or treating the benign esophageal stricture.Methods:During the esophageal ESD procedure,the mucosal and partial submucosal layer was directed.After the ESD procedure,the esophageal tissue in the remaining submucosal layer located in the inner side of the wound edge,referred to as normal esophageal(NE)tissue,was collected.And 1 week thereafter,post-operative esophageal(PE)tissue in the similar site was also obtained.Five cases in each group were enrolled.Differentially expressed lncRNA(DElncRNA)and mRNA(DEmRNA)in the NE and PE tissues were identified by using microarray chip analysis.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed.According to the KEGG enrichment,related DElncRNA and DEmRNA were confirmed and lncRNA-mRNA interaction network analysis was also performed.Quantitative RT-PCR(qRT-PCR)was then performed to validate the integrated results of the lncRNA and mRNA.Results:A total of 1978 lncRNAs and 30946 mRNAs were differentially expressed between the NE and PE tissues.Compared with the NE tissue,13835 mRNAs and 1273 lncRNAs were significantly up-regulated and 17111 mRNAs and 705 lncRNAs were significantly down-regulated in the PE tissue.Furthermore,the KEGG analysis showed that certain pathways were predominantly dysregulated,including the phospholipase D signaling,MAPK,focal adhesion,cGMP-PKG,and regulation of actin cytoskeleton pathways.According to the KEGG analysis,the related DE-mRNA(CXCL8,IL1B,LAMB3,SPP1,TNC,COL4A6,DGKB,CHRM3,ADCY5,KCNMA1)and DE-lncRNA(LOC105371569,LOC105377924,SCEL-AS1,LOC107985926,PGM5P4,MBNL1-AS1,LOC105372273)were confirmed.The interaction network analysis revealed that multiple lncRNAs and multiple mRNAs interacted with each other.The qRT-PCR evaluated the expression of related mRNA and lncRNA,which was consistent with the microarray results.Conclusion:We concluded that multiple lncRNAs and mRNAs play critical roles in the process of esophageal fibrosis after ESD,by phospholipase D signaling,MAPK,focal adhesion,cGMP-PKG,and regulation of actin cytoskeleton pathways.Our findings suggested that the corresponding targets identified in our study will provide evidence for new prevention and treatment strategies for post-ESD fibrosis and BES.Part 3 The mechnism of Mitomycin C for inhibiting esophageal fibrosis Background:Benign esophageal strictures(BES)frequently results from esophageal fibrosis.The transformation of fibroblasts into fibrocyte is an important cause of fibrosis.Its treatment is challenging.Our previous clinical studies have indicated the antifibrotic effect of mitomycin C(MMC),which could treat BES.LncRNA-ATB participated in the process of liver fibrosis and keloid.The aim of this study was to confirm whether MMC inhibit esophageal fibrosis and benign esophageal stricture by regulating lncRNA-ATB,miR-200b and the following target genes.Methods:In the present study,human esophageal fibroblast cells(HEFs)were treated with different concentrations of MMC(0,2,5,l0ug/ml)and time(24h and 48h).Comparing with the MMC group,MMC combining with different drugs,including lncRNA-ATB mimic,miR-200b inhibitor,autophagy promoter(rapamycin,RAPA),autophagy inhibitor(3-Methyladenine,3-MA)contribute to the cell viability and apoptosis.The cell viability was evaluated using CCK-8 assay.The cell apoptosis was evaluated by flow cytometric analysis.In addition,expression of apoptosis related proteins(caspase8 and caspase3),autophagy related proteins(LC3Ⅱ and ATG5)and fibrosis related proteins(α-SMA,collagen-1 and TGF-β)were evaluated by Western blot and immunofluorescence staining.Furthermore,autophagosome was observed by transmission electron microscope.Results:After MMC treatment on esophageal fibroblast,the expression of lncRNA-ATB was significantly down-regulated and miR-200b was significantly up-regulated in a time-and dose-dependent manner.The maximum concentration of MMC was 10 ug/ml and the time was 24h.Comparing with the MMC group,MMC comparing with RAPA,lncRNA-ATB mimic and miR-200b inhibitor could increase cell viability,inhibit cell apoptosis,decrease the expression of apoptosis related proteins(caspase8 and caspase3),increase the expression of autophagy related proteins(LC3Ⅱ and ATG5)and fibrosis related proteins(α-SMA,collagen-1 and TGF-β).Furthermore,the number of autophagosome increased by transmission electron microscope.MMC combining with 3-MA had opposite effect.Thus,lncRNA-ATB mimic,miR-200b inhibitor and RAPA may promote fibrosis and partially reverse the effect of MMC.On the other hand,MMC comparing 3-MA could enhance the anti-fibrotic effect.Conclusion:MMC inhibits fibrosis by regulating cell apoptosis and autophagy via inhibiting lncRNA-ATB and upregulating miR-200b.This study suggests that lncRNA-ATB and miR-200b could be ideal targets for prevention and treatment of esophageal fibrosis and other age-related fibrotic diseases.