| Background and Objectives: Oral submucous fibrosis(OSF)is a kind of oral potentially malignant disorders(OPMDs),which has the potential to develop into oral squamous cell carcinoma(OSCC).The mechanism of OSF malignant transformation is complex.Predicting and identifying the malignant transformation of OSF is an important research field.Long non-coding RNAs(lncRNAs)are RNAs with a length of more than 200 nucleotides,which act at the transcriptional and post-transcriptional levels.Many lncRNAs are tissue-specific,so the search for lncRNAs-related biomarkers has become a research hotspot in recent years.In addition,because lncRNAs are superior to proteins in tissue-specific,cell-network specific regulation and toxicity,lncRNAs that play a key role in disease may be potential therapeutic targets.The function and mechanism of lncRNAs in OSF and its malignant transformation are still far from elucidated.This study aims to identify lncRNAs related to the malignant transformation of OSF to OSCC,and preliminarily explore their functions and mechanisms.This will help to clarify the mechanism of OSF malignant transformation from the molecular level,provide potential early diagnosis and prognostic markers,and provide new ideas for its targeted therapy.Methods:1.Microarray data of the GSE125866 and GSE64216 datasets were downloaded from Gene Expression Omnibus(GEO).Differentially expressed gene analysis(DEGA)was performed by using “limma”packages and “edge R”.GO and KEGG pathway enrichment analyses were carried out by using Fisher’s exact test.Weighted gene co-expression network analysis(WGCNA)was performed by using“WGCNA” packages in “R” to identify the most significant modules related to the malignant transformation of OSF.Then,the intersection of DEGA and WGCNA results was taken to obtain lncRNAs and mRNAs related to the malignant transformation of OSF.LncRNA-related ceRNA networks were constructed by miRNAs-mRNAs and lncRNAs-miRNAs pairs.2.Based on the Cancer Genome Atlas(TCGA)database,lncRNAs associated with the prognosis and early diagnosis of OSCC in ceRNAs were screened by using Kaplan-Meier prognostic analysis model("survival " of the "R" package),Cox regression analysis model and ROC curve analysis("p ROC" package).A nomogram was constructed to quantitatively predict OSCC prognosis.Clinical tissue samples were used to verify the expression of candidate lncRNAs.External data sets were used to further verify the expression of candidate lncRNAs at the big data level.3.Gene Set Enrichment Analysis(GSEA)was performed by running the "GSEA" package in "JAVA" and "R" to predict the biological functions and mechanisms of candidate lncRNAs in the malignant transformation of OSF.4.Nucleocytoplasmic separation assay and fluorescence in situ hybridization(FISH)were used to confirm the subcellular localization of the candidate lncRNAs.RNA knockdown and overexpression,qPCR,western blot(WB),CCK-8 assay,flow cytometry assay,wound-healing assay,Transwell assay,cell derived xenograft(CDX)and dual luciferase gene reporter assay were performed to investigate the biological functions and mechanisms of the candidate lncRNAs.Results:1.26 upregulated mRNAs,51 downregulated mRNAs,7upregulated lncRNAs,and 13 downregulated lncRNAs with sequential changes from normal oral mucosa(NOM)to OSF to OSCC were identified to construct ceRNA networks.The upregulated-lncRNAmediated ceRNA network contains 3 lncRNAs,7 miRNAs,and 7mRNAs.The downregulated-lncRNA-mediated ceRNA network contains8 lncRNAs,25 miRNAs,and 20 mRNAs.A total of 11 lncRNAs that may play a role in the malignant transformation of OSF to OSCC were identified based on the ceRNA networks.2.Kaplan-Meier survival analysis suggested that 3 lncRNAs:LINC02147,RP11.108K3.1 and LINC01725 have potential prognostic values for OSCC.ROC analysis suggested that only LINC02147 and RP11.108K3.1 could be used as early diagnostic markers for OSCC.The expression of LINC02147 was confirmed by qPCR at clinical tissue levels that LINC02147 was sequentially downregulated from NOM to OSF to OSCC.LINC02147 was highly expressed in NOM and gradually decreased in OSF and OSF cancerous tissues(OSCC).The expression of LINC02147 was significantly lower in OSF and OSCC than in NOM(p<0.05),and significantly lower in OSCC than in OSF(p<0.05),which was consistent with the results of bioinformatics analysis.However,the expression of RP11-108K3.1 was not confirmed in clinical tissues by qPCR.Moreover,GSE160042 also validated the expression of LINC02147.Meanwhile,low LINC02147 expression,as an independent prognostic factor,predicted a poor prognosis for OSCC.A LINC02147signature-based nomogram successfully quantified each indicator’s contribution to the overall survival(OS)of OSCC.3.GSEA predicted that LINC02147 negatively regulates the basic processes of cell proliferation and differentiation in the malignant transformation of OSF,including mitotic cell cycle checkpoint,chromosome segregation,mitotic sister chromatid segregation,G1 to S cell cycle control,spindle assemble and phospholipid homeostasis.In addition,GSEA pathway enrichment map predicted that LINC02147 negatively regulated MCM pathway,mitochondrial pathway,and Rho-GTPase/ROCKs pathway in the malignant transformation of OSF.4.Nucleocytoplasmic separation assay and FISH showed the subcellular localization of LINC02147 was mainly cytoplasm in human oral mucosal fibroblasts(hOMFs).20 μg/m L arecoline significantly down-regulated the expression of LINC02147 in hOMFs(p<0.05).Knockdown of LINC02147 significantly increased the expression of TGF-β1,MCM2,MCM3,MCM5,α-SMA,COL1α1,FN1 and vimentin(p<0.05),and promoted the proliferation of hOMFs and activation of myofibroblasts.CCK-8 assay,flow cytometry assay,wound-healing assay,Transwell assay,and CDX assay demonstrated that knockdown LINC02147 promoted OSCC cell proliferation,metastasis,and invasion(p<0.05),while overexpression of LINC02147 inhibited OSCC cell proliferation,metastasis,and invasion(p<0.05).Dual luciferase gene reporter assays showed that hsa-miR-21-5p mimics could reduce the luciferase activity of LINC02147-WT+psi CHECK-2,the difference was statistically significant(p<0.05),but had no significant effect on the luciferase activity of LINC02147-MUT+psi CHECK-2(p > 0.05).hsa-miR-21-5p mimics could reduce the luciferase activity of MEIS1-WT+psi CHECK-2,the difference was statistically significant(p<0.05),but had no significant effect on the luciferase activity of MEIS1-MUT+psi CHECK-2(p > 0.05).Dual luciferase gene reporter assays confirmed that LINC02147-hsa-miR-21-5p-MEIS1 was consistent with the ceRNA theory: LINC02147 had direct interaction with hsa-miR-21-5p,and MEIS1 had direct interaction with hsa-miR-21-5p.Conclusions: In this study,we constructed lncRNA-related ceRNA networks associated with the malignant transformation of OSF and identified a novel biomarker: LINC02147.LINC02147 is a potential biomarker for the early diagnosis and prognosis in the malignant transformation of OSF to OSCC.LINC02147 acts as a tumor suppressor in OSF and its carcinogenesis by negatively regulating hOMFs proliferation and myofibroblast activation,negatively regulating OSCC cell proliferation,migration,and invasion.57 figures,12 tables,196 references... |
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