Down-regulated MicroRNA-27b-3p Is Involved In Epithelial Cell Apoptosis In Oral Lichen Planus By Targeting CypD

Objective Oral lichen planus(OLP)is commonly considered as a chronic inflammatory mucocutaneous disease with unknown etiology,which is being discussed as a potentially premalignant condition.Recent studies showed that alterations in apoptosis may play a role in the pathogenesis of OLP.Here,we examined the microRNA(miRNA)and mRNA profiling in patients with OLP and healthy controls(HC),aiming to assess a list of candidate miRNAs and genes,and provide evidence of on-target mechanisms.Material and method According to the inclusion and exclusion criteria,biopsies of oral buccal mucosa were collected from 2 patients with OLP and 2 healthy controls(HC).(1)Differentially expressed miRNAs(DEMs)and differentially expressed genes(DEGs)were screened with DESeq and edgeR software algorithm.Potential regulatory miRNAs and genes were obstained via constructing and analyzing the miRNA-mRNA networks.KEGG and GO database functional pathways/terms for DEGs were revealed using Fisher Exact Test and hypergeometric distribution analysis,respectively.(2)TdT-mediated dUTP nick end labeling(TUNEL)assay were performed to identify apoptotic cells in the epithelium of OLP and HC.The apoptosis index(AI)of the epithelium was measured in the large samples of OLP and HC.(3)Predicting the predicted target gene of miR-27b-3p,and then dual luciferase reporter assay,qPCR tests and western blot were performed to determine whether miR-27b-3p downregulated expression.(4)Deeply investigate the mechanisms of miR-27b-3p and its target geneaffecting apoptosis progression.Results(1)94 DEMs and 599 DEGs were detected in OLP compared to HC,based on which,we retrieved several potential miRNA-mRNA pairs.Identified DEGs were significantly enriched in ‘inflammatory' events and immune-related terms.(2)MiR-27b-3p is downregulated in 15 pairs of collected OLP samples and HC samples.MiR-27b-3p promotes apoptosis by affecting the expression level of apoptosis marker(caspase-3)and apoptotic cells rate through western blot(WB)and flow cytometry(FCM)analysis.(3)MiR-27b-3p suppresses CypD 3? untranslated region(3?-UTR),mRNA and protein expression.CypD inhibits apoptosis by affecting the expression level of apoptosis marker(caspase-3)and apoptotic cells rate through WB and FCM analysis.(4)Co-immunoprecipitation ? ELISA and western blot experiments confirmed that CypD interacted with Bcl2 and enhanced the limiting effect of Bcl2 on the release of cytochrome c(CytC)from mitochondria.Conclusion Our results identify a novel miR-27b-3p–CypD/Bcl2–CytC–caspase9–caspase3axis involved in the regulation of cell apoptosis through the CytC-dependent mitochondrial pathway,providing evidence of on-target mechanisms.We may shed light on a revolutionary theory that epithelial apoptosis in OLP could have its own regulatory system independent of T cell initiation.The involvement of miR-27b-3p in the pathogenesis of OLP and epithelial apoptosis makes it a high-priority therapeutic target.