| Objective: Vulvar lichen sclerosis(VLS)is a chronic inflammatory skin disorder characterized by atrophy of the labia majora,clitoral adhesion and introital stenosis.Molecular target treatment may be a promising therapeutic strategy for VLS.Micro RNAs(mi RNAs)are a large type of endogenous,small(~22 nucleotides)and single-stranded non-coding RNA molecules,which exerts the role of gene expression regulation by sequence-specific binding to 3' untranslated regions(3'UTRs)of theirs target messenger RNA(m RNAs).Increasing evidence has revealed that abnormal expression of mi RNAs is implicated in a wide range of human neoplasms and other pathological processes.Thus,a better understanding of the biological functions of mi RNAs may be helpful for developing effective therapies for human diseases.Mi R-155-5p has been reported to be up-regulated and act as an oncogene in numerous human cancers.However,the role of mi R-155 p in VLS remains unknown.This study aimed to explore the functional role of mi R-155-5p in VLS and clarify the potential molecular mechanisms involved.Methods: Different mi RNAs between the vulva lichen sclerosus and normal vulva skin were screened by mi RNA gene chip technology.Cell viability was identified by MTT assay.RT-PCR and western blot were used to determine the expression of mi R-155-5p,FOXO and CDKN1 B.Cell apoptosis was tested by Flow cytometry.Luciferase reporter assay was used for determining the targets of mi R-155-5p.Cell cycle was then examined using a flow cytometer and analyzed with Mod Fit software(BD Biosciences,San Jose,USA).Results: 1.Affymetrix mi RNA expression profiling technology screened 82 distinct mi RNA between VLS and normal vulvar skin.As evident from q RT-PCR analysis,VLS tissues exhibited higher expression levels of mi R-155-5p than matched normal vulvar tissues.Our finding indicates that mi R-155-5p may be involved in the pathogenesis and development of VLS.2.To investigate the potential role of mi R-155-5p in VLS,HFF-1 cells were transfected with mi R-155-5p mimics or mi R-155-5p inhibitor.The transfection efficiency was identified using q RT-PCR.As evident from MTT assays,cell proliferation was dramatically promoted by mi R-155-5p mimics,whereas mi R-155-5p inhibitor notably suppressed cell proliferation.Flow cytometry was performed to assess cell cycle after transfection with mi R-155-5p mimics or mi R-155-5p inhibitor.HFF-1 cells overexpressing mi R-155-5p exhibited a lower percentage of G1-phase cells and a higher percentage of S-phase cells than HFF-1 cells transfected with mimics-NC.A significant increase in the proportion of G1-phase cells and a notable decrease in the proportion of S-phase cells were observed in mi R-155-5p inhibitor treatment compared with inhibitor-NC group.These results suggest that mi R-155-5p promotes fibroblast cell proliferation and cell cycle progression.3.Target Scan and mi Randa algorithms were employed to predict the potential targets of mi R-155-5p.FOXO3 is known to be closely associated with cell proliferation;in addition,CDKN1 B has been frequently reported to exert crucial roles in cell cycle progression.Here,FOXO3 and CDKN1 B were selected as candidate targets of mi R-155-5p.To further verify whether FOXO3 and CDKN1 B are regulated by direct binding of mi R-155-5p to their 3'UTRs,luciferase reporter assays were performed.The 3'UTR fragments of FOXO3 and CDKN1 B containing mi R-155-5p targeting sequences and its corresponding mutant fragments were subcloned into firefly luciferase vectors,respectively.As obvious from luciferase reporter assays,luciferase activities of wild-type FOXO3 3'UTR vectors and wild-type CDKN1 B 3'UTR vectors were dramatically repressed by mi R-155-5p mimics,but enhanced by mi R-155-5p inhibitor;the alterations of luciferase activity were abolished after mutations in the putative binding sites of mi R-155-5p in the 3'UTRs of FOXO3 and CDKN1 B.Cells transfected with mi R-155-5p mimics were found to display lower m RNA levels of FOXO3 and CDKN1 B than cells treated with mimic-NC;furthermore,a dramatic increase in the m RNA levels of FOXO3 and CDKN1 B was observed in mi R-155-5p inhibitor treatment compared with inhibitor-NC group.Our data indicate that mi R-155-5p binds directly to the 3'UTRs of FOXO3 and CDKN1 B to repress their expression.FOXO3 and CDKN1 B m RNA levels in VLS tissues and corresponding normal vulvar tissues were detected using q RT-PCR.VLS tissues were observed to show lower m RNA levels of FOXO3 and CDKN1 B.Additionally,Pearson's correlation analysis demonstrated that mi R-155-5p expression was negatively correlated with FOXO3 and CDKN1 B expression in VLS tissues,respectively.4.FOXO signaling pathway is known to play critical roles in cell cycle regulation.Expression of FOXO1 and p130,important FOXO signaling pathway-associated proteins,was identified in HFF-1 cells by Western blot analysis.As obvious from Western blot,a significant decrease in the expression levels of FOXO1 and p130 was found in mi R-155-5p mimics treatment compared with mimics-NC group;a notable increase in the levels of FOXO1 and p130 was observed in HFF-1 cells transfected with mi R-155-5p inhibitor in comparison with inhibitor-NC group.These data indicate that mi R-155-5p represses FOXO signaling pathway.5.Given that FOXO3 and CDKN1 B are closely associated with cell proliferation and cell cycle,we lowered the expression of FOXO3 or CDKN1 B in HFF-1 cells using specific si RNAs,with a view to exploring the functional connection between mi R-155-5p and its targets,FOXO3 and CDKN1 B.The knockdown efficiency was evaluated via western blot analysis.As obvious from MTT assays,cell proliferation was notably promoted by the knockdown of FOXO3 or CDKN1 B compared with negative control group.A notable decrease in the proportion of G1-phage cells and a marked increase in the proportion of S-phage cells were detected in si-FOXO3 and si-CDKN1 B treatments compared with si-NC treatment.The data demonstrate that downregulation of FOXO3 or CDKN1 B has similar effects with mi R-155-5p overexpression in fibroblast cells.To further investigate the functional connection between mi R-155-5p and its targets,FOXO3 and CDKN1 B,HFF-1 cells were co-transfected with mi R-155-5p inhibitor and si-FOXO3 or si-CDKN1 B.The results showed that mi R-155-5p inhibitor significantly repressed cell proliferation and the inhibitory effect of mi R-155-5p inhibitor on cell proliferation was partially reversed by FOXO3 or CDKN1 B knockdown.The data suggest that mi R-155-5p exerts its suppresssive effect on cell proliferation partially by targeting FOXO3 and CDKN1 B.Conclusion: Taken together,our results indicate that mi R-155-5p promotes fibroblast cell proliferation and inhibits FOXO signaling pathway by negative modulation of both FOXO3 and CDKN1 B in VLS,and that mi R-155-5p may be used to be a potential therapeutic target for VLS. |
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