Screening And Functional Study Of Related MiRNAs Regulating HTERT Gene In Malignant Melanoma

Objective: The aim of this study is to detect human telomerase reverse transcriptase(hTERT)mRNA and its protein expression in formalin fixed paraffin-embedded(FFPE)tissues of malignant melanoma and piegmented nevus.Then analyze the relationship between hTERT mRNA and its protein,as well as the clinicopathological characteristics.Next,we predict and screen the related miRNAs regulating hTERT gene,analysis the relationship between candidate miRNAs and hTERT mRNA.The cell functional tests were performed to explore the effects on hTERT mRNA and its protein expression after over-expression or inhibition of related miRNAs.A direct target gene of miRNAs was analyzed by a dual luciferase reporter activity assay.Methods: Quantitative real-time polymerase chain reaction(qPCR)and immunohistochemistry(IHC)were performed to detecting hTERT mRNA and protein expression in 36 FFPE melanoma tissues and 36 ageand sex-matched pigmented nevi cases,respectively.Bioinformatics analysis was determined to predicting and screening candidate mi RNAs with the regulation of hTERT gene.The expressions of candidate miRNAs were verified by Custom mi RNA PCR Array.To investigate the biological functions,miRNAs mimics or inhibitors were transfected into melanoma A375 cells.The relative expression of miR-497-5p,miR-195-5p,miR-455-3p and hTERT mRNA were performed by qPCR.The protein expression of hTERT was detected by western blot.MTT and flow cytometry were employed respectively to detect cell proliferation ability,cell apoptosis and cycle.Transwell assay and wound healing assay were used to observe cell invasion and migration abilities.A dual luciferase reporter activity assay was used to explore the potential mechanism of related miRNAs regulating hTERT gene.Results: The expression levels of hTERT mRNA and its protein in melanoma were all higher than that in pigmented nevus tissues.The hTERT mRNA expression levels in distant metastasis patients were significantly higher than that in non-distant metastasis patients.Further more,there existed a positive correlation between hTERT mRNA and it's protein expression.14 candidate miRNAs were selected afterprediction and screening.5 differentially expressed miRNAs were found by Custom miRNA PCR Array.MiR-497-5p,mi R-195-5p and mi R-455-3p were significantly down-regulated,while miR-212-5p and miR-424-5p were significantly up-regulated in melanoma tissues compared with pigmented nevus control.What's more,hTERT mRNA and protein expression levels were inversely correlated with mi R-497-5p,miR-195-5p and miR-455-3p.Over-expression of miR-497-5p,miR-195-5p and mi R-455-3p inhibited A375 cells proliferation,migration,invasion,arrested cell cycle and induced cells apoptosis.Suppression of miR-497-5p,mi R-195-5p and miR-455-3p partially reversed the inhibitory effects.Over-expression of miR-497-5p,miR-195-5p and miR-455-3p decreased hTERT expression at both mRNA and protein levels.And miR-497-5p,miR-195-5p and miR-455-3p were involved in the occurrence and progression of melanoma by direct binding to 3'UTR region of hTERT gene.Conclusions: MiR-497-5p,miR-195-5p and miR-455-3p act as tumor suppressors by directly targeting hTERT 3'UTR region in melanoma.Therefore,miR-497-5p,miR-195-5p and miR-455-3p could be a potential targeted therapeutic choice for the treatment of melanoma.