MiR-497-5p, miR-195-5p and miR-455-3p function as tumor suppressors by targeting hTERT in melanoma A375 cells

Abstract

Background

hTERT gene plays an important role in melanoma, although the specific mechanism involved is unclear. The aim of this study was to screen and identify the relative miRNAs with the regulation of hTERT in melanoma.

Materials and methods

Quantitative real-time polymerase chain reaction (q-PCR) and immunohistochemistry were performed to detect hTERT mRNA and protein expression in 36 formalin-fixed paraffin-embedded melanoma tissues and 36 age- and sex-matched pigmented nevi cases, respectively. Bioinformatics analysis and custom miRNA polymerase chain reaction array were determined for predicting, screening and verifying miRNAs with the regulation of the hTERT gene. 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 was determined by q-PCR. The protein expression of hTERT was detected by Western blot. 3-(4,5-Dimethylthiazolyl-2-yl)-2,5-biphenyl tetrazolium bromide and flow cytometry were employed to detect cell proliferation ability, cell apoptosis and cell cycle. Transwell and wound healing assays were used to observe cell invasion and migration abilities. A direct target gene of miRNAs was analyzed by a dual luciferase reporter activity assay.

Results

MiR-497-5p, miR-195-5p, miR-455-3p were significantly downregulated, while hTERT was upregulated in melanoma tissues. hTERT expression level was inversely correlated with miR-497-5p, miR-195-5p and miR-455-3p. Overexpression of miR-497-5p, miR-195-5p and miR-455-3p inhibited A375 cell proliferation, migration and invasion, arrested the cell cycle, induced cell apoptosis and decreased hTERT expression at both mRNA and protein levels. Suppression of miR-497-5p, miR-195-5p and miR-455-3p partially reversed the inhibitory effects. Finally, hTERT was identified as a direct target of miR-497-5p, miR-195-5p and miR-455-3p.

Conclusions

MiR-497-5p, miR-195-5p and miR-455-3p act as tumor suppressors by targeting hTERT in melanoma A375 cells. Therefore, miR-497-5p, miR-195-5p and miR-455-3p could be potential targeted therapeutic choice for melanoma.