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Authors Chai L, Kang XJ, Sun ZZ, Zeng MF, Yu SR, Ding Y, Liang JQ, Li TT, Zhao J
Received 21 January 2018
Accepted for publication 2 March 2018
Published 3 May 2018 Volume 2018:10 Pages 989—1003
DOI https://doi.org/10.2147/CMAR.S163335
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Amy Norman
Peer reviewer comments 2
Editor who approved publication: Dr Antonella D'Anneo
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.
Keywords: melanoma, miR-497-5p, miR-195-5p, miR-455-3p, hTERT