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.

Keywords: hTERT; melanoma; miR-195-5p; miR-455-3p; miR-497-5p.

Figure 1

The relative expression levels of miR-497-5p (A), miR-195-5p (B), miR-455-3p (C), miR-424-5p (D) and miR-212-5p (E) in melanoma and pigmented nevi group tissues, respectively. Note: ***p<0.001, compared to control groups. Abbreviations: M, melanoma group; C, control (pigmented nevi group).

Figure 2

The relative expression of hTERT and miR-497-5p, miR-195-5p and miR-455-3p. Notes: (A) hTERT protein expression in melanoma and pigmented nevus tissues (magnification, ×100). (B) hTERT mRNA expression in melanoma and pigmented nevus tissues. hTERT mRNA expression level was inversely correlated with miR-497-5p (C), miR-195-5p (D) and miR-455-3p (E). Note: ***p<0.001, compared to control groups. Abbreviations: hTERT, human telomerase reverse transcriptase; M, melanoma group; C, control (pigmented nevi group).

Figure 3

hTERT protein expressions levels in melanoma cell lines and transfection efficiency. Notes: (A) hTERT protein expressions in A375 cells and M14 cells were detected by Western blot. The expression levels of miR-497-5p (B), miR-195-5p (C), and miR-455-3p (D) after transfection were determined by q-PCR. All data are presented as mean±SD, and all of these experiments were performed in triplicate. ***p<0.001, compared to control groups. Abbreviations: hTERT, human telomerase reverse transcriptase; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; q-PCR, quantitative real-time polymerase chain reaction.

Figure 4

The effects of overexpression of miR-497-5p (A), miR-195-5p (B) and miR-455-3p (C) on cells proliferation. Notes: All data are presented as mean±SD, and all of these experiments were performed in triplicate. ***p<0.001, compared to control groups. Abbreviations: NC, negative control; OD, optical density.

Figure 5

Overexpression of miR-497-5p, miR-195-5p and miR-455-3p arrested cell cycle and promoted cell apoptosis. Notes: (A–C) Cell cycle analysis was performed after transfected with miR-497-5p/miR-195-5p/miR-455-3p mimics and inhibitors. (D–F) The percentage of early/late/total apoptosis cells of each group after transfection. All data were presented as mean±SD, and all of these experiments were performed in triplicate. ***p<0.001, **p<0.01, *p<0.05, compared to control groups. Abbreviation: NC, negative control.

Figure 6

Overexpression of miR-497-5p, miR-195-5p and miR-455-3p inhibited cell migration and invasion abilities. Notes: (A–C) Micrographs of 0 and 24 h (left, 100×) and quantification data of relative wound area (right, 24 h) were analyzed by Image J software. (D–F) Quantification data of invasion cells (left) and micrographs of crystal violet-stained cells (right, 200×) are shown. All data are presented as mean±SD, and all of these experiments were performed in triplicate. ***p<0.001; *p<0.05, compared to control groups. Abbreviation: NC, negative control.

Figure 6

Overexpression of miR-497-5p, miR-195-5p and miR-455-3p inhibited cell migration and invasion abilities. Notes: (A–C) Micrographs of 0 and 24 h (left, 100×) and quantification data of relative wound area (right, 24 h) were analyzed by Image J software. (D–F) Quantification data of invasion cells (left) and micrographs of crystal violet-stained cells (right, 200×) are shown. All data are presented as mean±SD, and all of these experiments were performed in triplicate. ***p<0.001; *p<0.05, compared to control groups. Abbreviation: NC, negative control.

Figure 7

Overexpression of miR-497-5p (A), miR-195-5p (B) and miR-455-3p (C) decreased hTERT expression at both mRNA (left) and protein (right) levels. (D) Sequences of miR-497-5p, miR-195-5p, miR-455-3p, 3′UTR of hTERT are shown. The HEK 293T cells were cotransfected with miR-497-5p (E), miR-195-5p (F), miR-455-3p (G) mimics and luciferase reporter vectors containing a fragment of hTERT 3′UTR harboring either the binding sites (hTERT-3′UTR-wt) or a mutant sites (hTERT-3′UTR-mut). Notes: All data are presented as mean±SD, and all of these experiments were performed in triplicate. **p<0.01; ***p<0.001, compared to control groups. Abbreviations: hTERT, human telomerase reverse transcriptase; NC, negative control; wt, wild type; mut, mutant type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 7

Overexpression of miR-497-5p (A), miR-195-5p (B) and miR-455-3p (C) decreased hTERT expression at both mRNA (left) and protein (right) levels. (D) Sequences of miR-497-5p, miR-195-5p, miR-455-3p, 3′UTR of hTERT are shown. The HEK 293T cells were cotransfected with miR-497-5p (E), miR-195-5p (F), miR-455-3p (G) mimics and luciferase reporter vectors containing a fragment of hTERT 3′UTR harboring either the binding sites (hTERT-3′UTR-wt) or a mutant sites (hTERT-3′UTR-mut). Notes: All data are presented as mean±SD, and all of these experiments were performed in triplicate. **p<0.01; ***p<0.001, compared to control groups. Abbreviations: hTERT, human telomerase reverse transcriptase; NC, negative control; wt, wild type; mut, mutant type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.