Effects of miR-145-5p through NRAS on the cell proliferation, apoptosis, migration, and invasion in melanoma by inhibiting MAPK and PI3K/AKT pathways

Abstract

We aimed to detect the effects of miR-145-5p on the cell proliferation, apoptosis, migration, and invasion in NRAS-mutant, BRAF-mutant, and wild-type melanoma cells, in order to figure out the potential mechanisms and provide a novel therapeutic target of melanoma. RT-qPCR and western blot were used to detect the expression of miR-145-5p and NRAS in melanoma tumor tissues and cells, respectively. Luciferase assay was performed to determine whether miR-145-5p directly targeted NRAS. After transfecting miR-145-5p mimics, miR-145-5p inhibitors, NRAS cDNA and NRAS siRNA into CHL-1, VMM917 and SK-mel-28 cells, functional assays were used to detect the proliferation, apoptosis, invasion and migration, including MTT, flow cytometry, Transwell and wound healing assays. In addition, xenograft models in nude mice were also conducted to verify the role of miR-145-5p in vivo. MiR-145-5p was able to suppress proliferation, invasion, and migration of VMM917 and CHL-1 cells and induce apoptosis by inhibiting MAPK and PI3K/AKT pathways. However, aberrant expression of miR-145-5p and NRAS has little impact on the viability and metastasis of BRAF-mutant melanoma. The higher expression of miR-145-5p in xenograft models repressed the VMM917-induced and CHL-1-induced tumor growth observably and has little effect on SK-mel-28-induced tumor growth which was consistent with the results in vitro. Through targeting NRAS, miR-145-5p could suppress cell proliferation, invasion, and migration and induce apoptosis of CHL-1 and VMM917 melanoma cells by inhibiting MAPK and PI3K/AKT pathways.

Keywords: MAPK; NRAS; melanoma; miR-145-5p.

Figure 1

The expressions of miR‐145‐5p and NRAS mRNA in melanoma tumor tissues and cells. (A–B) Box‐whisker plots of relative miR‐145‐5p (A) and NRAS mRNA (B) expression in melanoma clinical specimens were determined by RT‐qPCR, respectively (bold lines represent median value). **P < 0.001 compared with expression in adjacent tissues. (C) Correlation of the expression levels of miR‐145‐5p and NRAS mRNA in melanoma tumor tissues. (D) Relative expression levels of miR‐145‐5p and NRAS mRNA in normal human epidermal melanocytes (NHEMs), and three melanoma cell lines (CHL‐1, VMM917, and SK‐mel‐28) were determined by RT‐qPCR. All data were presented as mean ± SD from three independent experiments. **P < 0.001 compared with miR‐145‐5p expression in NHEMs, ^## P < 0.001 compared with NRAS expression in NHEMs. NHEMs, normal human epidermal melanocytes.

Figure 2

MiR‐145‐5p directly targeted the 3'‐UTR of NRAS. (A) Human melanoma CHL‐1, VMM917, and SK‐mel‐28 cells were transfected with negative control, miR‐145‐5p mimics, and miR‐145‐5p inhibitor to manipulate the level of miR‐145‐5p. 48 h after infection and sorting, the transfection efficiency was examined by RT‐qPCR. (B) After transfection, the effect of miR‐145‐5p on NRAS expression was measured by RT‐qPCR. (C) Putative miR‐145‐5p complementary site in the 3'‐UTR of NRAS mRNA was shown. A mutated 3'‐UTR of NRAS mRNA for the miR‐145‐5p complementary site was generated. (D) HEK293T cells overexpressing miR‐145‐5p or negative control were cotransfected with luciferase reporter plasmid with either WT or MUT and luciferase reporter. 48 h later, luciferase activity was measured and luciferase activity was used as an internal reference. All data were presented as mean ± SD from three independent experiments. ^a,b,c P < 0.05 compared with empty group in CHL‐1, VMM917 and SK‐mel‐28 cells, respectively. ^d,e,f P < 0.05 compared with NC group in CHL‐1, VMM917, and SK‐mel‐28 cells, respectively. **P < 0.001 compared with NC group.

Figure 3

MiR‐145‐5p functions as a tumor suppressor in CHL‐1 and VMM917 melanoma cells. (A) After incubation with MTT, OD values of CHL‐1, VMM917, and SK‐mel‐28 cells were plotted against time series to determine the amount of MTT formazan. MiR‐145‐5p mimics repressed the cell proliferation in CHL‐1 and VMM917 cells, whereas the miR‐145‐5p inhibitor promoted. (B) Annexin V staining and Flow cytometry sorting was performed to detect the cell apoptosis rate of indicated cell lines. (C) Wound healing assay was used to detect migration of indicated cell lines. D Transwell assay was used to detect invasion of indicated cell lines. All data were presented as mean ± SD from three independent experiments. ^a P < 0.05 compared with empty group in CHL‐1 cells, ^b P < 0.05 compared with NC group in CHL‐1 cells, ^c P < 0.05 compared with empty group in VMM917 cells, ^d P < 0.05 compared with NC groups in VMM917 cells.

Figure 4

The antitumor effect of miR‐145‐5p in CHL‐1and VMM917 melanoma cells is mediated by NRAS. By cotransfecting NRAS cRNA with miR‐145‐5p mimics or NRAS siRNA with miR‐145‐5p inhibitor into CHL‐1 and VMM917 cells, the expression of NRAS was manipulated and the effects of NRAS expression on CHL‐1 and VMM917 cells were detected. (A) 48 hours after infection and sorting, the expression level of NRAS was measured using RT‐qPCR. (B) MTT assay was performed to detect the proliferation of indicated cell lines. (C) Annexin V staining and Flow cytometry sorting was performed to detect the cell apoptosis rate of indicated cell lines. (D) Wound healing assay was used to detect migration of indicated cell lines. (E) Transwell assay was used to detect invasion of indicated cell lines. All data were presented as mean ± SD from three independent experiments. ^a,b,c P < 0.05 compared with NC group, mimics+NRAS group, and inhibitor+siNRAS group in CHL‐1 cells, respectively. ^d,e,f P < 0.05 compared with NC group, mimics+NRAS group, and inhibitor+siNRAS group in VMM917 cells, respectively.

Figure 5

MiR‐145‐5p suppressed NRAS/BRAF/MEK/ERK pathway by targeting NRAS in CHL‐1 and VMM917 melanoma cells. (A) Western Blot analysis demonstrated that the levels of proteins in NRAS/BRAF/MEK/ERK pathway including NRAS, BRAF, MEK1/2, phosphorylation of MEK1/2, ERK1/2 and phosphorylation of ERK1/2 in indicated groups of CHL‐1 cells was regulated by miR‐145‐5p and NRAS. (B) Western Blot demonstrated that the levels of proteins in NRAS/BRAF/MEK/ERK pathway in indicated groups of VMM 917 cells was regulated by miR‐145‐5p and NRAS. (C–D) Semiquantitative analysis of the expression of proteins using ImageJ. All data were presented as mean ± SD from three independent experiments. ^a,b,c P < 0.05 compared with NC group, mimics+NRAS group, and inhibitor+siNRAS group in CHL‐1 cells, respectively. ^d,e,f P < 0.05 compared with NC group, mimics+NRAS group, and inhibitor+siNRAS group in VMM917 cells, respectively.

Figure 6

MiR‐145‐5p suppressed PI3K/AKT pathway by targeting NRAS in CHL‐1 and VMM917 melanoma cells. (A) Western Blot analysis demonstrated that the levels of proteins in PI3K/AKT pathway including PI3K(p110α), PI3K(p110β), AKT, phosphorylation of AKT(ser473 and thr308) and PTEN in indicated groups of CHL‐1 cells was regulated by miR‐145‐5p and NRAS. (B) Western Blot demonstrated that the levels of proteins in PI3K/AKT pathway in indicated groups of VMM 917 cells was regulated by miR‐145‐5p and NRAS. (C–D) Semiquantitative analysis of the expression of proteins using ImageJ. All data were presented as mean ± SD from three independent experiments. ^a,b,c P < 0.05 compared with NC group, mimics+NRAS group, and inhibitor+siNRAS group in CHL‐1 cells, respectively. ^d,e,f P < 0.05 compared with NC group, mimics+NRAS group, and inhibitor+siNRAS group in VMM917 cells, respectively.

Figure 7

The role of miR‐145‐5p on tumor growth in vivo. After CHL‐1‐induced, VMM917‐induced and SK‐mel‐28‐induced tumor formed in nude mices, miR‐145‐5p mimics, and negative control was injected intratumorally. (A) Tumor volume was measured every 2 days and tumors excised at day 21. Compared with NC groups, miR‐145‐5p efficiently decreased the tumor growth which was induced by CHL‐1 and VMM917 but could not affect SK‐mel‐28‐induced tumor growth. Scale bar = 1 cm. *P < 0.05 compared with CHL‐1‐induced NC group, whereas ^# P < 0.05 compared with VMM917‐induced NC group. All data were presented as mean ± SD from three independent experiments. (B) Mices were sacrificed at 21 days. Tumor was isolated and total protein was extracted from tumor issues. Western blot analysis was performed to measure the protein levels of NRAS, AKT, phosphorylation of AKT (ser473 and thr308), ERK1/2 and phosphorylation of ERK1/2. MiR‐145‐5p downregulated the expression of NRAS and attenuated phosphorylation of AKT and ERK1/2 in CHL‐1 and VMM917‐induced tumors.

Figure 8

Proposed mechanism on how miR‐145‐5p impacts on MAPK and PI3K/AKT pathways. Red lines represent the activating functions, whereas green lines show the inhibition. Straight lines represent direct effect, whereas curves act on phosphorylation.