MiR-5195-3p targets the PCBP2/PI3K/AKT pathway to inhibit melanoma cell proliferation and migration

Abstract

Although miR-5195-3p has been acknowledged for its tumor suppressor role in diverse cancer categories, its precise functions and mechanisms concerning melanoma have not been comprehensively elucidated. In this study, we employed quantitative reverse transcription PCR, Western blot analysis, and immunohistochemistry staining to investigate the expression patterns of miR-5195-3p and poly (rC) binding protein 2 (PCBP2) in melanoma tissues compared to adjacent tissues. Our findings revealed downregulation of miR-5195-3p and upregulation of PCBP2 in melanoma tissues. Through the implementation of a luciferase reporter assay, we successfully identified PCBP2 as a newly discovered target of miR-5195-3p in melanoma cells. Enforced expression of miR-5195-3p via mimics inhibited cell proliferation and migration in A375 and A2058 cells, as demonstrated by CCK-8 and transwell migration assays. In melanoma cells, reintroduction of PCBP2 partially reversed the inhibitory effects of miR-5195-3p overexpression. Treatment with LY294002, an inhibitor of the PI3K/AKT signaling pathway, also reversed the effects of PCBP2 in melanoma cells. Furthermore, our results suggest that miR-5195-3p inhibits the activation of the PI3K/AKT signaling pathway in melanoma by inhibiting PCBP2. In conclusion, our research has identified the miR-5195-3p targeting of the PCBP2-mediated PI3K/AKT signaling pathway as a potential therapeutic target for melanoma treatment.

Keywords: MIRN5195-3p; Melanoma; PI3K/AKT signaling; Poly(rC) binding protein 2.

Fig. 1

The expression levels of miR-5195-3p and PCBP2 in melanoma tissues. (A) The expression level of miR-5195-3p was determined in 45 paired melanoma tissues and adjacent tissues using quantitative reverse transcription PCR. ***p < 0.001, by Student's t-test; (B) Representative immunohistochemical staining of PCBP2 in melanoma and normal tissues (Scale bar: 100 μm; 250 × magnification). (C) Representative images of Western blot analysis on four paired tumor and normal tissues derived from melanoma patients were shown. **p < 0.01, by Student's t-test.

Fig. 2

Targeted binding of miR-5195-3p to PCBP2. (A) Sequence analysis of binding sites between miR-5195-3p and PCBP2. (B) Dual-luciferase reporter assay was performed in A375 and A2058 cells after co-transfection with miR-5195-3p mimics or miR-NC and PCBP2-WT or PCBP2-MUT plasmids. (C–D) The expression levels of PCBP2 mRNA and protein were determined in A375 and A2058 cells transfected with miR-5195-3p mimics or miR-NC. (E) The expression level of PCBP mRNA was determined in 45 paired melanoma tissues and adjacent tissues using quantitative reverse transcription PCR. (F) Spearman's correlation analysis was performed to assess the association between miR-5195-3p and PCBP2 mRNA expression in melanoma tissues. The experiments were replicated three times and presented as mean ± SD. **p < 0.01, ***p < 0.001, by Student's t-test.

Fig. 3

Overexpression of miR-5195-3p suppressed cell proliferation and migration in melanoma cells. A375 and A2058 cells were transfected with miR-5195-3p mimics or miR-NC for 48 h. (A–B) The expression level of miR-5195-3p was determined in transfected A375 and A2058 cells using quantitative reverse transcription PCR. (C–D) CCK-8 assay was performed to analyze the effects of miR-5195-3p overexpression on A375 and A2058 cell proliferation. (E) Cell migration was assessed in A375 and A2058 cells after miR-5195-3p overexpression by transwell assay. Scale bars = 50 μm. The experiments were replicated three times and presented as mean ± SD. **p < 0.01, ***p < 0.001, by Student's t-test.

Fig. 4

Overexpression of PCBP2 partially reversed the inhibitory effect of miR-5195-3p in melanoma cells. (A) Protein level of PCBP2 was measured in A375 and A2058 cells after transfection with pcDNA3.1-PCBP2 or pcDNA3.1. (B) Protein level of PCBP2 was detected in A375 and A2058 cells after co-transfected with pcDNA3.1-PCBP2 in combination with or without miR-5195-3p mimics. (C–D) CCK-8 assay was performed to analyze cell proliferation in A375 and A2058 cells after co-transfected with pcDNA3.1-PCBP2 in combination with or without miR-5195-3p mimics. (E) Cell migration was assessed in A375 and A2058 cells after co-transfected with pcDNA3.1-PCBP2 in combination with or without miR-5195-3p mimics. Scale bars = 50 μm. The experiments were replicated three times and presented as mean ± SD. **p < 0.01, ***p < 0.001, compared with miR-NC + pcDNA3.1; ##p < 0.01, ###p < 0.001, compared with miR-5195-3p mimics + pcDNA3.1, by ANOVA with Tukey's post hoc test.

Fig. 5

The effects of inhibited PI3K/AKT pathway on PCBP2-mediated cell proliferation and migration in melanoma cells. A375 and A2058 cells were transfected with 2.5 μg pcDNA3.1-PCBP2, followed by treatment with LY294002 (50 μmoL/l) for 48 h at 37 °C. (A–B) Cell proliferation rate was determined using CCK-8 assay in A375 and A2058 cells. (C) Transwell assay was applied to analyze cell migration ability in A375 and A2058 cells. Scale bars = 50 μm. The experiments were replicated three times and presented as mean ± SD. **p < 0.01, ***p < 0.001, compared with pcDNA3.1; #p < 0.05, ##p < 0.01, compared with pcDNA3.1-PCBP2, by ANOVA with Tukey's post hoc test.

Fig. 6

miR-5195-3p suppressed the PI3K/AKT signaling pathway in melanoma cells via PCBP2. (A) The protein expression levels of PCNA, E-cadherin, p-PI3K, PI3K, p-AKT and AKT were measured in A375 and A2058 cells after co-transfection with miR-5195-3p mimics and pcDNA3.1-PCBP2 or pcDNA3.1. **p < 0.01, ***p < 0.001, compared with miR-NC + pcDNA3.1; #p < 0.05, ##p < 0.01, compared with miR-5195-3p mimics + pcDNA3.1, by ANOVA with Tukey's post hoc test. (B) The protein expression levels of PCNA, E-cadherin, p-PI3K, PI3K, p-AKT and AKT were measured in A375 and A2058 cells transfected with pcDNA3.1-PCBP2, followed by treatment with LY294002. *p < 0.05, ***p < 0.001, compared with pcDNA3.1; #p < 0.05, ##p < 0.01, ###p < 0.001, compared with pcDNA3.1-PCBP2, by ANOVA with Tukey's post hoc test. The experiments were replicated three times and presented as mean ± SD.

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