Unlocking melanoma Suppression: Insights from Plasma-Induced potent miRNAs through PI3K-AKT-ZEB1 axis

Abstract

Introduction: Melanoma is a rare but highly malignant form of skin cancer. Although recent targeted and immune-based therapies have improved survival rates by 10-15%, effective melanoma treatment remains challenging. Therefore, novel, combinatorial therapy options such as non-thermal atmospheric pressure plasma (NTP) are being investigated to inhibit and prevent chemoresistance. Although several studies have reported the apoptotic and inhibitory effects of reactive oxygen species produced by NTP in the context of melanoma, the intricate molecular network that determines the role of microRNAs (miRNAs) in regulating NTP-mediated cell death remains unexplored.

Objectives: This study aimed to explore the molecular mechanisms and miRNA networks regulated by NTP-induced oxidative stress in melanoma cells.

Methods: Melanoma cells were exposed to NTP and then subjected to high-throughput miRNA sequencing to identify NTP-regulated miRNAs. Various biological processes and underlying molecular mechanisms were assessed using Alamar Blue, propidium iodide (PI) uptake, cell migration, and clonogenic assays followed by qRT-PCR and flow cytometry.

Results: NTP exposure for 3 min was sufficient to modulate the expression of several miRNAs, inhibiting cell growth. Persistent NTP exposure for 5 min increased differential miRNA regulation, PI uptake, and the expression of genes involved in cell cycle arrest and death. qPCR confirmed that miR-200b-3p and miR-215-5p upregulation contributed to decreased cell viability and migration. Mechanistically, inhibiting miR-200b-3p and miR-215-5p in SK-2 cells enhancedZEB1, PI3K, and AKT expression, increasing cell proliferation and viability.

Conclusion: This study demonstrated that NTP exposure for 5 min results in the differential regulation of miRNAs related to the PI3K-AKT-ZEB1 axis and cell cycle dysregulation to facilitate melanoma suppression.

Keywords: Differential regulation; Melanoma inhibition; Non-thermal plasma; miRNA sequencing.

Graphical abstract

Fig. 1

Melanoma growth inhibition by NTP. (A) Graph depicting viability of SK-2 cells after increasing exposure (0.5 min, 1 min, 1.5 min, 2 min, and 4 min) to NTP, (B) Graph depicting viability of SK-2, SK-31 and G-361 cells after NTP exposure (0, 3 min, and 5 min), (C) Histograms and a graph showing results of cell death (PI uptake) analysis using flow cytometry in SK-2 cells following NTP exposure, (D) Graphs showing gene expression of markers involved in cellular proliferation (c-Myc, Ki-67), survival (AKT), cell cycle (CDKN2A and p53) and cell death (Casp9) –by qPCR analysis, (E) Representative image and corresponding graph indicating results of clonogenic assay for SK-2 cells post 12 days of NTP exposure. (F) Representative images and a graph indicating SK-2 cell migration analysis using Incucyte scratch wound assay. NTP, nonthermal atmospheric pressure plasma; PI, propidium iodide; CDKN2A, cyclin-dependent kinase inhibitor 2A; CASP9, Caspase-9; Statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001) was determined using unpaired t-tests or Dunnett’s/Tukey’s multiple comparison tests.

Fig. 2

Global transcriptome analysis. (A) Schematic presentation of High throughput RNA sequencing for miRNA (small RNA seq) (B) Plot showing principal component analysis (PCA) of the control (con) and NTP-treated samples (C) Heat map showing differentially expressed miRNAs after NTP exposure (D, E) Scatter plots showing the spread of upregulated and downregulated miRNAs in 3 min NTP and 5 min NTP exposed samples respectively, and (F) Venn diagram indicating numbers of upregulated and downregulated miRNAs differentially regulated and overlapping in both NTP groups (left panel). Similar data has been shown using a bar graph (right panel). NTP, non-thermal plasma.

Fig. 3

Cellular process-based categorization of differentially regulated miRNAs. Graph and representative scatter plots showing differentially regulated miRNAs involved in biological processes in (A) 3 min NTP and (B) 5 min NTP groups. NTP, non-thermal plasma.

Fig. 4

Pathway enrichment analysis using the Mierturnet tool. Functional enrichment of differentially regulated miRNAs in 3 min NTP exposed melanoma cells. (A) KEGG enrichment for upregulated miRNAs, and (B) KEGG enrichment for downregulated miRNAs. KEGG, Kyoto encyclopedia of genes and genomes; NTP, non-thermal plasma.

Fig. 5

GO analysis of NTP-exposed miRNAs. Top significantly enriched GO pathway categories under ontology of biological processes in NTP exposed groups compared with control groups (A) 3 min only, and (B) 5 min only. GO, gene ontology; NTP, non-thermal plasma.

Fig. 6

NTP-induced cellular growth suppression is mediated through miR-200b-3p and miR-215-5p. (A) Bar graph showing top upregulated (red) and downregulated (green) identified from RNAseq analysis in 5 min NTP-exposed groups, (B) qRT-PCR analysis of upregulated (red) and downregulated (green) miRNAs in control and 5 min NTP-exposed SK-2 cell line, (C) Network analysis of upregulated miRNAs using mienturnet. (D) qRT-PCR analysis of the expression of miR-200b-3p and miR-215-5p levels in SK-2 cells following NTP-exposure where these miRNAs were inhibited. (E) Cellular viability of SK-2 cells after 5 min NTP treatment in the presence or absence of miR-200b-3p or miR-215-5p inhibitors. (F) Representative images and survival fraction graph illustrating the clonogenic assay conducted on SK-2 cells after NTP exposure in the presence or absence of given miRNAs inhibitors. (G) Representative images and graphs indicating cell migration analysis under similar conditions as panel F. Statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001) was determined using Sidak's or Tukey’s multiple comparison tests with one or two-way ANOVA. NC, normal control; NTP, non-thermal plasma.

Fig. 7

PI3K-AKT-ZEB axis in melanoma promotes cellular progression. (A) Expression of ZEB2 in normal skin and melanoma tissues obtained using protein atlas database (B) Protein expression levels of ZEB2 in Skin Cutaneous Melanoma (SKCM) samples analyzed by GEPIA2. (C) Stage-wise expression of ZEB2 was retrieved in melanoma, and a representative plot is shown using GEPIA2. (D) Graph showing survival analysis of SKCM samples with high and low ZEB2 levels was retrieved using OncoLnc webtool. (E, F) Flow cytometry analysis of the protein expression of AKT, PI3K, and ZEB1 in SK-2 cells in the presence or absence of miRNAs inhibitors following NTP treatment. Quantification of data has been shown in the representative graph in panel F. P3 gating is used to compare miR inhibitor groups with NTP treatment. (G) qRT-PCR analysis of the expression of AKT, PI3K, ZEB1, and CDKN2A under similar conditions as panel E. PI3K, phosphoinositide 3-kinases; SKCM, skin cutaneous melanoma; GEPIA2, gene expression profiling interactive analysis; ZEB1, Zinc finger E-box-binding homeobox 1; CDKN2A, cyclin-dependent kinase inhibitor 2A; CASP9, Caspase-9; Statistical significance (*p < 0.05, **p < 0.01 and ***p < 0.001) was determined using Tukey’s multiple comparison tests with two-way ANOVA.

Fig. 8

Analysis of downregulated miRNAs and their clinical relevance in SKCM patient survival. (A) Network enrichment of downregulated miRNAs, (B-G) Survival analysis plots of SKCM samples with high and low levels of hsa-miR-19a-3p, hsa-miR-129-5p, hsa-miR-340-3p, hsa-miR-151-3p, hsa-miR-18a-3p, and hsa-miR-340a-5p, respectively. (H) An illustration depicting the plausible action of NTP for melanoma growth inhibition. SKCM, skin cutaneous melanoma.