Upregulation of miR-34a-5p, miR-20a-3p and miR-29a-3p by Onconase in A375 Melanoma Cells Correlates with the Downregulation of Specific Onco-Proteins

Abstract

Onconase (ONC) is an amphibian secretory ribonuclease displaying cytostatic and cytotoxic activities against many mammalian tumors, including melanoma. ONC principally damages tRNA species, but also other non-coding RNAs, although its precise targets are not known. We investigated the ONC ability to modulate the expression of 16 onco-suppressor microRNAs (miRNAs) in the A375 BRAF-mutated melanoma cell line. RT-PCR and immunoblots were used to measure the expression levels of miRNAs and their regulated proteins, respectively. In silico study was carried out to verify the relations between miRNAs and their mRNA targets. A375 cell transfection with miR-20a-3p and miR-34a-5p mimics or inhibitors was performed. The onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. The cytostatic effect of ONC in A375 cells was mechanistically explained by the sharp inhibition of cyclins D1 and A2 expression level, as well as by downregulation of retinoblastoma protein and cyclin-dependent-kinase-2 activities. Remarkably, the expression of kinases ERK1/2 and Akt, as well as of the hypoxia inducible factor-1α, was inhibited by ONC. All these proteins control pro-survival pathways. Finally, many crucial proteins involved in migration, invasion and metastatic potential were downregulated by ONC. Results obtained from transfection of miR-20a-3p and miR-34a-5p inhibitors in the presence of ONC show that these miRNAs may participate in the antitumor effects of ONC in the A375 cell line. In conclusion, we identified many intracellular downregulated proteins involved in melanoma cell proliferation, metabolism and progression. All mRNAs coding these proteins may be targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p, which are in turn upregulated by ONC. Data suggest that several known ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of onco-suppressor miRNAs. Notably, miRNAs stability depends on the upstream regulation by long-non-coding-RNAs or circular-RNAs that can, in turn, be damaged by ONC ribonucleolytic activity.

Keywords: AXL; CREB; Fra1; HIF1α; PDK1; SIRT1; SOX2; cMet; cyclin A2; cyclin D1; ribonuclease.

Figure 1

Immunoblot data attesting the onconase (ONC) inhibitory effects on cell cycle-related protein expression. A375 melanoma cells were cultured in the presence or absence of 1 µM ONC for 48 or 72 h. Left: immunoblots showing the expression levels of cell cycle-related proteins; right: histograms reporting the mean values ± S.D. of protein expression level measured by densitometry and deriving from three to four independent experiments. All comparisons were performed vs. each control sample after normalization with β-actin expression; * p < 0.05, ** p < 0.01. pRB, phosphorylated form of retinoblastoma protein; pCDK2, phosphorylated form of cyclin-dependent kinase-2; P21/Cip1; p27/Kip1; p16/Ink4A.

Figure 2

ONC effects on the expression of proteins involved in cell proliferation signaling and metabolism. A375 melanoma cells were cultured for 48 h in the presence or absence of 1 µM ONC. Left: immunoblots showing the expression levels of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) and their active forms, and of many enzymes involved in metabolism; right: histograms reporting the mean values ± S.D. of the protein expression level measured by densitometry and deriving from three to four independent experiments. All comparisons were performed vs. each control sample after normalization with enolase-1 (ENO1) and phospho-glucomutase-2 (PGM2) expression; * p < 0.05, ** p < 0.01. HIF1α, hypoxia inducible factor 1 alpha; PDK1, pyruvate dehydrogenase kinase 1; ALDO A, aldolase A; G6PD, glucose-6-phosphate dehydrogenase; LDHA, lactate dehydrogenase A; pPLM2, phosphorylated form of pyruvate kinase M-2.

Figure 3

Immunoblot data attesting the ONC inhibitory effects on the expression of proteins involved in cell migration, invasion and tumor progression. A375 melanoma cells were cultured for 48 h in the presence or absence of 1 µM ONC. Left: immunoblots showing the expression levels proteins involved in cell invasive potential; right: histograms reporting the mean values ± S.D. of the protein expression level measured by densitometry and deriving from three to four independent experiments. All comparisons were performed vs. each control sample after normalization with β-actin expression; * p < 0.05, ** p < 0.01. ZO1, zonula occludens protein-1; SIRT1, sirtuin 1; SOX2, SRY (sex determining region Y)-box 2; uPAR, urokinase plasminogen activator receptor; CREB, cAMP response element-binding protein.

Figure 4

Relative expression of miRNAs after 48 h incubation of A375 cells with ONC. Cells were cultured for 48 h after 1 µM ONC administration in the culture medium. Red color bars refer to the onco-suppressor miRNAs that were upregulated by ONC at a statistically significant level; the blue ones refer instead to miRNAs whose expression level was not significantly different from the one relative to the untreated control. The mean values ± S.D. of miRNAs expression level measured by RT-PCR and deriving from three independent experiments are shown. All comparisons were performed vs. each control sample after normalization to miR-191 expression; * p < 0.05, ** p < 0.01.

Figure 5

miRNA-target interaction. Top: table shows predicted and validated miRNA-target interactions. Bottom: Venn diagram for miR-20a-3p, miR-29a-3p, and miR-34a-5p. Genes targeted by all three miRs: CDK2, CDKN1A, MAPK1, SIRT1, STAT3.

Figure 6

Immunoblots and densitometric data representing the correlation between mesenchymal–epithelial transition factor (cMet), tyrosine-protein kinase receptor UFO (AXL), Fos-related antigen 1 (Fra1) and cyclin A2 protein level and miR-34a-5p and miR-20a-3p. Left panel: A375 melanoma cells were treated for 48 h with or without ONC; expression of cMet, AXL, and Fra1 proteins. Central panel: effects of the overexpression (72 h) of miR-34a-5p and miR-20a-3p on cMet, AXL, Fra1 and cyclin A2 protein level in untreated cells. Right panel: effects of miR-34a-5p and miR-20a-3p inhibitors on cMet, AXL, Fra1 and cyclin A2 protein levels on ONC-treated A375 cells transfected with 50 nM miRNAs inhibitors for 72 h. In the upper part of each panel, immunoblots show the expression levels of the proteins. In the lower part, histograms report the relative mean values ± S.D. of protein expression level measured and deriving from three independent experiments. All comparisons were performed vs. each control sample after normalization with LDHA expression; * p < 0.05, ** p < 0.01.