Multitargeting activity of miR-24 inhibits long-term melatonin anticancer effects

Abstract

We have previously shown that melatonin exerts tumor suppressor activities by inducing the p38-p53 axis. This occurred within a few hours while no data are available on how melatonin pathway can be sustained on the long term. Here we show that miR-24, which has been demonstrated to target genes involved in the DNA repair process, targets p38, p53, PML and H2AX simultaneously. We show that long-term treatment with melatonin can decrease miR-24 levels post-transcriptionally, which pairs with a long-wave regulation of genes involved in cell proliferation, DNA damage, RNA metabolism and cell shape and transformation. Moreover, we show that melatonin can inhibit cell proliferation and migration, at least in part, by downregulating miR-24. Furthermore, we propose the involvement of hnRNP A1, which is downregulated by melatonin and involved in miRNA processing, in the regulation of miR-24 levels by melatonin. We conclude showing that miR-24 is upregulated in colon, breast and head and neck datasets and its levels negatively correlate with overall survival.

Keywords: PML; RNA-Seq; melatonin; miR-24; p53.

Figure 1. miR-24 targets the melatonin-p53 pathway

(A) Graphs show the sequence coverage, as well as the signal intensity, of H2AFX (H2AX) and MAPK12 (p38-γ) in both vehicle and melatonin treated HCT 116 cell line. (B) HCT 116 and MCF-7 cells have been treated with melatonin for 24, 48 and 72 hours. The ratio between miR-24 and RNU49 levels, normalized to their respective untreated controls, are indicated in the graphs. (C) HCT 116 cells have been transfected with either negative control (NC) or miR-24 mimic and the indicated Dual Luciferase reporters. Values for NC are set to 1. *p < 0,01. (D, E) HCT 116 and MCF7 cells have been transfected with the indicated mimic (D) or LNA (E) and subjected to quantitative Real-Time PCR. Values have been normalized to NC or LNA. (F, G) Cells have been transfected with the indicated mimic (F) or LNA (G) and cell extracts have been subjected to immunoblot with the indicated antibodies. Numbers indicate signal quantification normalized to NC or LNA-NC.

Figure 2. Melatonin induces the expression of the p53-pathway and alleviates genomic instability caused by miR-24

(A) HCT 116 and MCF-7 cell lines have been treated or not with melatonin for 72 hours and the expression levels of the indicated mRNAs were assessed by quantitative Real-Time PCR. Values have been normalized to untreated control. *p < 0,01. (B) HCT 116 cell lines stably expressing miR-Vec or (C) miR-Vec-24 have been pre-treated with either vehicle or melatonin, then DNA damage has been induced as indicated (CDDP: Cisplatin, Doxo: Doxorubicin, UVB: Ultra Violet B rays). Cells have been subjected to comet assay. Histograms show the percentage of comets.

Figure 3. Melatonin impairs the capability of miR-24 to induce cell proliferation and migration

(A) Cells were transfected with the indicated LNA and allowed to proliferate. Percentage of cells relative to control is indicated in the graph. *p < 0,05. (B) HCT 116 and (C) HCC1143 cells were transiently transfected with either NC or mimic-24 and subjected to transwell migration assays in the absence and in the presence of melatonin. Histograms show the percentage of invading cells. *p < 0,001. (D) HCT 116 cells were transfected with the indicated LNA and subjected to wound healing assay. Histograms show the percentage of wound opening. *p < 0,05. (E) HCT 116 were transfected with the indicated LNA and subjected to transwell migration assay. Histograms show the percentage of invading cells. *p < 0,01. (F) HCT 116 cells have been treated with 5 μM SB202190 for 2 hours and then subjected to transwell migration assay. *p < 0,05. (G) HCC1143 cells have been subjected to wound healing assay in the absence and in the presence of SB202190 and melatonin. Histograms show the percentage of wound opening. *p < 0,05.

Figure 4. Regulation of miR-24 levels by melatonin

(A) HCT 116 and MCF-7 cells have been either pretreated or not with Luzindole 15 minutes before melatonin treatment and miR-24 levels have been quantified 72 h later by quantitative Real-Time PCR. Histograms show miR-24/RNU49 levels relative to untreated control. *p < 0,01. (B) HCT 116 cells have been treated with melatonin for 72 hours in the absence and in the presence of 5 μM SB202190. Histograms show miR-24/RNU49 levels relative to untreated control. *p < 0,01. (C) HCT 116 and MCF-7 cells were treated with melatonin for 72 h. Levels of expression of the indicated miRNAs were assessed by quantitative Real-Time PCR. (D) HCT 116 and MCF-7 cells were treated with melatonin for 72 h. Histograms show the ratio between pre-miR-24–1 and pri-miR-24–1 and the ratio between pre-miR-24–2 and pri-miR-24–2 levels. *p < 0,01.

Figure 5. hnRNP A1 is involved in the regulation of miR-24 levels

(A) HCT 116 and MCF-7 cells were treated with melatonin for 72 h and protein extracts were subjected to immunoblot with the indicated antibodies. Numbers indicate the densitometry ratio between hnRNP A1 and 20Sα5 signals. (B) HCT 116 cells were transfected with the indicated plasmids and treated with melatonin for 72 h, miR-24 levels were assessed by quantitative Real-Time PCR. *p < 0,01. (C) HCT 116 and MCF-7 were transfected with the indicated siRNAs. 72 h post transfection, cell lysates were subjected to immunoblot with the indicated antibodies. Numbers indicate the densitometry ratio between hnRNP A1 and tubulin or nucleolin signals, normalized to siGFP.

Figure 6. Clinical association of miR-24 with survival and recurrence in cancer patients

(A) Box plot showing miR-24 median value in 314 cancer cases and 11 controls belonging to the Pan-Cancer Colon and rectal adenocarcinoma datasets. (B-C) Box plot showing miR-24 median value in 208 basal-like cancers and 116 controls (METABRIC) and 90 basal-like cancers and 83 controls (TCGA) (B) and association between expression levels of miR-24 and disease-free survival evaluated by Kaplan-Meier analysis in the METABRIC dataset, comprising 1284 cases and 116 controls (C). (D–E) Box plot showing miR-24 median value in 121 cancer cases and 66 controls (D) and association between expression levels of miR-24 and recurrence-free survival evaluated by Kaplan-Meier analysis (E) in our HNSCC casuistry [61].

Figure 7. Model of melatonin activities

By binding to its receptors (MT1/MT2), Melatonin induces p38 expression. With a fast kinetics (green arrow), Melatonin induces phosphorylation of p53 and H2AX thereby promoting DNA repair and inhibiting cell proliferation. Long-term activation of Melatonin receptors sustains p38 activation and leads to inhibition of hnRNP A1 (red arrow) thereby causing a decrease in miR-24, which results in p53, PML, H2AX and p38 activity reduction. Through downregulation of hnRNP A1 and miR-24, melatonin impairs the migrating capability of cancer cells.