microRNA editing in seed region aligns with cellular changes in hypoxic conditions

Abstract

RNA editing is a finely tuned, dynamic mechanism for post-transcriptional gene regulation that has been thoroughly investigated in the last decade. Nevertheless, RNA editing in non-coding RNA, such as microRNA (miRNA), have caused great debate and have called for deeper investigation. Until recently, in fact, inadequate methodologies and experimental contexts have been unable to provide detailed insights for further elucidation of RNA editing affecting miRNAs, especially in cancer.In this work, we leverage on recent innovative bioinformatics approaches applied to a more informative experimental context in order to analyze the variations in miRNA seed region editing activity during a time course of a hypoxia-exposed breast cancer cell line. By investigating its behavior in a dynamic context, we found that miRNA editing events in the seed region are not depended on miRNA expression, unprecedentedly providing insights on the targetome shifts derived from these modifications. This reveals that miRNA editing acts under the influence of environmentally induced stimuli.Our results show a miRNA editing activity trend aligning with cellular pathways closely associated to hypoxia, such as the VEGF and PI3K/Akt pathways, providing important novel insights on this poorly elucidated phenomenon.

Figure 1.

Average modification levels have been calculated for each replicate in normoxia and in each hypoxia time point. (A) The colors used for the rows are summarized in the ‘volume’ icon on the left of the table in the figure, with green representing low levels and red representing high levels. Fold-changes (in linear scale) for each microRNA at each time point relative to normoxia control have been reported as obtained by Camps et al. (45) (clear rows). Pearson's correlation (r), with relative P-value, was calculated between the average modification level and the linear fold-change for each miRNA. (B) Plotting of observed average modification levels over time-course for each selected A-to-I edited miRNA.

Figure 2.

Venn diagrams of predicted mRNA target sets for the WT and A-to-I ED miRNAs.

Figure 3.

Effects of A-to-I miRNA seed editing on targeting. (A) c-MET 3′ UTR binding sites for miR-27a-3p, ED (yellow spot) and WT (blue spot), along with corresponding deletions (lightening bolt). (B) Luciferase assay for pGL3-MET 3′ UTR WT construct co-transfected with miR-27a-3p WT, miR-27a-3p ED or negative scramble miRNA control (Scr) in HeLa cells (error bars: ±). (C) Luciferase assay for pGL3-MET 3′ UTR WT/mut and pGL3-MET 3′ UTR ED/mut constructs co-transfected with miR-27a3p ED or negative control (Scr) in HeLa cells. (D) Luciferase assay for pGL3-EGFR 3′ UTR WT construct co-transfected with miR-27a-3p WT, miR-27a-3p ED or negative controls (Scr) in HeLa cells. (E and F) c-MET and EGFR expression by western blot in A549 cells transfected with miR-27a-3p ED, miR-27a-3p WT or negative control (Scr) and harvested after 48 h, with graphs for c-Met/Tubulin or EGFR/Tubulin ratio quantification. (G) qRT-PCR of WT and ED miR-27a-3p respectively after miR transfection in A549 cells as control for Figure 3E and F. (H) EGFR expression as in (45).

Figure 4.

Functional enrichment of differentially expressed targets. (A) General scheme representing the functional enrichment workflow: differentially expressed genes with a significant adjusted P-value (BH < 0.01) in each hypoxia time point relative to normoxia were separated according to whether or not belonging to predicted miRNA targetome (WT or ED) for each miRNA, followed by MCF7-specific functional enrichment by Ingenuity Pathway Analyzer (IPA) software. (B) IPA analysis on VEGF and PI3K/Akt pathways in MCF7 cell line. Graphs show -log (P-value) over time course for both WT (purple) and A-to-I ED (green) predicted miRNA targets. Bars represent the level of significance for the indirect involvement in the considered pathways for WT and A-to-I ED miRNAs, respectively. P-values represent the significance of the set of target genes (which and how many) involved in a given pathway for each miRNA (WT and ED, respectively). The red dotted line represents the significance threshold level (-log(p), where P = 0.05).