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. 2018 Jan 1;8(4):1106-1120.
doi: 10.7150/thno.19904. eCollection 2018.

MiR-205-5p and miR-342-3p cooperate in the repression of the E2F1 transcription factor in the context of anticancer chemotherapy resistance

Xin Lai  1 , Shailendra K Gupta  2   3 , Ulf Schmitz  4   5 , Stephan Marquardt  6 , Susanne Knoll  6 , Alf Spitschak  6 , Olaf Wolkenhauer  2   7 , Brigitte M Pützer  6 , Julio Vera  1
Affiliations

MiR-205-5p and miR-342-3p cooperate in the repression of the E2F1 transcription factor in the context of anticancer chemotherapy resistance

Xin Lai et al. Theranostics. .

Abstract

High rates of lethal outcome in tumour metastasis are associated with the acquisition of invasiveness and chemoresistance. Several clinical studies indicate that E2F1 overexpression across high-grade tumours culminates in unfavourable prognosis and chemoresistance in patients. Thus, fine-tuning the expression of E2F1 could be a promising approach for treating patients showing chemoresistance. Methods: We integrated bioinformatics, structural and kinetic modelling, and experiments to study cooperative regulation of E2F1 by microRNA (miRNA) pairs in the context of anticancer chemotherapy resistance. Results: We showed that an enhanced E2F1 repression efficiency can be achieved in chemoresistant tumour cells through two cooperating miRNAs. Sequence and structural information were used to identify potential miRNA pairs that can form tertiary structures with E2F1 mRNA. We then employed molecular dynamics simulations to show that among the identified triplexes, miR-205-5p and miR-342-3p can form the most stable triplex with E2F1 mRNA. A mathematical model simulating the E2F1 regulation by the cooperative miRNAs predicted enhanced E2F1 repression, a feature that was verified by in vitro experiments. Finally, we integrated this cooperative miRNA regulation into a more comprehensive network to account for E2F1-related chemoresistance in tumour cells. The network model simulations and experimental data indicate the ability of enhanced expression of both miR-205-5p and miR-342-3p to decrease tumour chemoresistance by cooperatively repressing E2F1. Conclusions: Our results suggest that pairs of cooperating miRNAs could be used as potential RNA therapeutics to reduce E2F1-related chemoresistance.

Keywords: Chemotherapy resistance; E2F1; Kinetic modelling.; MicroRNA; Molecular dynamics simulation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
E2F1 _targeting miRNAs. (A) In total 26 miRNAs were predicted to be post-transcriptional regulators of E2F1 expression. The figure shows the location of the binding sites and highlights putatively cooperating miRNAs (the transparent boxes on top). The transparent box indicates the location of the miRNAs that could putatively cooperate with miR-205-5p in repressing E2F1. (B) Conservation of miRNA binding sites. The green track in this UCSC genome browser snapshot shows the PhastCons score (100 vertebrate conservation track) indicating the evolutionary conservation of miRNA binding sites in the 3' UTR of E2F1. The genomic region shown includes binding sites of miR-205-5p and putatively coopering miRNAs (black bars). A multiple sequence alignment of some related species is shown in the bottom track. The E2F1 gene is locate on the negative strand. While the 3′ UTR sequence in (A) is in 5′ to 3′ orientation, the reading direction in the UCSC genome browser snapshot (B) is opposite to that.
Figure 2
Figure 2
Intermolecular H-bonds between mRNA and miRNAs in RNA triplexes during 1,000 ps MDS run. (A) The solid lines indicate the number of H-bonds when both miRNAs are associated with E2F1 mRNA, while the extended dotted line shows the number of H-bonds between the remaining miRNAs (underlined) and the mRNA. For each triplex, the underlined miRNA first disassociates from the mRNA at the time point indicated by the correspondingly coloured arrow. (B) Interactions between the E2F1 3′ UTR (grey), miR-342-3p (green) and miR-205-5p-5p (red) in a 3D model. The intermolecular H-bonds between the three RNA species are shown as green dotted lines. The simulation also indicates that miR-205-5p and miR-342-3p interact with each other through H-bonds (red circle), which may contribute to the better stability of this triplex over others.
Figure 3
Figure 3
Simulations of E2F1 repression by cooperating miRNA pairs. The plots show the expression levels of E2F1 (0: silencing; 1: non-repression) for different combinations of the miRNA transcriptional activation (denoted by formula image and formula image in the model). The nominal value of the miRNA transcriptional activation is 1, and values smaller than 1 indicate down-regulation while values greater than 1 represent up-regulation of the miRNA. The four reference points represent normal expression of miRNAs (dot), strong up-regulation of one miRNA (triangles) and moderate up-regulation of both miRNAs (diamond). The numbers denote the gain of E2F1 repression efficiency while increasing miRNA expression levels.
Figure 4
Figure 4
miR-205-5p and miR-342-3p cooperate in the regulation of E2F1. (A) Quantification of miRNA levels after individually or co-transfecting miR-205-5p and miR-342-3p expression plasmids into H1299 and SK-Mel-147 cells. (B) Relative luciferase activity after co-transfection of E2F1-3' UTR (0.5 μg) and miR-205-5p/miR-342-3p plasmids into H1299 and SK-Mel-147 cells. (C) Western blot showing E2F1 protein expression after single or combined miR-205-5p/miR-342-3p overexpression in H1299 and SK-Mel-147 (top). The E2F1 protein levels (data) were quantified and compared with model simulations (model) showing the cooperative E2F1 repression by miR-205-5p and miR-342-3p (bottom). Actin was used as loading control and for normalising the protein expression data. Plasmid concentrations for miRNA expression are indicated in the corresponding figures. The details of the model simulation can be found in Supplementary Material. (D) Multidose combinations of miR-205 and miR-342 for repressing E2F1. Relative luciferase activity was measured after co-transfection of E2F1-3' UTR and miR-205-5p/miR-342-3p plasmids into H1299 and SK-Mel-147 cells. For the individual treatments, 0.25 µg, 0.5 µg, or 1 µg of miR-205 or miR-342 plasmid were transfected into cells, and for the combined treatment, 0.25 µg, 0.5 µg, or 1 µg of both miRNA plasmids were co-transfected. (E) The table shows the calculated fraction affected (fa) by the individual miRNA or combined miRNA treatments and the corresponding combination indexes (CI) of the combined treatments (synergism: CI < 1; antagonism: CI > 1). The estimated parameter values can be found in Table S5. Data shown in the bar plots are mean ± SD (n = 3).
Figure 5
Figure 5
Effects of cooperative E2F1 repression on chemoresistance. (A) Biochemical reaction network underlying the kinetic model of E2F1-mediated drug resistance. (B) Illustration of a typical model simulation, accounting for the temporal dynamics of E2F1 protein, miR-205-5p, Harakiri protein (Hrk), BCL2 protein and the population of tumour cells (TC) before (shaded area) and after drug administration (white area). (C) Model predicted values of E2F1, miR-205-5p and the ratio between p73 and DNp73 for different combinations of E2F1 and miR-342-3p expression levels, whose corresponding variables FSE2F1 and FSmiR342 were iteratively modified in the specified intervals. The highlighted and indexed areas correspond to the description in the main text. (D) Predicted tumour cell population size in non-genotoxic drug stimulation conditions (left) and predicted fraction of surviving tumour cells 48 h after genotoxic drug administration (right). The enclosed numbers indicate the transition of tumour growth rate under different biological conditions.
Figure 6
Figure 6
The effect of miR-205-5p and miR-342-3p on tumour cell chemosensitization. (A) Cisplatin (cDDP) treatment of chemoresistant H1299 with single miR-205-5p (0.5 µg or 1 µg plasmid), miR-342-3p (0.5 µg or 1 µg plasmid) or combined miRNA (0.25 µg or 0.5 µg each plasmid) overexpression. Twenty-four hours post transfection, cisplatin was added to the cell culture medium at a concentration of 20 µM. For apoptosis detection, live-cell assays were performed using Hoechst 33342 staining. Fluorescent cells were visualized by microscopy and apoptotic cells were counted from seven areas. Chemosensitization is indicated as a relative increase of apoptosis in cisplatin-treated cells compared to untreated cells (set as 1). Data shown in the bar plot are mean ± SD (n = 3). (B) The table shows the calculated fraction affected (fa) by the individual miRNA or combined miRNA treatments and the corresponding combination indexes (CI) of the combined treatments. fa is denoted by the relative increase of apoptotic tumour cells that is normalized to the treatment with cisplatin only (i.e., ctrl in +cDDP) and divided by the maximum increase of apoptosis in all listed treatments (synergism: CI < 1; antagonism: CI > 1). The estimated parameter values can be found in Table S6. (C) Western blot analyses show protein levels of E2F1 as well as BCL2 and Bax as survival and apoptosis markers. Actin was used as loading control and for normalisation to quantify protein bands (top of each immunoblot).
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