https://orcid.org/0000-0002-4176-6710
Figures
Abstract
Translation is a crucial regulatory mechanism involved in several diseases, including cancer, where pro-inflammatory conditions within the microenvironment have been shown to modulate the translation of specific mRNAs. In the present study, we focused on the regulation of insulin growth factor-like family member 1 (IGFL1) in MCF7 breast cancer cells in response to pro-inflammatory IL-1β and observed an induction of both transcription and translation. We characterized the 3’ untranslated region as regulatory hub for the post-transcriptional regulation and identified a distinct G-rich region to confer the IL-1β-dependent translational increase. Our study therefore provides new insights into the translation regulation of IGFL1 in the context of an inflammatory tumor microenvironment.
Citation: Cardamone G, Flohr M, Raue R, Bode I, Meyer SP, Hauns S, et al. (2026) Enhanced IGFL1 translation in response to IL-1β is controlled by distinct 3’UTR elements. PLoS One 21(6): e0342288. https://doi.org/10.1371/journal.pone.0342288
Editor: Georg Stoecklin, Medical Faculty Mannheim, University of Heidelberg, GERMANY
Received: January 20, 2026; Accepted: June 9, 2026; Published: June 25, 2026
Copyright: © 2026 Cardamone et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the Deutsche Forschungsgemeinschaft (https://www.dfg.de/) (SCHM 2663/7-1 [TS] and BA 2168/25-1 [RB]), as well as by FFF Nachwuchsforscher-Förderung (https://www.uni-frankfurt.de/60800758/Forschung) [GC]. The funders played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Post-transcriptional processes, including translation regulation, are increasingly recognized as key regulatory mechanisms in several disease contexts, including cancer [1,2] and inflammatory diseases [3,4]. Tumor progression is largely controlled by the tumor microenvironment (TME) [5], and the TME is characterized by infiltrating immune cells, including macrophages, that orchestrate the inflammatory, tumor-promoting properties of the TME [6]. The secretion of cytokines and other mediators within the TME has been demonstrated to affect translation, mainly by modulating pathways affecting mTOR kinase activity [7,8]. Pro-inflammatory cytokines, such as interleukin (IL)-6, IL-1α, and IL-1β, appeared to also affect the translation of specific mRNA [9,10]. Concerning IL-1β, it was demonstrated to directly regulate the translation of THBD and EGR2 mRNAs in A459 human lung adenocarcinoma and MCF7 breast tumor cells, respectively [11,12].
The focus of the present work was the characterization of IGFL1, which was previously shown to be involved in breast cancer and in inflammatory diseases [13,14]. The IGFL1 gene encodes the insulin growth factor (IGF)-like family member 1 (IGFL1) protein, belonging to the IGF-like family, consisting of 4 genes (IGFL1 to IGFL4) and two pseudogenes (IGFL1P1 and IGFL1P2) [15]. IGFL1 expression was first detected in ovary and spinal cords [15] and was shown to be upregulated in skin from psoriasis patients and in primary keratinocyte cultures treated with tumor necrosis factor (TNF)-α [14]. Furthermore, IGFL1 expression was detected in various breast cancer cells [13,16]. Concerning IGFL1 expression regulation, it was found to be modulated by the long non-coding RNA IGF-like family member 2 antisense RNA 1 (IGFL2-AS1) [16]. In detail, Wang and colleagues [13] showed that the expression of both IGFL2-AS1 and IGFL1 was increased through KLF transcription factor 5 (KLF5) upon TNF-α stimulation in basal-like breast cancer cells and that IGFL2-AS1 contributed to KLF5-dependent IGFL1 upregulation. With respect to IGFL1 post-transcriptional regulation, IGFL2-AS1 was proposed to act as a sponge for a microRNA (miRNA) binding to the IGFL1 3’ untranslated region (UTR) [13].
In this work, we identified IL-1β as a novel stimulus to induce IGFL1 transcription and translation in MCF7 breast cancer cells and characterized its post-transcriptional regulation to be mediated by a specific, G-rich region within its 3’UTR.
Materials and methods
Chemicals
All chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany), if not stated otherwise. Recombinant human IL-1β was purchased from PeproTech (Hamburg, Germany).
Cell culture
MCF7 cells were purchased from ATCC-LGC Standards GmbH (Wesel, Germany) and were cultured in RPMI 1640 GlutaMAX medium (Gibco, Dreiech, Germany) supplemented with 10% heat-inactivated fetal calf serum (Capricon Scientific, Ebsdorfergrund, Germany), 1% sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were grown at 37 °C in a humidified atmosphere with 5% CO2.
RNA isolation, reverse transcription, and quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated using TRIzol (Thermo Fisher Scientific, Dreieich, Germany) according to the manufacturer’s instructions. RNA was reverse transcribed with the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), and qPCR analyses were accomplished by using PowerUp SYBR Green Master Mix on QuantStudio 3 and 5 PCR Real-Time Systems (Thermo Fisher Scientific). Primers were obtained from Biomers (Ulm, Germany) and are listed in S1 Table in S1 File.
Transcription inhibition using actinomycin D
3 x 105 MCF7 cells were seeded in 6-well plates, and the following day were treated with IL-1β (50 ng/mL) 4 h before de novo transcription was blocked by the addition of actinomycin D (10 µg/mL). RNA was isolated using TRIzol according to the manufacturer’s instructions, either before (= 0 h) or 1, 2, and 4 h after the administration of actinomycin D and reverse transcription and qPCR analyses were performed as previously described.
Polysomal fractionation
MCF7 were subjected to polysomal fractionation as described previously [12]. Briefly, 7.5 x 106 MCF7 cells were seeded in a 15 cm dish, one day prior to polysome fractionation and treated with IL-1β (50 ng/mL) 4 h prior to harvest. Subsequently, cells were incubated with 100 µg/mL cycloheximide (CHX, Carl Roth, Karlsruhe, Germany) for 10 min, washed with PBS/CHX (100 µg/mL), and lysed in 750 µL polysome lysis buffer (140 mM KCl, 20 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.5% NP-40, 0.5 mg/mL heparin, 1 mM DTT, 100 U/mL RNasin (Promega, Walldorf, Germany), 100 µg/mL CHX). After pelleting the cell debris (16,000 x g, 5 min, 4 °C), 600 µL of the cell lysates were layered onto 11 mL of 10–50% continuous sucrose gradients. Gradients were centrifuged at 35,000 rpm for 2 h at 4 °C without brake using a SW40 rotor in an Optima L-90K Ultracentrifuge (Beckman Coulter, Brea, CA, USA). The gradient was collected into 1-mL fractions using a Gradient Station (BioComp Instruments, Fredericton, Canada), and UV absorbance was measured at 254 nm. RNA was precipitated by adding 1/10 volume of sodium acetate (3 M) and 1 volume of isopropyl alcohol. RNA was further purified using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. RNA obtained from polysomal fractions was reverse transcribed and quantified by qPCR as described above.
Plasmid constructs
The psiCHECK-2 vector (Promega) was digested with NheI or with AsiSI and NotI (New England Biolabs, Frankfurt am Main, Germany) to insert either the IGFL1 5’UTR and/or IGFL1 3’UTR, and IGFL1 3’UTR truncated versions, respectively. All the inserts were amplified from human cDNA using appropriate PCR primer couples and the PCR product was purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). The fragments were inserted into the linearized vector with the In-Fusion HD Cloning Kit (Takara Bio Europe, Saint-Germain-en-Laye, France) according to the manufacturer’s protocol. The construct 1–413 Δ207–247 was obtained by a cloning reaction of two fragments. All the other constructs containing either a deletion or a point mutation were obtained by site-directed mutagenesis, by means of the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Waldbronn, Germany) following the manufacturer’s instructions.
All plasmids were purified using the NucleoSpin plasmid kit (Macherey–Nagel) and were verified by Sanger sequencing (SeqLab-Microsynth, Göttingen, Germany).
All primers are listed in S1 Table in S1 File.
Transient transfection and luciferase reporter assay
5 x 104 MCF7 cells were seeded in 24-well plates 24 h prior to transfection and were transfected with 250 ng of plasmid using jetPRIME (Polyplus, Illkirch-Graffenstade, France), as described by the manufacturer.
Cells were harvested 48 h after transfection. IL-1β-treated cells were treated with 50 ng/mL IL-1β during the last 4 h. Cells were lysed in 100 µl Passive Lysis Buffer (Promega) and the activities of firefly/renilla luciferase were measured by using the Dual-Luciferase Reporter Assay System (Promega) on a Spark multimode microplate reader (Tecan, Männedorf, Switzerland). Renilla luciferase activity was normalized to the corresponding firefly luciferase activity, that served as internal transfection control.
RNA secondary structure predictions
RNA secondary structures were predicted using RNAfold [17] with default parameters and subsequently drawn with the RNA visualization tool VARNA [18].
Statistical analyses
Statistical analyses were performed with GraphPad Prism v10.6.0 (GraphPad Software, San Diego, CA, USA). Data are reported as means ± SEM of at least three independent experiments. Normal distribution was assessed using the D’Agostino & Pearson test, Anderson-Darling test, Shapiro-Wilk test, and Kolmogorov-Smirnov test. If residuals were assumed to be not normally distributed based on all four tests, data were log-transformed before statistical testing. Statistically significant differences were calculated using paired t-test or two-way ANOVA (either with Tukey’s, Šídák’s, or Dunnett’s multiple comparisons test).
Results
IL-1β induces IGFL1 expression transcriptionally and translationally
IGFL1 mRNA expression was shown earlier to be induced by TNF-α in basal-like breast cancer cells [13]. To assess, if other inflammatory mediators of the tumor microenvironment might affect IGFL1 expression in breast tumor cells as well, we stimulated MCF7 breast cancer cells with IL-1β, IL-6, IL-10, and TNF-α (50 ng/mL, 4 h). While TNF-α induced IGFL1 expression as expected, neither IL-6 nor IL-10 affected IGFL1 levels (S1A Fig in S1 File). Interestingly, IGFL1 mRNA expression increased even more pronouncedly upon IL-1β stimulation in comparison to TNF-α (Fig 1A). IGFL1 expression showed a time-depend, cumulative increase over the 4 h stimulation with IL-1β (S1B Fig in S1 File). To assess whether the elevated IGFL1 expression in response to IL-1β might be due to increased IGFL1 mRNA stability, we blocked de novo mRNA synthesis by adding the transcription inhibitor actinomycin D (10 µg/mL) at the end of IL-1β treatment and followed IGFL1 mRNA levels for up to 4 h. Yet, IGFL1 mRNA stability did not increase upon IL-1β treatment (Fig 1B). Since we previously observed that IL-1β commonly affects translational processes in a macrophage-generated tumor microenvironment [12], we next assessed the impact of IL-1β on IGFL1 translation by polysome profiling. While IL-1β treatment did not alter global translation (Fig 1C) or translation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as compared to untreated controls (Fig 1D), IGFL1 mRNA distribution in response to IL-1β treatment significantly increased in the late polysomal fractions (8−10), which contain the efficiently translated mRNAs (Fig 1E). Concomitantly, IGFL1 mRNA abundance decreased in the sub-polysomal (1−3) and early polysomal fractions (4−7). Of note, CXCL8, a transcript known to be upregulated by IL-1β stimulation [19], showed the expected increase in mRNA expression (S2A Fig in S1 File) with no increase in mRNA stability (S2B Fig in S1 File) and slight changes in translation efficiency (S2C Fig in S1 File).
MCF7 cells were treated with IL-1β (50 ng/mL) for 4 h. (A) IGFL1 mRNA expression was measured by RT-qPCR and normalized to GAPDH expression (n = 6). (B) Transcription was blocked by the addition of actinomycin D (10 µg/mL) at the end of the IL-1β treatment and mRNA expression was followed for up to 4 h by RT-qPCR analyses (n = 3). (C, D, E) Translational status of IGFL1 was assessed by polysomal fractionation analysis. (C) Left panel: UV profiles obtained for the sucrose gradients during fractionation for the untreated control and IL-1β-treated cells are shown (representative tracks of five independent experiments). Peaks corresponding to 40S, 60S, and 80S ribosomes and polysomes are depicted. Sub-polysomal (sub, fractions 1-3), early (fractions 4-7), and late (fractions 8-10) polysomal fractions are shown. Right panel: the area under the curve (AUC) was calculated for each fraction and depicted as % of the total for sub, early, and late polysomal fractions. GAPDH (D) and IGFL1 mRNA (E) distribution across the gradients was analyzed by RT-qPCR (n = 5). The distribution across all the fractions and for sub, early, and late polysomal fractions are shown in the left and right panel, respectively. All data were presented as means ± SEM and statistically analyzed using paired t-test (A) or two-way ANOVA with Šídák’s multiple comparisons test (B-E); ** p < 0.01, *** p < 0.001 compared to respective untreated controls.
Thus, IL-1β apparently not only increases IGFL1 mRNA expression without altering the mRNA stability, but also enhances IGFL1 translation efficiency.
IL-1β-dependent changes in IGFL1 translation are controlled by a distinct part of the 3’UTR
Since translation regulatory mechanisms commonly involve the 5’UTR of transcripts, but can also be affected by the 3’UTRs [20,21], we asked if the UTRs might contribute to the IL-1β-dependent translational regulation of IGFL1 as well. To this end, we cloned the IGFL1 5’ and 3’UTRs into the psiCHECK-2 reporter vector up- and downstream of the renilla luciferase coding region, respectively, and transfected the resulting vectors into MCF7 cells. Surprisingly, insertion of the 5’UTR neither altered the luciferase activity compared to the empty vector under control conditions nor in response to IL-1β (Fig 2). In contrast, insertion of the IGFL1 3’UTR significantly increased the luciferase activity compared to both the empty vector and the vector including the 5’UTR already under basal conditions. Moreover, the 3’UTR-containing vector was markedly more responsive to IL-1β stimulation. Noteworthy, inserting 5’ and 3’UTRs in combination resulted in an intermediate phenotype.
IGFL1 5’UTR and 3’UTR and a combination of both were inserted into the psiCHECK-2 vector upstream and downstream of the renilla luciferase coding region, respectively. Firefly luciferase served as internal transfection control. MCF7 cells were transfected with the psiCHECK-2 vector (empty vector) or with the psiCHECK-2 vector containing either IGFL1 5’UTR or 3’UTR or both. Renilla (RL) and firefly luciferase (FL) activities were determined 48 h after transfection with or without treatment with IL-1β (50 ng/mL) during the last 4 h. Data were normalized to the empty vector control, presented as means ± SEM (n = 5), and statistically analyzed using two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001 compared to the respective empty vectors, §§ p < 0.01, §§§ p < 0.001 compared to the same condition in the 5’UTR + 3’UTR vector.
To get further insights into the role of the 3’UTR in the translational regulation of IGFL1, we aimed to identify the exact region within the 3’UTR of IGFL1 responsible for the IL-1β-induced increased translation. Therefore, we systematically shortened the IGFL1 3’UTR within the luciferase vector system. Specifically, we generated constructs representing 75% (1−309), 50% (1−206), and 25% (1−103) of the full length (1−413) IGFL1 3’UTR, plus two constructs representing intermediate sizes (1−279 and 1−247) (Fig 3A). Under basal, i.e., unstimulated, conditions (Ctr), the increase in reporter activity appeared to correlate rather directly with the length of the 3’UTR, i.e., the shorter the 3’UTR fragment the lower the respective activity. Conversely, the IL-1β-elicited increase of the 3’UTR-dependent luciferase activity remained comparable to the vector containing the full length 3’UTR for all constructs incorporating at least the first 247 nucleotides of the 3’UTR. Further reduction of the IGFL1 3’UTR length to 206 nucleotides led to a substantial drop of the IL-1β response (Fig 3A). These findings suggested that the region between nucleotides 207 and 247 might be relevant for the IL-1β-dependent activation. Modelling of the 2D structure of the entire IGFL1 3’UTR reveals that this region encompasses a full, small hairpin structure (nucleotides 211−228) and one arm of a larger hairpin structure (nucleotides 231−284) (Fig 3B). To validate the role of this region and to further rule out a pure length effect, we deleted nucleotides 207−247 in the full length 3’UTR-containing vector (Fig 3B; green marks). Indeed, deletion of nucleotides 207−247 (Δ207−247) (S3 Fig in S1 File; upper left panel) significantly reduced the IL-1β-induced luciferase activity compared to the full length 3’UTR-bearing vector (Fig 3C). Next, we deleted only nucleotides 235−247, corresponding to a G-rich region embedded within the larger hairpin structure (Fig 3B; yellow marks). Strikingly, deletion of this restricted area led to a similar reduction in the IL-1β-induced luciferase activity compared to the deletion of nucleotides 207−247, indicating that this G-rich region might be involved in the IL-1β responsiveness of IGFL1 translation.
MCF7 cells were transfected with the psiCHECK-2 empty vector or with the psiCHECK-2 vector containing either the full length IGFL1 3’UTR or parts of it. Renilla (RL) and firefly luciferase (FL) activities were determined 48 h after the transfection with or without treatment with IL-1β (50 ng/mL) during the last 4 h. (A) Schematic representation of the progressive 3’UTR deletion constructs generated in the psiCHECK-2 vector downstream of the renilla luciferase coding region and their associated luciferase reporter activities. Data were normalized to the empty vector, presented as means ± SEM (n = 4), and statistically analyzed using two-way ANOVA with Tukey’s multiple comparisons test; *** p < 0.001; ### p < 0.001 compared to the IL-1β-treated full length 3’UTR vector (1−413). (B) RNA structure of the full length IGFL1 3’UTR (1−413 full length). The depicted structure was generated by VARNA [18]. Nucleotides 207−247 are highlighted in green, nucleotides 235−247 in yellow with a green rim. (C) Luciferase reporter data from MCF7 cells transfected with the psiCHECK-2 vector containing the full length IGFL1 3’UTR (1−413 full length), the IGFL1 3’UTR deleted of nucleotides 207−247 (1−413 Δ207−247), and the IGFL1 3’UTR deleted of nucleotides 235−247 (1−413 Δ235−247). Data were normalized to the full length 3’UTR control vector, presented as means ± SEM (n = 4), and statistically analyzed using two-way ANOVA with Dunnett’s multiple comparisons test; # p < 0.05, ## p < 0.01 compared to the IL-1β-treated full length 3’UTR vector.
Impact of a G-rich region on IGFL1 translation-regulatory properties
Detailed inspection of the G-rich region revealed that it in fact includes two distinct G-rich motifs, GGGGG at position 235−239 and AGGGA at position 243−247 (Fig 4A). We therefore decided to delete each motif individually (Δ235−239 and Δ243−247) in the full length IGFL1 3’UTR construct (S3 Fig in S1 File; lower panels). Single deletions of the two motifs equally attenuated the IL-1β-responsiveness of the IGFL1 3’UTR (Fig 4B), implying an equal importance of these two G-stretches. Strikingly, deletion of the entire region (Δ235−247) (S3 Fig in S1 File; upper right panel) yielded a similar reduction as deletion of the single motifs. As both motifs were predicted to be part of the same hairpin structure, we aimed to assess, if the loss of IL-1β responsiveness might be due to a loss of this RNA structural element. Therefore, we introduced point mutations within each motif, predicted to alter the local RNA structure of the IGFL1 3’UTR. Specifically, we mutated guanine (G) at position 238 in the lower part of the hairpin structure to a cytosine (C) and G at position 245 to a thymine (T) in the upper part both predicted to lead to massive local structural rearrangement, i.e., a loss of the hairpin between nucleotides 231 and 284. Importantly, both mutations were predicted to leave the structures encompassing nucleotides 1−173 and 284−413 of the full length 3’UTR unaltered, only disrupting the structure in the region in between (Fig 4C; upper panels). To unambiguously validate the potential role of this specific structural element, we further introduced corresponding reverse mutations (G238C_C277G; G245T_C269A) predicted to restore the RNA secondary structure to the original IGFL1 full length 3’UTR structure (Fig 4C; lower panels). Introduction of the structure-disrupting mutations indeed attenuated the IL-1β-induced increase in luciferase activity (Fig 4D). Surprisingly though, the reverse mutations did not rescue the IL-1β-induced luciferase activity compared to the structure-disrupting mutations. Since restoring the predicted RNA secondary structure to the original IGFL1 full length 3’UTR structure did not rescue the IL-1β-responsiveness, the IL-1β-dependent increase in IGFL1 translation likely does not depend on structural elements within the 3’UTR. Moreover, the isolated introduction of nucleotides 207−247 directly after the stop codon of the regulated renilla luciferase coding region did not recapitulate the IL-1β responsiveness of the full length 3’UTR (S4 Fig in S1 File).
(A) RNA structure of the full length IGFL1 3’UTR. Nucleotides 235−247 are highlighted in yellow, nucleotides 235−239 in blue, and nucleotides 243−247 in red. The depicted structure was generated by VARNA [18]. (B) MCF7 cells were transfected with the psiCHECK-2 vector containing either the full length IGFL1 3’UTR or 3’UTRs subjected to deletions. Renilla (RL) and firefly luciferase (FL) activities were determined 48 h after the transfection with or without treatment with IL-1β (50 ng/mL) during the last 4 h. Data were normalized to the full length 3’UTR control vector, presented as means ± SEM (n = 4), and statistically analyzed using two-way ANOVA with Dunnett’s multiple comparisons test; ## p < 0.01, ### p < 0.001 compared to the IL-1β-treated full length 3’UTR vector. (C) RNA structures of the full length IGFL1 3’UTR with structure-disrupting and respective rescue mutations. The depicted structures were generated by VARNA [18]. Structure-disrupting (G(238)>C) and respective rescue mutations (C(277)>G) are circled in dark blue; structure-disrupting (G(245)>T) and respective rescue mutations (C(269)>A) are circled in red. (D) MCF7 cells were transfected with the psiCHECK-2 vector containing either the full length IGFL1 3’UTR or 3’UTRs subjected to structure-disrupting and respective rescue mutations. Renilla (RL) and firefly luciferase (FL) activities were determined 48 h after the transfection with or without treatment with IL-1β (50 ng/mL) during the last 4 h. Data were normalized to the full length 3’UTR control vector, presented as means ± SEM (n = 4), and statistically analyzed using two-way ANOVA with Dunnett’s multiple comparisons test; ### p < 0.001 compared to the IL-1β-treated full length 3’UTR vector.
Taken together, our results indicate that the IL-1β-dependent increase in IGFL1 translation is not mediated by potential RNA structures within the 3’UTR, but rather by regulatory G-rich sequence elements.
Discussion
In the present study, we demonstrate that IL-1β increases IGFL1 mRNA transcription and translation in MCF7 breast cancer cells. We further identify the IGFL1 3’UTR as regulatory hub for its post-transcriptional regulation and provide evidence that the IL-1β responsiveness of IGFL1 translation is mediated by a G-rich region within the 3’UTR.
While IGFL1 has been increasingly recognized as a biomarker in several tumor types as well as in inflammatory diseases [22–26], little is known about its exact function. Similarly, the details of its regulation remain largely elusive. In fact, the pro-inflammatory cytokine TNF-α is the only reported IGFL1-inducing stimulus so far, both in the context of psoriasis and in breast cancer cells [13,14]. Here, we extend the panel of IGFL1 regulating stimuli to IL-1β. Specifically, we observed an IL-1β-dependent induction of IGFL1 expression in breast cancer cells, both at the level of IGFL1 mRNA expression and translation. This is in contrast to earlier reports, where IGFL1 expression was shown to remain not affected by IL-1β in primary keratinocytes [14]. These differences point to a marked tissue- or cell type-specific regulation of IGFL1, and, along the same lines, the cell type-specificity of the responses to IL-1β is well-characterized [27–29].
We further identified the 3’UTR rather than the 5’UTR of IGFL1 to contribute to its translational regulation. While 5’UTRs are well-established in the regulation of translation initiation, 3’UTRs have also been extensively recognized as important players in the regulation of post-transcriptional processes, including mRNA stability, localization, and translation regulation [30]. 3’UTRs indeed may harbor regulatory sequences including microRNA and RNA-binding protein binding sites as well as structural elements affecting translation regulation [31]. Of note, IGFL1 was previously shown to be regulated post-transcriptionally by the long non-coding RNA IGFL2-AS1, in part by impinging on the 3’UTR of IGFL1 [13]. Specifically, IGFL2-AS1 was proposed to compete with IGFL1 for the binding of miR4795-3p, which is predicted to bind at nucleotide 383 of IGFL1 3’UTR. In contrast, we identified the region between nucleotides 207−247 as crucial for the response to IL-1β. Moreover, IGFL2-AS1 was not or barely detectable in MCF7 cells [13,16]. Instead, we identified two adjacent G-rich motifs (GGGGG and AGGGA) at positions 235−239 and 243−247 of the IGFL1 3’UTR, which appeared to equally contribute to the IL-1β-dependent post-transcriptional induction of IGFL1. Indeed, deletion of the single motifs markedly attenuated the IL-1β-dependent induction of IGFL1. Moreover, while single nucleotide mutations within each motifs, predicted to disrupt the 3’UTR local structure, reduced the IL-1β responsiveness, reversing the local structural changes with respective rescue mutations did not overcome the attenuating effects. This observation rather speaks against the relevance of RNA structures suggesting a sequence-mediated regulatory mechanism in the case of the IL-1β-dependent, post-transcriptional induction of IGFL1. It can be envisioned that the two G-rich motifs may be bound by trans-acting factors responsible for regulating translation. Indeed, the two motifs, GGGGG at position 235−239 and AGGGA at position 243−247, were identified as hotspots for RNA binding proteins (RBPs) as they contained putative binding sites for numerous proteins, as predicted by ATtRACT [32] (S5 Fig in S1 File). Interestingly, while deletion of the single motifs markedly attenuated the IL-1β-dependent induction of IGFL1 translation, combined deletion of both motifs displayed no additional effect. Thus, not only are both protein binding sites equally important, but they also appear to be functionally connected. This finding is in line with the concept that many RBPs bind to bipartite motifs, i.e., two short motifs (usually 3-mers) separated by a spacer of 1−10 nucleotides [33]. This aligns perfectly with the two GGG 3-mers separated by four to six nucleotides within the 3’UTR of IGFL1. In this case, the deletion of one motif would be sufficient to impair the bipartite binding of the protein, thus, causing the reduction in the activity alike to our observation. Further studies are needed to identify the exact RBPs interacting with this bipartite motif. As a side note, both single nucleotide mutations, which were aimed at disrupting the 3’UTR structure, also happened to target the respective GGG 3-mers and similarly reduced the IL-1β responsiveness. Noteworthy, while several G-rich motifs are found across the entire 3’UTR of IGFL1, the proposed bipartite motif is the only G-rich context within the identified regulatory region of the 3’UTR (207−247). The fact that the isolated bipartite bearing region, when introduced directly after the renilla luciferase coding region, did not recapitulate the IL-1β responsiveness of the full length 3’UTR is in line with proposed involvement of RBPs, as the close proximity to the coding region, might prevent binding of regulatory RBPs due to steric interference of the translating ribosomes.
In conclusion, we provide evidence that IGFL1 can be regulated translationally by IL-1β through a 3’UTR-mediated mechanism. In particular, we identify a distinct region within the IGFL1 3’UTR, which contains G-rich motifs contributing to the IL-1β-mediated response. It will be interesting to see if the identified mode of regulation is responsible for the cell type-specificity of the IGFL1 regulation, which might also allow for therapeutic targeting of IGFL1 translation in tumors or inflammatory diseases by blocking the G-rich bipartite motif, e.g., with specific antisense oligonucleotides.
Supporting information
S1 File. Supplementary Information.
S1 Table. Primers used in this study; S1 Fig. IGFL1 mRNA expression upon TNF-α, IL-6, IL-10, and IL-1β stimulation; S2 Fig. Effect of IL-1β on CXCL8 mRNA expression, mRNA stability, and translation; S3 Fig. Predicted structures of the deletion constructs; S4 Fig. Translation-regulatory activity of the isolated G-rich region of the IGFL1 3’UTR; S5 Fig. RBP prediction using ATtRACT.
https://doi.org/10.1371/journal.pone.0342288.s001
(PDF)
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