Introduction

Endometriosis (EMs) is a long-term ailment marked by pelvic discomfort and infertility (Giudice 2010; Taylor et al. 2021), impacting approximately 5–10% of women in their childbearing years globally (Simoens et al. 2012). Despite its prevalence, there is a significant lack of awareness surrounding the disease, resulting in prolonged diagnostic journey that can span over a period of 4 to 11 years, with 65% of women receiving misdiagnoses at first (Greene et al. 2009). Furthermore, the standard diagnostic tool, laparoscopy, can be prone to inaccuracies when identifying EMs (Taylor et al. 2021). Hence, a holistic, clinically oriented approach is essential for ensuring precise and timely diagnosis of this condition (Agarwal et al. 2019; Chapron et al. 2019). Diving deeper into the biological and chemical processes of EMs and pinpoingting possible treatment targets are imperative for advancing treatment strategies.

RNA-binding proteins (RBPs) are vital regulators of gene expression and are capable of interacting with numerous transcription factors, forming an extensive regulatory network involved in maintaining cellular homeostasis (Gebauer et al. 2021). Insulin like growth factor II mRNA binding proteins (IGF2BPs) comprises IGF2BP1, IGF2BP2 and IGF2BP3, which are pivotal in disease development through their regulation of mRNA stability and translation. These IGF2BPs modulate key regulatory factors involved in cell division and metabolism, exerting a significant impact on disease progression (Degrauwe et al. 2016; Elcheva et al. 2023). Insulin like growth factor II mRNA binding protein 3 (IGF2BP3), also recognized as IMP3, KOC and VICKZ3, serves as a tumorigenic protein implicated in various cancers (Wan et al. https://github.com/zhengrongbin/MEBOCOST). The findings revealed that glutamine, which maintaind cell life activities in stromal cells, primarily originated from macrophages (Fig. 5A). Furthermore, by conducting spatial transcriptome analysis of the GEO dataset (GSE213216), we observed the expression of the critical enzyme GLS1 involved in glutamine metabolism within endometrial lesions (Fig. 5B), and the data was visualized using the Seurat package. We also investigated the expression of GLS1 in EC and EN using another GEO dataset (GSE105764) (Fig. 5C). Additionally, insights from the GSE231938 dataset revealed a positive association between GLS1 and IGF2BP3 in non-small cell lung cancer, prompting further exploration into the interplay among GLS1, IGF2BP3 and UCA1. After altering the expression levels of UCA1 and IGF2BP3, we observed changes in both glutamine consumption and GLS1 expression levels (Fig. 5D–F). In order to delve deeper into the underlying mechanism of the UCA1 and IGF2BP3 interaction in the regulation of GLS1 expression, a rescue experiment was conducted. The results demonstrated that down-regulation of UCA1 effectively prevented the increase in GLS1 expression induced by IGF2BP3 in eESCs (Fig. 5G–I). Based on previous studies, it has been suggested that the IGF2BP3 protein selectively bind to the conserved sequences "UGGAC" or "UUUUAAA" present in the 3'UTR. To investigate the potential protein-RNA interactions between IGF2BP3 and GLS1, we performed computational analysis using the starBase website (Fig. 5J). For the RNA pull-down experiment, a 3'UTR fragment labeled with biotin of GLS1 containing the predicted binding pattern was used. The outcomes revealed a notable increase in the binding of the IGF2BP3 protein to the GLS1 3'UTR sequence as compared to the negative control (Fig. 5K). Our study discovered a positive association among the expression levels of GLS1, IGF2BP3 and UCA1 in 9 patients with EMs (Fig. 5L). To further investigate the impact of the UCA1/IGF2BP3 interplay on the stability of GLS1 mRNA, we conducted RNA stability assays. The alterations in both UCA1 and IGF2BP3 affected the stability of GLS1 mRNA. Additionally, rescue experiments indicated that reducing the expression of UCA1 counteracted the increased stability of GLS1 mRNA observed in eESCs overexpressing IGF2BP3 (Fig. 5M). Overall, these findings provided compelling evidence that underscored the crucial function of UCA1 in the IGF2BP3-mediated prolongation of GLS1 mRNA half-life.

Fig. 5
figure 5

IGF2BP3 up-regulated GLS1 expression in eESCs by interacting with UCA1. A The GEO dataset (GSE179640) using the mebocost algorithm in EC revealed that stromal cells uptake glutamine from macrophages to sustain cellular metabolic activity. B Single-cell spatial omics form the GEO dataset (GSE213216) through spatial transcriptomic analysis demonstrated the expression distribution of GLS1 mRNA in EC. C The volcano plot using another GEO dataset (GSE105764) showed the expression of GLS1 between EN and EC. D, E The spectrophotometer was used to determine the glutamine uptake content in eESCs that were transfected with IGF2BP3-overexpressing plasmid and IGF2BP3-silenced lentivirus (D), as well as UCA1-overexpressing plasmid and UCA1-silenced lentivirus (E). F The spectrophotometer was used to measure the Glutamine uptake content in eESCs that were transfected with a lentivirus containing a non-targeting control sequence or UCA1-silenced lentivirus and co-transfected with IGF2BP3-overexpressing plasmid or matching control plasmid. G, H Western blot assays showed the expression of GLS1 in eESCs transfected with IGF2BP3-overexpressing plasmid and IGF2BP3-silenced lentivirus (G) as well as UCA1-overexpressing plasmid and UCA1-silenced lentivirus (H). I Western blot assays were executed to measure the levels of GLS1 expression in eESCs of different transfection ways as before. J Predicted binding motifs for IGF2BP3. K Verified the association between IGF2BP3 protein and the GLS1 3' UTR regions by RNA pull assays and western blot. L Correlation analysis was conducted to examine the mRNA expression of IGF2BP3 and GLS1, as well as UCA1 and GLS1, in EC. M qRT-PCR was utilized to detect the half-life of GLS1 in eESCs subjected to various treatments involving IGF2BP3 and UCA1. These treatments included transfection of an IGF2BP3-overexpressing plasmid and an IGF2BP3-silenced lentivirus, as well as transfection of a UCA1-overexpressing plasmid and a UCA1-silenced lentivirus. Additionally, a negative control lentivirus or UCA1-silenced lentivirus was co-transfected with an IGF2BP3-overexpressing plasmid or the corresponding control plasmid

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IGF2BP3 exerts its pro-proliferative and pro-migration effect in eESCs through GLS1

GLS1 plays a pivotal role in various cancers, particularly in ovarian cancer and endometrial cancer (Wu et al. 2021). Subsequently, siRNAs were transfected into eESCs to assess their efficiency (Fig S2B). The inhibitor BPTES was employed to inhibit GLS1 function by reducing its enzyme activity. Results from the rescue experiments indicated that both knockdown of GLS1 and treatment with BPTES resulted in inhibited proliferation (Fig. 6A, B) and migration (Fig. 6C, D) of eESCs. Importantly, these effects were mediated by IGF2BP3, suggesting that IGF2BP3 promoted eESC progression by regulating GLS1 function.

Fig. 6
figure 6

IGF2BP3 exerted its pro-proliferative effect in eESCs via GLS1. A, B The proliferation of eESCs overexpressing IGF2BP3 combined with si-GLS1 or BPTES was assessed through Edu assays and CCK-8 assays. C, D Wound healing assays and transwell migration assays revealed that eESCs with IGF2BP3-overexpression exhibited increased migration compared to cells with GLS1-silenced or demonstrate with BPTES

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UCA1 promotes GLS1 expression by stabilizing c-MYC levels

Numerous researches have shown that UCA1 was crucial in c-MYC. The western blot outcomes revealed that both UCA1 and IGF2BP3 positively regulated the expression of c-MYC (Fig. 7A, B). Further investigation using actinomycin D delved into the involvement of UCA1 and IGF2BP3 in maintaining c-MYC stability. The results indicated that knockdown of IGF2BP3 and UCA1 led to a reduction in c-MYC mRNA stability (Fig. 7C). Moreover, UCA1 was shown to interact with c-MYC protein through RNA pull-down and RIP assays (Fig. 7D, E). Based on the data presented, it was assumed that UCA1 has the capability to bind to c-MYC levels post-transcriptionally. It was speculated that this binding could offer protection against the degradation of c-MYC. When eESCs were exposed to the protein synthesis inhibitor CHX, overexpression of UCA1 was observed to prevent the breakdown of c-MYC protein (Fig. 7F). Furthermore, the connection of UCA1 and c-MYC was confirmed in the nucleus via a FISH-IF assay (Fig. 7G). Previous studies have presented compelling evidence demonstrating that c-MYC had the ability to bind to the specific region of GLS1 promoter, leading to a significant augmentation in its expression. In this context, the upregulation of UCA1 resulted in elevated mRNA expression of GLS1, a response was found to be reversible upon the inhibition of c-MYC (Fig. 7H). Collectively, these findings indicated that UCA1 facilitated glutamine metabolism in eESCs through the IGF2BP3/c-MYC/GLS1 signaling pathway (Fig. 7I).

Fig. 7
figure 7

UCA1 promoted GLS1 expression by stabilizing c-MYC levels. A, B Quantified the protein abundance of c-MYC in eESCs transfected with UCA1-overexpressing plasmid and UCA1-silenced lentivirus (A), and IGF2BP3-overexpressing plasmid and IGF2BP3-silenced lentivirus (B). C Used qRT-PCR to evaluate the expression of c-MYC mRNA in eESCs transfected with UCA1-silenced lentivirus and IGF2BP3-silenced lentivirus, subsequently managed with actinomycin D (5 μg/ml) for the specified durations. D The relationship between c-MYC protein and UCA1. E RIP assay. F Used western blot to explore the influence of CHX treatment on UCA1-overexpressing plasmid mediated changes in c-MYC protein levels. G RNA-FISH combined with Immunofluorescence staining experiments showed that UCA1 (red) located together with c-MYC (green) in the nucleus of eESCs. H Western blot was performed to measure the expression of GLS1 in eESCs. I A graphical representation was created to depict the regulation of the IGF2BP3/UCA1/c-MYC/GLS1 axis in EM

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Blocking the UCA1/IGF2BP3/GLS1 axis inhibited the progression of EMs in vivo

To assess the influence of GLS1 on the progression of EMs in vivo, EMs mice models were established. All of the mice were separated into two categories: the control group and the BPTES group (Fig. 8A). HE staining of uterus and ectopic lesions demonstrated a significant decrease in disease severity in the BPTES group (Fig. 8B–D). When compared to the control group, the BPTES group showed reductions in both lesion weight and size (Fig. 8E). To validate the roles of IGF2BP3 and UCA1 in EMs in vivo, subcutaneous xenograft nude mice models were established. However, due to the limited proliferative capacity of eESCs, an immortalized cell line (hEM15A) was used instead. In line with their in vitro findings, down-regulation of IGF2BP3 and UCA1 in hEM15A cells resulted in a decrease in subcutaneous xenograft growth, weight and volume, as well as a corresponding reduction in the weight of the nude mice.

Fig. 8
figure 8

Blocking the UCA1/IGF2BP3/GLS1 axis inhibited the progression of EM in vivo. A The circuit diagram for the creation of the mouse EM model. Created by BioRender.com. B HE staining of GLS1 in uterine tissues and ectopic lesions in the models following treatment with either DMSO or BPTES. Scale bar: 100 µm. C, D Visible lesions of the model of EM after treatment. E The weight of ectopic lesions. (F-I) Representative images of subcutaneous tumors from mice (F, G) injected with control lentivirus cells (left), IGF2BP3-silenced lentivirus cells (middle) or UCA1-silenced lentivirus cells (right). The mean weight of tumors was measured (H); body weight growth curves (I) and tumor volume growth curves (J) were plotted every 4 days until 24 days. K The levels of GLS1 protein and Ki-67 index of different groups by IHC. Scale bar: 50 μm

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Immunohistochemistry experiments revealed that the down-regulation of IGF2BP3 and UCA1 led to decreased expression levels of GLS1 and Ki67 (Fig. 8K). In conclusion, these in vivo studies validated that IGF2BP3 and UCA1 promoted the proliferation of hEM15A cells. These findings provided a basis for further exploration of a novel combination therapy for EMs.

Discussion

EMs is a long-term inflammatory gynecologic disease characterized by the presence of ectopic endometrial tissue. It has been believed to primarily affect the pelvic, but recent studies have found that it can affect liver and adipose tissue metabolism, resulting in widespread inflammation throughout the body. Changes in gene expression within the brain have also been observed, which contribute to heightened pain sensitivity and the development of emotional disorders (Taylor et al. 2021). Although the exact cause of endometriosis is still unclear, researchers have been investigating the role of RBPs in its development. RBPs have the ability to recognize multiple RNA transcripts and form regulatory networks that are crucial for maintaining cellular balance. Dysfunction of RBPs has been implicated in development of various diseases. One particular RBP, IGF2BP3, has been linked with metabolic reprogramming in various diseases like gastric cancer, lung cancer, and glioma (Lin et al. 2023; Wang et al. 2023; Fang et al. 2023). Our observations have shown that the metabolism of glutamine in endometriotic cells undergoes reprogramming, resulting in heightened activity of important metabolic enzymes. Previous studies have confirmed the involvement of different transcription factors (TFs) and signaling pathways in the reprogramming of glutamine metabolism in other diseases (Najumudeen et al. 2021). However, the extent of their contribution to endometriosis remains uncertain. Based on our research, we have discovered that the IGF2BP3/UCA1/c-MYC/GLS1 axis was crucial to stimulate the proliferation and migration of endometriotic cells through its regulation of glutamine metabolism. IGF2BP3 is an RNA-binding protein that plays a vital role in regulating the stability of mRNAs. Increased expression of IGF2BP3 contributed to the development of tubular cell fibrosis by directly binding to β-catenin mRNA, enhancing its stability and preventing degradation (Song et al. 2023). In prostate cancer, a circular RNA called hsa_cir_0003258 bound to IGF2BP3 in the cytoplasm, leading to increased stability of HDAC4 mRNA and promoting cancer metastasis (Yu et al. 2016; ** et al. 2023). Glutamine metabolism and its associated metabolic pathways, such as those involving glutamine carriers, glutaminase, amino acid transferase, and oxidative stress balance, play a vital role in supporting the survival of cancer cells (** et al. 2023; Matés et al. 2020). Previous research has established the crucial role of GLS1 in the metabolic behaviors of cancer cells, facilitating fast growth, cell viability, and immune escape (Chen et al. 2023). However, the presence and influence of GLS1 in eESCs remain unexplored. In this research, we discovered that IGF2BP3 could enhance the proliferation and glutamine metabolism of GLS1-mediated cell in eESCs through its interaction with UCA1. Moreover, preceding studies have indicated that c-MYC interacts with the promoter sequences of GLS1 to iniciate transcriptional activition (Liu et al. 2019; Zeng et al. 2023). In accordance with the evidence concerning GLS1, we noted that the mRNA expression of c-MYC can be elevated by UCA1 and IGF2BP3. Remarkably, this investigation revealed a direct interaction between UCA1 and c-MYC protein, leading to its stability. This interaction consequently resulted in enhanced GLS1 transcription and subsequent activation of glutamine metabolism. As a whole, these collective outcomes revealed that UCA1 drived the promotion of glutamine metabolism mediated by GLS1 in eESCs through its interaction with IGF2BP3 and c-MYC. Nevertheless, the mechanism by which UCA1 binds to IGF2BP3 and influences the stability of downstream target mRNA is an aspect that we will thoroughly investigate in the future studies.

Conclusion

All in all, it was observed that the expression of IGF2BP3 was detected in EMs. It was found to enhance the proliferation and migration of EMs. In terms of mechanism, we firstly revealed that IGF2BP3 bond directly to UCA1, thereby contributing to the stability of GLS1 mRNA levels.

Furthermore, it was discovered that UCA1 enhanced the function of IGF2BP3 through direct binding, without influencing its expression. Additionally, UCA1 was observed to interact with and maintain mRNA and protein levels of c-MYC. Moreover, it was also demonstrated to activate the expression of GLS1 by facilitating its transcription. This study offered a novel perspective on the underlying mechanisms of EMs and presented new possibilities for develo** innovative treatment strategies.