RNA-Binding Protein IGF2BP1 Enhances mRNA Stability and Translation Efficiency of INHBA to Promote the Invasion and Migration of Esophageal Squamous Cancer Cells


 BackgroundMetastasis are mainly responsible for the death of patients with advanced esophageal squamous cell carcinoma (ESCC). At present, there is no targeted drug for the treatment of ESCC in clinic practice. The present study aims to investigate the roles and implication of IGF2BP1 overexpression in ESCC.MethodsIGF2BP1 protein expression was assessed by immunohistochemistry (IHC), and the mRNA abundance of IGF2BP1 and INHBA were analyzed with TCGA datasets and by RNA in situ hybridization (RISH). Cell viability, migration, invasion and in vivo metastasis assays were performed to explore the roles of IGF2BP1 in ESCC. RNA immunoprecipitation sequencing (RIP-seq) and mass spectrometry were applied to identify the targets and interacting proteins of IGF2BP1, respectively. RIP-PCR, RNA-pulldown, immunofluorescence (IF), gene specific m6A PCR and RNA stability assay were used to uncover the molecular mechanism of IGF2BP1 dysregulation. The methylation level of IGF2BP1 promoter region was detected by MSP-PCR. BTYNB, a small molecular inhibitor which could block the binding of IGF2BP1 to c-Myc mRNA, was evaluated for the inhibition effect on the malignant phenotypes of ESCC cells.ResultsIGF2BP1 overexpression was detected in ESCC tissues and associated with depth of tumor invasion. Knockdown of IGF2BP1 inhibited ESCC cell invasion and migration as well as tumor metastasis. Importantly, INHBA was identified as a direct target of IGF2BP1 in ESCC cells, which had a role in promoting the malignant phenotypes. TCGA data and RISH analyses showed that the mRNA level of INHBA was upregaluted in ESCC tissues as well. Mechanistically, IGF2BP1 bound and stabilized INHBA mRNA and then enhanced its translation, leading to an activation of Smad2/3 signaling. Ras GTPase-activating protein-binding protein 1 (G3BP1) was recruited by IGF2BP1 to participate in activating the signaling process, which was inhibited by the IGF2BP1 inhibitor BTYNB. Of note, IGF2BP1 mRNA expression in ESCC cells was negatively correlated with the level of its promoter methylation.Conclusions﻿IGF2BP1 overexpression promotes the invasion and migration of ESCC cells by up-regulating TGF-β-Smad2/3 pathway through enhancing INHBA mRNA stability and translation, providing a potential therapeutic target for ESCC treatment.


Abstract Background
Metastasis are mainly responsible for the death of patients with advanced esophageal squamous cell carcinoma (ESCC). At present, there is no targeted drug for the treatment of ESCC in clinic practice. The present study aims to investigate the roles and implication of IGF2BP1 overexpression in ESCC.
Methods IGF2BP1 protein expression was assessed by immunohistochemistry (IHC), and the mRNA abundance of IGF2BP1 and INHBA were analyzed with TCGA datasets and by RNA in situ hybridization (RISH). Cell viability, migration, invasion and in vivo metastasis assays were performed to explore the roles of IGF2BP1 in ESCC. RNA immunoprecipitation sequencing (RIP-seq) and mass spectrometry were applied to identify the targets and interacting proteins of IGF2BP1, respectively. RIP-PCR, RNA-pulldown, immuno uorescence (IF), gene speci c m 6 A PCR and RNA stability assay were used to uncover the molecular mechanism of IGF2BP1 dysregulation. The methylation level of IGF2BP1 promoter region was detected by MSP-PCR. BTYNB, a small molecular inhibitor which could block the binding of IGF2BP1 to c-Myc mRNA, was evaluated for the inhibition effect on the malignant phenotypes of ESCC cells.

IGF2BP1 overexpression was detected in ESCC tissues and associated with depth of tumor invasion.
Knockdown of IGF2BP1 inhibited ESCC cell invasion and migration as well as tumor metastasis.
Importantly, INHBA was identi ed as a direct target of IGF2BP1 in ESCC cells, which had a role in promoting the malignant phenotypes. TCGA data and RISH analyses showed that the mRNA level of INHBA was upregaluted in ESCC tissues as well. Mechanistically, IGF2BP1 bound and stabilized INHBA mRNA and then enhanced its translation, leading to an activation of Smad2/3 signaling. Ras GTPaseactivating protein-binding protein 1 (G3BP1) was recruited by IGF2BP1 to participate in activating the signaling process, which was inhibited by the IGF2BP1 inhibitor BTYNB. Of note, IGF2BP1 mRNA expression in ESCC cells was negatively correlated with the level of its promoter methylation.
Conclusions IGF2BP1 overexpression promotes the invasion and migration of ESCC cells by up-regulating TGF-β-Smad2/3 pathway through enhancing INHBA mRNA stability and translation, providing a potential therapeutic target for ESCC treatment.

Background
Esophageal cancer is one of the major malignancies threatening human health. Esophageal squamous cell carcinoma (ESCC) accounts for more than 90% cases of esophageal cancer in China. Most patients are diagnosed with advanced stage and the 5-year survival rate was only about 30% [1] due to invasive growth and distal metastasis. However, the molecular mechanisms underlying the invasion and metastasis of ESCC are still not fully understood, and so far there is no effective targeted drug for clinical treatment. Therefore, it is an urgent need to identify the key molecules affecting the invasion and metastasis of ESCC.
Moreover, IGF2BP1 has been proved as a N6-methyladenosine (m 6 A) reader, which recognizes and binds m 6 A-modi ed mRNA and thus enhances their stability [14,15]. Additionally, IGF2BP1 is termed as oncofetal protein due to its space-time speci c expression pattern: predominantly expressed in embryonic development and suppressed in most of adult tissues, while re-expressed in multiple tumor types [16].
Several researches reported that, BTYNB, a selective inhibitor, can impair cell proliferation in vitro by blocking IGF2BP1 binding to its oncogenic transcripts [17,18]. Altogether, these studies suggest that IGF2BP1 might be a promising biomarker and therapeutic target.
We found a IGF2BP1 remarkable upregulation in ESCC tissues by IHC. But there is no information available for the role of IGF2BP1 in ESCC. In this study, we focus on the roles of IGF2BP1 overexpression on malignant phenotypes and the underlying mechanisms in ESCC cells, aiming to explore the possibility of IGF2BP1 as the biomarker and therapeutic target for the disease.

Materials And Methods
Tissue specimens and cell lines ESCC and operative margin tissues from a total of 311 patients were procured from surgical resection specimens. All of the specimens were residual specimens collected after sampling for pathological diagnosis. All the patients received no treatment prior to surgery, and signed separate informed consent forms for sample collection and molecular analysis. Immunohistochemistry (IHC) IHC assay was conducted as reported previously [19]. Slides were incubated with primary antibody IGF2BP1(ab184305, 1:1000, Abcam) at 4°C overnight. The tissue microarrays were scanned with Nano Zoomer digital pathology biopsy scanner (HAMAMATSU, Japan). Immunoreactive scores were calculated as multiplying the scores of staining intensity and percentage of positive cells. The intensity was scored as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong); the proportion of positive cells was scored as follows: 0 (negative), 1 (1%-20%), 2 (21%-50%), 3 (51%-100%).

Plasmids and cloning
Cloning strategies including plasmids, oligonucleotides used for PCR were summarized in Additional le 1: Table S1,2. All constructs were validated by sequencing.

Transfection and lentiviral transduction
Cells were transfected with siRNA (GenePharma, China) or overexpression plasmids using Lipofectamine 3000 Transfection Reagent (L3000015, Thermo) according to the manufacturer's instructions. Lentivirus expressing IGF2BP1-shRNA (shIGF2BP1) or no-silencing-shRNA (shNS) as negative control were used to infect ESCC cells, and the stable cell strains were selected by puromycin (1 µg/mL, Gibco) for one week. The siRNA or shRNA sequence were provided in Additional le 1: Table S2,3.

Co-immunoprecipitation-based mass spectrometry (Co-IP-MS)
Non-denaturing lysis buffer (P0013, Beyotime) was employed to isolate the total protein lysate. 1 µg of IP antibody or rabbit IgG (B900610, Proteintech) was used to immunoprecipitated 1 µg of total proteins overnight at 4°C with rotation. Subsequently, 50 µL protein A/G magnetic beads (MedChemExpress) were introduced into the mixture and incubated in rotation for 2 h at 4°C. The beads were rinsed four times with washing buffer after which they were eluted in 20 µL 1×loading buffer and denatured at 100℃ for 10 min. Protein samples were then subjected to WB assay or SDA-PAGE followed by coomassie staining and sent to Shanghai Applied Protein Technology Co. Ltd. for mass spectrometry analysis.. Cell viability and colony formation assay 1×10 3 cells were seeded on 96-well plates (per well with 3 replicates in each group), and the cell viability was quanti ed every 24 h using Cell Counting Kit-8 (CK-04, Dojindo, Japan) according to the manufacturer's instruction. Absorbance was measured at 450 nm using by Elx 808 Microplate Reader (BioTek, USA). For colony formation assay, 1×10 3 cells were seeded on 6-well plates (per well with 3 replicates in each group), xed and stained using 0.1% crystal violet after treatment with indicated doze of BTYNB for 7-14 days.

Cell apoptosis analysis
Cells treated with 10 µM BTYNB for 48 h were digested, collected and stained with uorescent-labeled Annexin V and PI using Annexin V, FITC Apoptosis Detection Kit (AD10, Dojindo). Then the apoptotic cells were identi ed by ow cytometry.

Wound-healing assay
Cells were seeded in six-well plates and grew until reached full con uence. 10 µl tips were used to scratch a wound vertically, and the scraped cells were removed by washing with PBS. The scratches were observed and photographed at 0, 12, 24, and 48 h after being made. Cellular migration and invasion assays 8 µm pore 24-well Transwell plate (Corning, USA) was employed to assess the migration and invasion ability of ESCC cells. Cells were seeded into the upper compartments at a density of 1×10 5 /well in 200 µL serum-free media, and the lower chamber were added 700 µL RPIM-1640 media with 30% fetal bovine serum as a chemoattractant. After incubation 36 hours (KYSE30) or 24 hours (TE1) at 37°C, the cells were xed in methanol and acetone xation solution (1:1, v/v) and stained with 0.1% crystal violet (Sigma). The polycarbonate membranes were then cut, cells removed from apical side, and placed on slide, followed by scanning and imaging with Nano Zoomer digital pathology biopsy scanner (HAMAMATSU, Japan). The percentage of stained cell area were measured by ImageJ (Ver. 1.51j8, NIH, USA) in three random elds.

Xenograft assay
Four-week-old female BALB/c nude mice (HFK Bioscience Co., LTD, Beijing, China) were purchased and randomly divided into two groups by body weight (10 per group). The mice were injected with 1×10 6 KYSE30 cells stably expressing shIGF2BP1 or shNon-Silencing (shNS) via the tail vein. Eight weeks later, the mice were sacri ced and the whole lung tissues were separated and xed in Bouin's Fixative Solution (PH0976, Phygene). Then the number of lung metastases was counted, and the lung tissues were embedded in para n, cut into 3 µm sections, and stained with hematoxylin and eosin (H&E).
All the animal experiments were approved by the Animal Center of the Institute of National Cancer Center/Cancer Hospital, CAMS & PUMC (NCC2019A016).
Quantitative and Semi-quantitative RT-PCR Total RNA was isolated using RNApure Tissue & Cell Kit (CW0506, Cwbiotech) following the manufacturer's instructions, and cDNA was synthesized using HiFiScript cDNA Synthesis Kit (CW2569M, Cwbiotech). Then quantitative real-time PCR was performed using TB Green™ Premix Ex Taq kit (RR420A, TaKaRa) on an ABI QuantStudio DX real-time PCR System (ABI, USA). GAPDH was used as loading control, and the relative expression levels of mRNA were assessed through the comparative threshold cycle method (2 −ΔΔCt ). All primers used in this study are listed in Additional le 1: Table S4. RNA co-Immunoprecipitation combined with highthroughput sequencing (RIP-seq) RIP was performed using EZ-Magna RIP Kit (17-701, Millipore). Brie y, 2×10 7 cells were harvested and re-suspended in complete RIP Lysis Buffer (RIP lysis buffer supplemented with protease inhibitor cocktail and RNase inhibitor) on ice. one tenth of the supernatant of RIP lysate was saved as input. 5 µg of IGF2BP1 antibody (RN007P, MBL) or negative control normal rabbit IgG (PP64B, Millipore) was conjugated to protein A/G magnetic beads (MedChemExpress), followed by incubation with the RIP lysate in RIP Immunoprecipitation buffer (RIP wash buffer supplemented with 0.5 M EDTA and RNase inhibitor) for 4 h at 4°C. Then the immunoprecipitate was digested by proteinase K buffer. RNA was nally puri ed with TRIzol reagent (Invitrogen) and analyzed by qPCR or RNA-seq (Wuhan Seqhealth Tech Co. Ltd.). The sequences of primers for qPCR are described in Additional le 1: Table S4.
Biotin RNA pull-down assay Cell lysates were prepared in pulldown buffer (150 mM KCL, 0.5% NP-40, 25 mM Tris-HCl pH7.4, 0.5 mM DTT, supplemented with proteasome inhibitors and 80 U/mL RNase inhibitors) and incubated with biotinlabelled DNA probes at 4℃ for 2 h. Then the RNA-protein complex was immunoprecipitated with streptavidin magnetic beads (MedChemExpress) after an additional 2 h incubation at 4℃. The complex was divided into two equal portions. One was added to TRIzol reagent for RNA isolation followed by RT-PCR; the other was eluted with 1× protein loading buffer for Western blotting. 1/50 of the cell lysates were saved as input for RT-PCR and WB analysis. The sequences of biotin-labelled DNA probes against INHBA were provided in Additional le 1: Table S5. RNA stability assay ESCC cells stably expressing shIGF2BP1 or shNS were seeded into 12-well plates to get 80% con uency after 24 h. Then cells were treated with actinomycin D (ActD, 5 µg/mL) for 0, 2, 4 h, respectively. Total RNA was then extracted and analyzed by quantitative real-time PCR, and the relative level of INHBA mRNA at each time point was quanti ed, and GAPDH was used as loading control. The mRNA half-life was estimated according to the previous description [20].
Gene-speci c m 6 A qPCR 100 µg of total RNA was extracted and one tenth of the RNA was saved as the input control. Protein A/G Magnetic Beads (MCE) were prewashed and incubated with 5 µg of anti-m 6 A antibody (ABE572, Millipore) or rabbit IgG (PP64B, Millipore) for 2h at 4°C with rotation. Then the antibody-conjugated beads were mixed with total RNA in 1 × binding buffer supplemented with RNase inhibitors for additional 2 h at 4°C with rotation. The methylated mRNAs were eluted with elution buffer (10 mL 0.1 M DTT, 0.44 g NaCl, 2.5 mL pH7. 5

Statistical analysis
IBM SPSS Statistics 23.0 software was applied for data analysis, and P < 0.05 was considered as statistically signi cant. Fisher's exact test was used to assess the IHC scores difference between ESCC tissues and adjacent non-cancerous specimens. The correlation between protein expression level and clinicopathological parameters was analyzed by Pearson Chi-square test. Comparison between two groups was performed by independent sample T test, and one-way ANOVA test was used for multiple comparisons. RStudio software (1.1463) was used for GO and Pathway enrichment analysis.
Additional Materials and Methods are described in Additional les.

Results
1. IGF2BP1 is highly expressed in ESCC tissues and is associated with depth of tumor invasion We examined the protein expression level of IGF2BP1 in 311 cases of ESCC tissues and 9 cases of adjacent normal tissues on TMA by IHC technique. The results showed that IGF2BP1 was highly expressed in ESCC tissues (155/311, 49.8%), but was barely be detectable in normal esophageal epithelia (Fig. 1A, Table 1). And the positive staining was predominant in the cytoplasm of ESCC cells. A higher IGF2BP1 expression level was positively correlated with the depth of tumor invasion (T 1 − 2 versus T 3 − 4 ), but no signi cant differences were found in other clinicopathological features, such as sex, age, histologic grade, lymph node metastasis and clinical stage (Table 1). Furthermore, RNA-seq data obtained from the TCGA database revealed that the mRNA expression level of IGF2BP1 was elevated in ESCC specimens compared with that in normal tissues (Fig. 1B), which was consistent with the IHC results.

IGF2BP1 promotes ESCC cells migration, invasion and metastasis
To identify the role of IGF2BP1 in ESCC, the expression of IGF2BP1 in 11 ESCC cell lines was detected by Western blot. We found that IGF2BP1 had a higher expression in KYSE30, KYSE450, KYSE140 and TE1, but was almost undetectable in KYSE70, KYSE150 and TE10 cells ( Fig. 2A). KYSE30 and TE1 were applied to the functional study as cell models. After transient knockdown of IGF2BP1 mediated by small interfering RNA (siRNA), there was no signi cant change in cell growth with seven days compared with the control group (Fig. 2B). However, the migration and invasion ability of both the two cell lines were substantially repressed after transfection in the transwell (Fig. 2C) and wound healing assays (Fig. 2D).
Next, KYSE30 cells stably expressing shRNA of IGF2BP1 (shIGF2BP1) or non-silencing shRNA (shNS) conducted by lentivirus infection were injected to nude mice via the tail veins. The formation of lung metastatic tumors was observed in both groups six weeks later, but the number of lung metastases in the shIGF2BP1 group was signi cantly reduced, and the metastatic nodules were noticeably small (Fig. 2E).

IGF2BP1 increases INHBA mRNA stability, promoting ESCC cells migration and invasion
Given that IGF2BP1 is an RNA binding protein, we performed RIP-seq in KYSE30 cells to identify its potential RNA targets. The results showed that most of the IGF2BP1-binding sites were highly enriched in coding regions (CDS) compared to the input control, indicating that IGF2BP1 mainly regulates gene expression by binding CDS (Additional le 2: Figure S1). We selected and veri ed several genes related to cell migration and invasion through GO analysis. Notably, the mRNA abundance of INHBA was remarkably reduced after IGF2BP1 knockdown in KYSE30, KYSE450 and TE1 cells (Fig. 3A). RIP-PCR and RNA-pulldown using biotin-labeled DNA probe against INHBA mRNA further con rmed the interaction between IGF2BP1 protein and INHBA transcript (Fig. 3B, C). Furthermore, silencing IGF2BP1 downregulated the protein expression of INHBA and Smad2/3 in KYSE30 and TE1 cells (Fig. 3D).
Previous studies have shown that IGF2BP1 can recognize N6-methyladenosine (m 6 A) and enhance mRNA stability and translation in an m 6 A-dependent manner [14,15]. Therefore, it is highly possible that IGF2BP1 stabilized the mRNA of INHBA after their binding. RNA stability assay showed that the half-life of INHBA mRNA was signi cantly shortened after knockdown of IGF2BP1 in KYSE30 and TE1 cells (Fig.   3E). As the m 6 A writers-METTL3 or METTL14 was repressed by siRNAs, the INHBA protein levels were decreased in both cell lines without affecting IGF2BP1 (Fig. 3F). Gene-speci c m 6 A qPCR further con rmed that INHBA was regulated by m 6 A modi cation (Fig. 3G).
INHBA is a member of the transforming growth factor β (TGF-β) superfamily, which is closely associated with tumor invasion and metastasis. We employed transwell assay to assess the effects of INHBA on invasive and migratory phenotypes in ESCC cells. We observed that cell invasion and migration were inhibited after knockdown of INHBA by siRNA (Fig. 3H), while transient overexpression of INHBA in cells with stably knockdown of IGF2BP1 partially restored the inhibition (Fig. 3I). Western blotting showed that Smad2/3 expression was repressed by IGF2BP1 depletion in KYSE30 and TE1 cells, while the IGF2BP1 knockdown-induced Smad2/3 decrease was reversed by INHBA overexpression (Fig. 3J).

IGF2BP1 activates INHBA-Smad2/3 signaling by recruiting G3BP1
It has been reported that IGF2BPs interacts with other RNA-binding proteins (RBPs) to regulate mRNA targets [21,22]. For further elucidating the functional mechanism of IGF2BP1 in ESCC cells, we adopted Co-IP-MS to investigate interactive partners of IGF2BP1 (Additional le 3: Figure S2). A total of 227 potential proteins were identi ed, 47% of them participate in RNA regulation. GO and Pathway enrichment analysis revealed that most of them involved in RNA processing, localization, stability, metabolism, and transport and RNA splicing (Fig. 4A-B).
We selected G3BP1 as a candidate owing to its oncogenic role in other tumors and the involvement in RNA binding. Endogenous G3BP1 was immunoprecipitated by IGF2BP1 antibody using Co-IP assay, and vice versa (Fig. 4C). Immuno uorescence by confocal microscopy con rmed the cytoplasmic colocalization of G3BP1 and IGF2BP1 in KYSE30 and TE1 cells (Fig. 4D). Moreover, Western blotting showed that silencing G3BP1 by siRNA led to a decrease of INHBA and Smad2/3, and a slight reduction in IGF2BP1 (Fig. 4E).

INHBA is signi cantly upregulated in ESCC, HNSC and invasive breast cancer
We then analyzed the TCGA transcriptome sequencing datasets, and observed that the mRNA of IHBA was also increased in ESCC tissues (Fig. 5A), which was consistent with its functional role in ESCC and posttranscriptional regulation by IGF2BP1. RISH on tissue microarrays further con rmed the RNA-Seq results. More importantly, INHBA was mainly distributed in the peripheral tumor cells of cancer nests and the stroma of ESCC tissues but negative in the normal esophageal epithelia and stroma (Fig. 5B). Besides, in HNSC and invasive breast cancer, INHBA mRNA was signi cantly elevated as well based on TCGA datasets (http://maplab.imppc.org/wanderer/) (Fig. 5C).

Small molecule inhibitor BTYNB signi cantly inhibits the invasion, migration and proliferation of ESCC cells in vitro
Currently, there are no inhibitors in clinical trials that directly target IGF2BP1. Previous studies reported a small molecule drug, BTYNB, could inhibit the binding of IGF2BP1 to c-Myc mRNA [17,23]. BTYNB also impairs cell proliferation in vitro by blocking β-TRCP1, E2F and other transcripts [17,18]. So, it is reasonable to speculate that BTYNB might interfere with the IGF2BP1-driven malignant phenotypes in ESCC cells. We tested BTYNB with transwell assay by adding the drug into the lower chamber, and found that the migration and invasion of KYSE30 and TE1 cells were signi cantly inhibited in 36 h or 24 h, respectively (Fig. 6A). Meanwhile, cell viability and colony formation were impaired in a dose-dependent manner ( Fig. 6B-C). Besides, a 48 h exposure to BTYNB increased the number of apoptotic cells (Fig. 6D), and INHBA and Smad2/3 expression were decreased after BTYNB treatment (Fig. 6E).

Elevated expression of IGF2BP1 in ESCC cells is associated with hypomethylation of its rst intron
We further detected possible dysregulation of IGF2BP1 in other types of SCCs, and also found a higher IGF2BP1 mRNA in HNSCC, LUSC, and CESC compared with that in the corresponding normal tissues (http://maplab.imppc.org/wanderer/, Fig. 7A). Moreover, we observed a high degree of consistency between the mRNA and protein levels of IGF2BP1 in 10 ESCC cell lines (Fig. 7B). And interestingly, according to the HPA (Human Protein Atlas) database, IGF2BP1 was almost absent in normal esophageal tissues and other normal tissues except in the embryo and reproductive system (Fig. 7C). To uncover the mechanism of IGF2BP1 mRNA upregulation in ESCC cells, we examined the methylation status of this gene. Three pairs of primers (methylated and unmethylated primers) were designed for detecting distinct CG sites in the rst intron of IGF2BP1, and MSP-PCR was conducted in both high and low IGF2BP1 expression cell lines, respectively. The results showed that IGF2BP1 gene was hypomethylated in cell lines with high IGF2BP1 expression, but hypermethylated in low IGF2BP1 cell lines except for TE10 (Fig. 7D).

Discussion
The insulin-like growth factor-2 mRNA-binding protein family (IGF2BPs), composed of IGF2BP1, IGF2BP2 and IGF2BP3, has a crucial role in early embryonic development. IGF2BP1 and IGF2BP3 are demonstrated as oncofetal proteins, because they are silent in adult organs, except in reproductive system [13,16,24]. In contrast, IGF2BP2 is the only expressive IGF2BP in most adult tissues. It has been observed that IGF2BP1 and IGF2BP3 are re-expressed in many types of tumors, and IGF2BP2 was also found to be excessive in malignances due to genomic ampli cation according to pan-cancer analysis with TCGA data. Growing evidences support the pro-oncogenic roles of these RNA-binding proteins in cancer progression by in uencing their RNA target fate [25].  6 A-modi ed mRNAs and promotes their stability in a m 6 A dependent manner [33]. We indeed observed that INHBA mRNA was m 6 A modi ed, and the turnover of INHBA was m 6 A dependent. So it is probably that mRNA methylation is required in the regulation of INHBA by IGF2BP .
Like other RNA-binding proteins (RBPs), IGF2BP1 participates in forming ribonucleoprotein (RNP) granules that regulate mRNA translation, localization, and turnover [21]. We identi ed 107 proteins related to RNA processing by mass spectrometry, accounting for 47% of all interactive proteins of IGF2BP1 in ESCC cells. We found that G3BP1 was a partner of IGF2BP1 and contributed to regulate INHBA-Smad2/3 signaling positively. G3BP1 contains two C-terminal motifs (associated with RNA binding) and an RNA recognition motif (RRM). It has been demonstrated that G3BP1 promotes stress-induced RNA granule interactions to preserve polyadenylated mRNA [34]. Meanwhile, G3BP1 involved in protein degradation by stably associated with USP10 deubiquitinase [35]. Therefore, G3BP1 might activate the IGF2BP1-INHBA-Smad2/3 signaling either by promoting protein stability or by enhancing RNA stability. The speci c detail on how G3BP1 facilitates IGF2BP1 to elevate INHBA still need further investigation.
In view of a oncofetal pattern of expression, IGF2BP1 could be a promising target, making it possible to speci cally target tumor cells without disturbing the noncancerous tissues. Several studies have demonstrated that BTYNB, a structure-speci c inhibitor, could block the binding of IGF2BP1 to its oncogenic target mRNA, thus disrupt their interaction [17,18]. We evaluated the e cacy of BTYNB in vivo, and found that the typical malignant phenotypes of high IGF2BP1 expression ESCC cells were sharply repressed, and apoptotic cells were increased after BTYNB exposure. At the molecular level, BTYNB treatment led to an attenuated IGF2BP1-INHBA-Smad2/3 signaling. These data imply that IGF2BP1 is a potential molecular target for the therapy of ESCC.
Little information is available about how the expression of IGF2BP1 is modulated at the transcriptional and post-transcriptional level. It has been proposed that IGF2BP1 transcription was induced by β-catenin [36] and enhanced by c-     (green) was detected by immuno uorescence staining by using laser confocal microscopy, DAPI was used to stain nuclei (blue). Scale bar = 30 μM. E. Western blot analysis of IGF2BP1, INHBA, Smad2/3 in ESCC cells transfected with NC-siRNA or G3BP1 siRNA. GAPDH was used as loading control.