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. 2024 Feb 10;42:101904. doi: 10.1016/j.tranon.2024.101904

RNA-binding protein IGF2BP2 suppresses metastasis of clear cell renal cell carcinoma by enhancing CKB mRNA stability and expression

Junwu Ren a,1, Bo Huang a,b,1, Wei Li c, Yongquan Wang d, Xiaojuan Pan a, Qiang Ma a, Yuying Liu a, Xiaolin Wang a, Ce Liang a, Yuying Zhang a, Shimin Wang a, Feifei Yang a, Haiping Li a, Hao Ning a, Yan Jiang a, Changhong Qin a, Ai Ran a, Bin Xiao a,⁎
PMCID: PMC10867445  PMID: 38341962

Highlights

  • •

    The RNA-binding protein IGF2BP2 is down-regulated in ccRCC tissues and cell lines, and IGF2BP2 inhibits ccRCC metastasis in vivo and in vitro.

  • •

    IGF2BP2 enhances CKB expression by binding to CKB mRNA and stabilizing its mRNA, thereby inhibiting ccRCC metastasis.

  • •

    Our findings demonstrate a novel insight for IGF2BP2 on ccRCC tumorigenesis via affecting mRNA stability.

Keywords: Clear cell renal cell carcinoma, IGF2BP2, CKB, mRNA stability, RNA binding protein

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer, with a highly aggressive phenotype and poor prognosis. RNA binding proteins (RBPs) play crucial roles in post-transcriptional gene regulation and have been implicated in tumorigenesis. RBPs have the potential to become a new therapeutic target for ccRCC. In this study, we screened and validated that insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) as an RBP, was down-regulated in ccRCC tissues and cell lines. Functionally, we verified that IGF2BP2 significantly suppressed the migration and invasion ability of ccRCC in vitro and in vivo. Mechanistically, RIP-seq and actinomycin D experiments results showed that IGF2BP2 enhanced the expression of Creatine Kinase B (CKB) by binding to CKB mRNA and enhancing its mRNA stability. Thus, IGF2BP2 inhibited ccRCC metastasis through enhancing the expression of CKB. Taken together, these finding suggests that IGF2BP2 is a novel metastasis suppressor of ccRCC and may serve as a potential therapeutic target.

Introduction

Renal cell carcinoma (RCC) is one of the most common malignancies of the urogenital system [1,2]. Clear cell renal cell carcinoma (ccRCC) is the most common type of RCC which accounts for 70–75 % with high morbidity and mortality in urogenital tumors [2], [3], [4]. Surgery is an effective treatment for ccRCC, but approximately 30 % of patients with distant metastasis prone to relapse after surgery treatment [5,6]. Traditional chemoradiotherapy is largely ineffective in the treatment of ccRCC [5,7]. Furthermore, molecular-targeted drugs like sunitinib and pazopanib, which are utilized for the management of metastatic ccRCC, exhibit limited clinical efficacy [8], [9], [10], [11]. The lack of effectiveness can be attributed to an incomplete understanding of the pathogenesis underlying ccRCC metastasis. Therefore, it is imperative to investigate the etiopathogenesis of ccRCC in order to identify potential therapeutic targets, explore personalized treatment regimens, and improve patient prognosis.

RNA-binding proteins (RBPs), which constitute 6–8 % of protein-coding genes. Substantial evidence indicates their pivotal roles in crucial cellular processes, including cellular transport, subcellular localization, and cell differentiation [12,13]. Additionally, RBPs are engaged in almost every aspect of post-transcriptional regulation, supervise the formation and function of transcripts while maintaining cell homeostasis. Mechanistically, RBPs regulate RNA splicing, polyadenylation, mRNA stability, mRNA localization, and translation through interactions with RNAs or other proteins [14,15]. IGF2BP2 is a recognized RBP that has been identified to regulate multiple biological processes [16], [17], [18]. It has been reported to acts as an oncogene in colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, exerting its function through post-transcriptional gene regulation [19], [20], [21], [22]. For instance, IGF2BP2 activates the Warburg effect and influences colorectal cancer tumorigenesis by stabilizing the ZFAS1/OLA1 axis [23]. Moreover, our previous study has validated IGF2BP2 regulated ccRCC migration via interaction with a circular RNA (circRNA) [24]. Nevertheless, the function and relevant molecular mechanisms of IGF2BP2 in ccRCC remain elusive.

In this study, we found IGF2BP2 was significant down-regulated in ccRCC. Functional experiments have substantiated that IGF2BP2 inhibits ccRCC migration and invasion both in vitro and in vivo. We confirmed CKB was a binding target of IGF2BP2 through RIP-seq assay. Furthermore, IGF2BP2 promoted CKB expression by binding to CKB mRNA and stabilizing its mRNA, thereby inhibiting tumor metastasis. In summary, our findings demonstrated that IGF2BP2 plays a novel and pivotal role in ccRCC metastasis by regulating CKB expression.

Materials and methods

Cell lines and cell culture

The human ccRCC lines Caki-1, RCC-JF, 786-O, ACHN and human normal renal epithelial cells 293T were obtained from Meisen CTCC (Zhejiang, China). Caki-1 were cultured in McCoy's 5A medium (Basal Media, Shanghai, China), 293T and ACHN in DMEM medium (Basal Media, Shanghai, China), RCC-JF and 786-O in RPMI-1640 medium (Basal Media, Shanghai, China) supplemented with 10 % FBS [25]. All cells were cultured in a 37 °C incubator with 5 % CO2.

Patients and tissue samples

A total of 136 pairs of ccRCC tissues and adjacent non-tumor tissues were obtained from Department of Urology, Southwest Hospital of the Army Military Medical University. The samples were stored in RNA later (ThermoFisher, USA). Clinical characteristics were listed in Table S1. All samples were collected with informed consent from patients and the study was approved by the Ethics Committee of the Southwest Hospital of the Army Military Medical University (Approval number: KY2020121).

Plasmids, siRNAs and cell transfection

The full-length IGF2BP2 fragment was cloned into pcDNA3.1(+) (Tsingke, China) to establish pcDNA3.1-IGF2BP2 vector. The siRNAs were obtained from GenePharma (Shanghai, China) and Tsingke Biotechnology (Beijing, China). Plasmids were transfected with NEOFECT™ DNA transfection reagent (Neofect biotech, Beijing, China). The siRNAs were transfected with Lipo8000™ Transfection Reagent (Beyotime, Shanghai, China) and Lipofectamine 2000 (Thermo, USA). The shRNAs and siRNAs sequence information was shown in Table S2.

Western blot analysis

ccRCC cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail and 1 % PMSF. Samples were separated with 10 % SDS-PAGE. Protein was transferred to PVDF membranes and incubated overnight at 4 °C with primary antibodies. Followed by incubation with a secondary antibody at room temperature for 1 h. Primary antibodies used in the article included: anti-IGF2BP2 antibody (SAB5701598, Millipore, USA), CKB Monoclonal antibody (66,764, Proteintech, Wuhan, China), GAPDH antibody (AF0006, Beyotime, Shanghai, China), anti-β actin mAb (TA-09, ZSGB-BIO, Beijing, China).

Quantitative real-time PCR

Total RNA was extracted by TRIzol reagent (Takara, Japan) and cDNA was reverse transcribed with PrimeScript™ RT reagent Kit (Takara, Japan). qRT-PCR assay was performed to detect relative gene expression levels by using 2 × SP qPCR Mix (Bioground, China). All primers were synthesized by Tsingke Biotechnology (Beijing, China). The primer sequences information was presented in Table S3.

Migration and invasion assays

After 2 days of transfection in a six-well plate, 200 μL of ccRCC cells suspension were added to the upper chamber of the transwell (3 × 104 cells for migration and 6 × 104 cells for invasion assays). Then, 500 μL of complete medium was added to the lower chamber of the transwell. After 20 h incubation, cells were fixed with 4 % paraformaldehyde for 10 min and stained with crystal violet for 10 min, photographed and counted using Image J software. Then, the cell layer was scratched after 48 h of transfection in a six-well plate. Wound width was observed at three time points: 0 h, 6 h and 12 h, and wound width was counted using Image J software.

RNA immunoprecipitation sequencing (RIP-Seq)

RIP assays were performed using the Magna RNA-binding protein immunoprecipitation kit (Millipore, USA), according to the manufacturer's protocols. The immunoprecipitated RNA in Caki-1 cells was verified by qRT-PCR. Furthermore, the remaining RNA was performed by Sinotech Genomics Co. Ltd (Shanghai, China) for RNA immunoprecipitation sequencing.

RNA sequencing (RNA-Seq)

In order to screen for the downstream target genes of IGF2BP2, we extracted RNA from Caki-1 with the IGF2BP2 down-regulation compared with the control groups. The mRNA sequencing was performed by Tsingke Biotechnology Co. Ltd (Beijing, China).

Constructing stable cell line

The sh-NC represents Caki-1 cells transfected with lentiviruses containing unrelated sequences, serving as the negative control and the sh-IGF2BP2 represents Caki-1 cell lines with stable knockdown IGF2BP2. The sh-NC and sh-IGF2BP2 lentiviruses were synthesized by Oligobio Co. Ltd (Beijing, China) and the sequences were provided in Table S2. Caki-1 cells were infected with lentiviruses at three different MOI values and stable transfectants were selected using puromycin. Subsequently, the stable IGF2BP2 knockdown and the negative control Caki-1 cell lines were established.

Animal metastasis model

Nude mice were anesthetized via intraperitoneal injection of 2 % sodium pentobarbital. Subsequently, the skin was disinfected using 75 % alcohol. Following laparotomy, each nude mouse was injected with 200 μL Caki-1 stable transfectant cell suspension (1 × 106 cells/ nude mouse, sh-NC n = 3, sh-IGF2BP2 n = 3) into their spleen. The blood vessels were ligated, and the spleen was excised and the incision sutured. After 52 days of post-operative observation, significant ascites had observed in the abdomens of the nude mice. The livers were excised and photographed. The H&E staining analysis of liver tissue was performed by Servicebio Co. Ltd (Wuhan, China).

mRNA stability assays

The si-NC and si-IGF2BP2 were transfected into 12-well plates. After 24 h, the medium was replaced with medium containing actinomycin D (10 μg/mL) or DMSO. Total RNA was collected at 0 h, 6 h, 9 h and 12 h after actinomycin D treatment. CKB mRNA relative expression level were detected by qRT-PCR [26].

Statistical analysis

One-way analysis of variance, Student's t-test, and Chi-square test were employed to compare the means among different groups. The statistical differences were calculated using SPSS 18.0 and GraphPad Prism 7.0. The data are presented as mean ± standard deviation (SD). Experiments were independently replicated at least three times. Statistical significance was defined as P < 0.05.

Results

IGF2BP2 exhibits significant down-regulation in ccRCC tissues

To identify differentially expressed RBPs in ccRCC, we conducted an intersection analysis between the GEPIA 2 database (Table S4) and the identified RBP genes (Table S5). Notably, IGF2BP2 ranked at the forefront of down-regulated RBP gene (Fold change=−1.944) (Fig. 1A). Furthermore, GEPIA 2 database analysis showed a significant decrease in IGF2BP2 mRNA expression in ccRCC tissues compared to adjacent tissues (Fig. 1B-C). We then validated IGF2BP2 expression in clinical tissues. qRT-PCR and western blot assays showed that IGF2BP2 was down-regulated in ccRCC tissues (Fig. 1D-F). We also analyzed the expression of IGF2BP2 in the normal human renal epithelial cell line and ccRCC cell line. Consistently, IGF2BP2 expression was down-regulated in all ccRCC cell lines, albeit to varying degrees. Moreover, Caki-1 and RCC-JF cell lines are shown as the highest relative expression of IGF2BP2. Therefore, Caki-1 and RCC-JF cells were selected for subsequent functional experiments (Fig. 1G).

Fig. 1.

Fig 1

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IGF2BP2 is down-regulated in ccRCC. (A) Schematic representation illustrating the screening process for differentially expressed RNA-binding proteins in ccRCC, along with a corresponding heatmap. (B-C) IGF2BP2 mRNA level in ccRCC patients was analyzed using the GEPIA 2 database. (D-E) IGF2BP2 mRNA expression was detected in ccRCC paired tissues (n = 136) by qRT-PCR. (F) Western blot assay was used to detect protein expression of IGF2BP2 in 8 pairs of tissues. (G) Expression level of IGF2BP2 was detected in different cell lines. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance.

Though analyzing the correlation between IGF2BP2 expression and potential clinical significance, we observed that IGF2BP2 expression was not significantly associated with distant metastasis, WHO/ISUP stage, or N stage. Unexpectedly, IGF2BP2 expression was higher in ccRCC tissues with advanced T stage (T2a-T4 vs T1a-T1b) (Fig. S1A). Moreover, GEPIA 2 database showed that higher IGF2BP2 expression was associated with lower overall survival (OS) and disease-free survival (DFS) in ccRCC patients (Fig. S1B). Our finding indicates that IGF2BP2 is significantly down-regulated in ccRCC tissues and cell lines. However, its expression is not significantly correlated and consistent with clinical characterise.

IGF2BP2 inhibits ccRCC cell migration and invasion in vitro

To investigate the function of IGF2BP2 in vitro, IGF2BP2 was respectively knockdown or overexpression in Caki-1 and RCC-JF cells. Subsequently, the altered expression levels were validated by qRT-PCR and western blot assay. The result showed that IGF2BP2 mRNA and protein levels was significantly silenced or overexpressed (Fig. 2A). Next, we performed a series of in vitro experiments to evaluate the function of IGF2BP2. Transwell assay demonstrated that IGF2BP2 knockdown significantly enhanced migration and invasion ability in vitro, whereas IGF2BP2 overexpression significantly suppressed migration and invasion in vitro (Fig. 2B-E). Furthermore, wound healing assays also indicated that IGF2BP2 inhibited the migratory capacity of ccRCC cells (Fig. 2F-G). However, IGF2BP2 had no significant effect on the proliferation of ccRCC cells as assessed by CCK-8 and clone formation assays (Fig. S2A-B). The above-mentioned results demonstrate that IGF2BP2 exerts an inhibitory effect on the migration and invasion of ccRCC cells in vitro.

Fig. 2.

Fig 2

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IGF2BP2 inhibits ccRCC cell migration and invasion in vitro. (A) Knockdown and overexpression efficiency of IGF2BP2 were evaluated in Caki-1 and RCC-JF cells. (B-C) Cell migration ability was detected by transwell migration assay. (D-E) Cell invasion ability was detected by transwell invasion assay. (F-G) Cell migration ability was detected by wound healing assay. Bars, 100 μm; *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance.

IGF2BP2 suppresses the liver metastasis of ccRCC in vivo

To further investigate the biological function of IGF2BP2 in vivo. We established a liver metastasis model in nude mice using Caki-1 cell stably transfected with shRNA and control vectors. As shown in Fig. 3A. qRT-PCR results showed that IGF2BP2 knockdown efficiency reaches approximately 75 % for all three different MOIs (Fig. 3B). Next, we injected Caki-1 cells with stable IGF2BP2 silencing into the spleen of nude mice. After 52 days, we found that sh-IGF2BP2 group exhibited a significantly higher number of liver metastatic foci in mice compared to the control group (Fig. 3C-E). Similarly, H&E staining confirmed that sh-IGF2BP2 group showed more liver metastatic nodules compared to the control group (Fig. 3F). Furthermore, to better evaluate the function of IGF2BP2 metastasis in vivo. We established a lung metastasis model via tail vein injection in nude mice. The results showed that no obvious metastatic foci were observed in the lungs (Fig. S3A). However, liver metastatic foci were observed in the nude mice model based on tail vein injection and the sh-IGF2BP2 group exhibited a significantly higher number of liver metastatic nodules compared to the control group (Fig. S3B-C). In summary, our findings suggest that IGF2BP2 can inhibit liver metastasis of ccRCC cells in vivo.

Fig. 3.

Fig 3

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IGF2BP2 suppresses ccRCC metastasis in vivo. (A) Schematic representation of the liver metastasis mouse model established through spleen injection. The schematic was drawn by Biorender (https://www.biorender.com). (B) IGF2BP2 knockdown efficiency in Caki-1 cells upon lentiviral infection at three distinct MOI levels. (C) Representative in vivo imaging of mice. (D) Representative photographs of whole liver tissues. (E) Statistical analysis was conducted to assess liver metastases in mice. (F) Representative photographs of hematoxylin-eosin (H&E) staining of liver metastatic nodules. Bars, 200 μm; *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance.

IGF2BP2 upregulates CKB expression by enhancing CKB mRNA stability

To further investigate the molecular mechanism of IGF2BP2, we identified downstream target genes by integrating the data from three databases: differentially expressed genes after silencing IGF2BP2 in Caki-1 cells (Table S6) (log2FC<−0.5), enriched genes from IGF2BP2 RIP-seq in Caki-1 cells (Table S7), and differentially expressed genes in ccRCC based on GEPIA 2 datebase (log2FC<−1). We ultimately identified four potential downstream target genes regulated by IGF2BP2, including Creatine Kinase B (CKB), HomeoBox B9 (HOXB9), Transforming Growth Factor Beta Receptor 3 (TGFBR3), Actin Filament Associated Protein 1 Like 2 (AFAP1L2) (Fig. 4A-B). qRT-PCR and western blot assay confirmed that CKB mRNA and protein expression level was significantly decreased upon IGF2BP2 knockdown. Conversely, CKB mRNA and protein expression was significantly increased upon IGF2BP2 overexpression (Fig. 4C-F). However, HOXB9 and TGFBR3 mRNA levels were not significantly regulated by IGF2BP2 (Fig. S4A-B). The above results suggest that IGF2BP2 indeed regulates the CKB expression level. Consequently, we promptly investigated the mechanism underlying IGF2BP2-mediated CKB regulation. Given that IGF2BP2 can bind and affect the stability of downstream mRNA to regulate target genes expression level [27], [28], [29], we performed RIP experiments and confirmed the interaction between IGF2BP2 protein and CKB mRNA (Fig. 4G). Actinomycin D assay demonstrated that the half-life period of CKB mRNA was significantly lower upon IGF2BP2 knockdown than the control group in Caki-1 and RCC-JF cells (Fig. 4H). Above data suggest that IGF2BP2 can increase CKB expression by binding to CKB mRNA and enhancing its stability.

Fig. 4.

Fig 4

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IGF2BP2 upregulates CKB expression by enhancing CKB mRNA stability. (A) Volcano plot of differentially expressed protein-coding genes after the IGF2BP2 knockdown. (B) Screening strategy for target genes of IGF2BP2. (C-F) qRT-PCR and Western blot assay were performed to verify the regulation of CKB expression upon IGF2BP2 knockdown or overexpression in Caki-1 and RCC-JF cells. (G) RIP assay was employed to detect the relative enrichment of CKB in association with IGF2BP2 in Caki-1 cells. (H) qRT-PCR was used to assessed the stability of CKB mRNA after IGF2BP2 knockdown in Caki-1 and RCC-JF cells treated with actinomycin D. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance.

CKB suppresses ccRCC migration and invasion in vitro

To investigate the effect of CKB on the migration and invasion in ccRCC cells. Initially, through analysing GEPIA 2 databases, we identified that CKB was significantly down-regulated in ccRCC tissues (Fig. 5A). Next, we evaluated CKB mRNA expression in 61 paired ccRCC tissues and verified the significant downregulation of CKB in ccRCC tissues (P <0.01), with nearly 92 % (56/61) of these tissues displaying down-regulated expression (Fig. 5B). Consistently, CKB protein expression was down-regulated in 8 ccRCC tissues compared to adjacent tissues (Fig. 5C). The CKB gene is located on chromosome 14q32.33 and consists of seven exons [30]. CKB has been reported to exhibit abnormal expression in malignant tumors like prostatic cancer and breast cancer and play a pivotal role in tumorigenesis [31,32]. However, it's function has not been reported in ccRCC. To investigate the biological role of CKB in ccRCC, we designed siRNA targeting CKB. qPCR and western blot results demonstrated that CKB mRNA and protein levels was successfully knockdown (Fig. 5D). Transwell and wound healing assays showed that a significant enhancement in the migration and invasion capabilities of both ccRCC cell lines upon CKB knockdown (Fig. 5E-J). In summary, CKB exerts an inhibitory effect on the metastatic potential of ccRCC cells in vitro.

Fig. 5.

Fig 5

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CKB suppresses ccRCC migration and invasion in vitro. (A) GEPIA 2 database was utilized to analyze CKB mRNA expression level in ccRCC. (B-C) qRT-PCR and Western blot assay to verify CKB expression level in ccRCC tissues compared to adjacent tissues. (D) Knockdown efficiency of CKB were evaluated in Caki-1 and RCC-JF cells. (E-H)Cell migration and invasion assays were utilized to evaluate the influence of CKB knockdown on ccRCC cells migration capacity. (I-J) Wound healing assay was used to investigate the changes of migration ability of Caki-1 and RCC-JF cells after CKB knockdown. Bars, 100 μm; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance.

IGF2BP2 inhibits ccRCC migration and invasion by regulating the expression of CKB

To validate whether IGF2BP2 could affect ccRCC tumorigenesis by regulating CKB, we transfected IGF2BP2-overexpressing Caki-1 and RCC-JF cells with siRNAs targeting CKB or a negative control. Rescue assays confirmed that the inhibition of migration and invasion induced by overexpressing IGF2BP2 in ccRCC cells was significantly reversed by CKB knockdown (Fig. 6A-B). qRT-PCR and western blot assays demonstrated that IGF2BP2 overexpression significantly counteracted the decreased CKB expression induced by CKB knockdown (Fig. 6C-D). In summary, these findings demonstrate that IGF2BP2 might inhibit ccRCC cell metastasis by regulating CKB expression.

Fig. 6.

Fig 6

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IGF2BP2 suppresses ccRCC migration and invasion by regulating the expression of CKB. (A-B) Transwell migration rescue assay was used to detect the effect of co-transfection with IGF2BP2 overexpression vector and CKB knockdown siRNA on cell migration and invasion. (C-D) qRT-PCR and Western blot assay was used to detect the effect of co-transfection with IGF2BP2 overexpression and CKB knockdown on the CKB level in Caki-1 and RCC-JF. (E) Graphic abstract showing that IGF2BP2 regulates CKB expression and inhibits ccRCC metastasis by affecting the stability of CKB. The schematic was drawn by Biorender (https://www.biorender.com). Bars, 100 μm; *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance.

Discussion

RBPs play crucial roles in post-transcriptional gene regulation and have been implicated in various cellular processes, including tumorigenesis. Researches have demonstrated significant dysregulation of RBPs expression in ccRCC tumorigenesis [14]. Therefore, the identification of differentially expressed RBPs in ccRCC holds great significance for the potential development of therapeutic targets for tumors. In this study, we screened 55 RBPs with differential expression in ccRCC. Notably, IGF2BP2 was identified as one of the most significantly down-regulated RBPs in ccRCC. Numerous studies have reported IGF2BP2 as an up-regulated oncogene in cancer, such as breast cancer, colorectal cancer, glioma cancer, hepatocellular cancer, and lung cancer [19,[33], [34], [35], [36]]. However, we validated the significant down-regulation of IGF2BP2 in ccRCC tissues by qRT-PCR and western blot assays. Consistently, it was found to be significantly down-regulated in ccRCC through GEPIA 2 database and GSE14994 dataset [37]. But the expression of IGF2BP2 was not significantly correlated and consistent with clinical characterize in our current study, indicating the complexity of IGF2BP2 in the progression of ccRCC.

The biological functions of IGF2BP2 in tumors have been reported to possess various molecular mechanisms. For instance, IGF2BP2 as a m6A reader protein can bind to and stabilize ubiquitin-conjugating enzyme E2D1 (UBE2D1) mRNA, leading to promote the breast cancer progression [38]. IGF2BP2 is able to activate endothelial cells to promote angiogenesis and metastasis of lung adenocarcinoma [39]. IGF2BP2 promotes aerobic glycolysis and cell proliferation in PDAC by directly binding to and stabilizing GLUT1 mRNA in pancreatic cancer [40]. However, the biological role of IGF2BP2 in ccRCC is still elusive. In our previous study, we elucidated that IGF2BP2 directly interacted with circTNPO3, which involved in ccRCC migration inhibition [24]. Thus, the complex and diverse role of IGF2BP2 is worthy for further investigation. In this study, we found that IGF2BP2 exerts a suppressive impact on ccRCC cells metastatic potential rather than proliferation. In addition, IGF2BP2 has been found to be implicated in drug resistance and metabolism in carcinoma [23]. IGF2BP2 promotes aerobic glycolysis by stabilizing HK2 mRNA in glioblastoma [41]. IGF2BP2 can interact with LDHA mRNA, contributing to enhanced glycolytic metabolism and docetaxel resistance in prostate cancer [22]. Hence, the other functions of IGF2BP2 in ccRCC need to be further explored in the future.

To identify the target genes of IGF2BP2 in ccRCC, we intersected the results of RNA bound to IGF2BP2 from RIP-Seq, differentially expressed genes after silencing IGF2BP2, and differentially expressed genes in the TCGA database. Subsequently, a series of experiments confirmed that IGF2BP2 bound to CKB mRNA to enhance its stability, and then promoted the expression of CKB. Several studies have reported significant differences in CKB expression in malignancies such as prostate cancer and breast cancer, emphasizing its crucial role in tumor progression [31,32]. Nevertheless, the biological function and mechanism in ccRCC remain unclear. We observed a significant down-regulation of CKB expression in ccRCC tissues compared to adjacent tissues. CKB suppressed ccRCC migration and invasion and was required for IGF2BP2 to inhibit ccRCC metastasis in vitro. In fact, the regulation of IGF2BP2 on target mRNA is not always a stabilizing effect, but may also be an inhibitory effect. Our previous study found IGF2BP2 can interact with SERPINH1 mRNA and destabilize it [24]. Moreover, IGF2BP2 can regulate target gene expression as one of m6A readers [19,42,43]. Therefore, further investigation is needed to determine whether IGF2BP2 regulates CKB expression by recognizing its m6A-modification.

Conclusions

In summary, we identified the down-regulation of IGF2BP2 in ccRCC. Functionally, IGF2BP2 effectively suppressed the migration and invasion of ccRCC cells in vivo and in vitro. Mechanistically, IGF2BP2 upregulated CKB expression by binding to CKB mRNA and enhancing its mRNA stability (Fig. 6E). Our study provides new insights into the downstream regulatory mechanisms of IGF2BP2.

CRediT authorship contribution statement

Junwu Ren: Writing – original draft, Software, Methodology, Investigation, Data curation, Conceptualization. Bo Huang: Software, Methodology, Investigation, Funding acquisition. Wei Li: Validation, Funding acquisition. Yongquan Wang: Resources. Xiaojuan Pan: Validation, Investigation. Qiang Ma: Validation, Methodology. Yuying Liu: Investigation. Xiaolin Wang: Investigation. Ce Liang: Validation. Yuying Zhang: Validation, Investigation. Shimin Wang: Validation. Feifei Yang: Validation. Haiping Li: Validation. Hao Ning: Validation. Yan Jiang: Validation. Changhong Qin: Validation. Ai Ran: Investigation. Bin Xiao: Writing – review & editing, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Ethics approval

Patient tissue collection was approved by the Ethics Committee of the Southwest Hospital of Army Medical University, Chongqing (Approval No.: KY2020121). All animal experiments were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of Chongqing Medical University, Chongqing (Approval No.:2022013).

Funding

This study was supported by the National Natural Science Foundation of China (82373001, 82073254, 81872392), Chongqing Talents-Exceptional Young Talents Project (CQYC202005044, CSTC2021YCJH-BGZXM0094), Chongqing Natural Science Foundation Innovation and Development Joint Fund (CSTB2022NSCQ-LZX0043), Science and Technology Research Project of Chongqing Municipal Education Commission (KJZD-K202100405), Future Medical Youth Innovation Team Project of Chongqing Medical University (W0042), Graduate Tutor Team Construction Project of Chongqing (CQMUDSTD202210), Top Graduate Talent Cultivation Program of Chongqing Medical University (BJRC202105), Natural Science Foundation of Chongqing (CSTC2020JCYJ-MSXMX0337, CSTC2022JXJL120012).

Data availability

All data generated or analyzed during the course of this study are included in this article or are available from the corresponding author upon reasonable request.

Acknowledgments

We sincerely thank the laboratory of Professor Yongquan Wang, Department of Urology, Southwest Hospital, Army Medical University for providing tissue samples.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.101904.

Appendix. Supplementary materials

mmc1.docx (1.1MB, docx)
mmc2.xlsx (1.5MB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (1.1MB, docx)
mmc2.xlsx (1.5MB, xlsx)

Data Availability Statement

All data generated or analyzed during the course of this study are included in this article or are available from the corresponding author upon reasonable request.

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