IGF2BP2 promotes ovarian cancer growth and metastasis via upregulating CKAP2L protein expression in a m6A-dependent manner

doi:10.21203/rs.3.rs-2124570/v1

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IGF2BP2 promotes ovarian cancer growth and metastasis via upregulating CKAP2L protein expression in a m6A-dependent manner

https://doi.org/10.21203/rs.3.rs-2124570/v1

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Ovarian cancer (OC) is the second leading cause of gynecologic cancer death in women around the world. N6-methyladenosine (m⁶A) is the most abundant internal modification on eukaryotic RNA. Human insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), as an m⁶A reader, can enhance mRNA stability and promote translation by recognizing m⁶A modifications. Its carcinogenic effect has been demonstrated in colon cancer, hepatocellular carcinoma, pancreatic cancer and other tumors. Here, we demonstrated that there was widespread dysregulation of m⁶A modification in OC tissues. The m⁶A modification, mRNA and protein level of IGF2BP2 were significantly elevated in OC. Overexpression of IGF2BP2 facilitated OC cell proliferation, migration, invasion in vitro and accelerated tumor growth and metastasis in vivo. Mechanistically, CKAP2L was a target mRNA of IGF2BP2. Unlike previous studies, IGF2BP2 promoted CKAP2L translation depending on m⁶A modification rather than affect mRNA and protein stability. Knockdown of CKAP2L rescued the oncogenic effect of IGF2BP2 in OC cells. In conclusion, this study unveiled the oncogenic role of IGF2BP2 potentially through promoting the translation of CKAP2L in a m⁶A dependent manner.

Ovarian cancer

N6-methyladenosine

IGF2BP2

CKAP2L

translation

Ovarian cancer (OC) is the eighth leading cause of cancer-related death in women worldwide[1], with the highest mortality rate among gynecological cancers in the United States[2]. Due to the asymptomatic nature of OC, most patients are diagnosed at an advanced stage, with high recurrence rate and poor prognosis[3]. Although surgical treatment, chemotherapy and immunotherapy have made progress in recent years, the 5-year survival rate of OC is still less than 50%[4]. Therefore, it is necessary for us to study the pathogenesis of OC and search for effective therapeutic targets.

Since the launch of the Human Epigenome Project in 2003, epigenetic modifications of DNA and histones have been progressed in stages[5, 6]. In recent years, RNA epigenetic modifications have received increasing attention. As a major class of RNA epigenetic modifications, N6-methyladenosine (m⁶A) is the most abundant internal modification on eukaryotic mRNA[7]. m⁶A accounts for more than 60% of all RNA modifications, and about 25–33% of the mRNA contains at least one m⁶A modification[8–10]. m⁶A modification occurring in the consensus motif RRACH (R = A or G, H = A, C, or U) is a dynamic and reversible process. It is well known that m⁶A modification is catalyzed by m⁶A methyltransferase complex (METTL3, METTL14)[11], removed by demethylases (FTO, ALKBH5)[12, 13], and recognized by readers (YTH domain family, IGF2BPs, HnRNPs)[14–18].

Emerging evidence confirm that the m⁶A modification regulates multiple biological functions, such as RNA processing, nuclear export, translation, degradation and RNA-protein interactions[19–23]. Besides, the dysregulation of m⁶A modification is closely related to the initiation and development of several cancers, including pancreatic cancer, hepatocellular carcinoma and colorectal cancer[24–29]. As an m⁶A reader, IGF2BP2 usually modulates mRNA stability and promotes translation by recognizing m⁶A in the 3’UTR of mRNA[15], and its carcinogenic effects have been demonstrated in various cancers[30–34]. In OC, there are only few reports on the regulation of m⁶A modification in OC progression[35]. The differential m⁶A profile in OC has not been revealed, and the role and molecular mechanism of IGF2BP2 are still unclear.

Here, we demonstrated that there is a wide range of m⁶A modification disorder in OC. IGF2BP2 is abnormally upregulated in OC and plays an important role in the development and metastasis of OC. What’s more, we found that IGF2BP2 can promote CKAP2L translation depending on m⁶A modification rather than affect mRNA and protein stability.

Overview of the m⁶A methylation map in OC

To investigate the role of m⁶A modification in the development of OC, we performed Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) analysis in 3 cases of high-grade serous ovarian cancer (HGSOC) tissues and 3 cases of normal ovarian tissues (Figure 1A). Comparing with normal ovarian tissues, 2398 upregulated m⁶A peaks on 2102 genes and 2347 downregulated m⁶A peaks on 2308 genes were identified in HGSOC (Figure S1A). The m⁶A modifications were predominately located in protein-coding transcripts and enriched in the coding sequence (CDS) of mRNA or exon of non-coding RNA and the 3' UTR (Figure 1B-C). The distribution of m⁶A peaks was consistent in both groups of tissues. Next, we explored the different mRNA expressions in HGSOC. RNA expression profiles (the input results) showed that the expression of 1166 mRNA was downregulated, and that of 1429 mRNA was upregulated (Figure S1B). Combined analysis showed that 225 mRNA were significantly upregulated with hypermethylated m⁶A peaks (29%; hyper-up), 151 mRNA were upregulated with hypomethylated m⁶A peaks (19%; hypo-up), while 176 mRNA were significantly downregulated with hypermethylated m⁶A peaks (17%; hyper-down), 312 mRNA were downregulated with hypomethylated m⁶A peaks (35%; hypo-down) (Figure 1D). KEGG analysis revealed that these differentially expressed genes were enriched in several cancer-related pathways such as pathways in cancer, proteoglycans in cancer and Rap1 signaling pathway (Figure S1C). Finally, we analyzed the expression of genes encoding the classical m⁶A writers, readers and erasers. The results showed that the expression of WTAP, FTO and YTHDC2 were significantly decreased, while the expression of IGF2BP2 and IGF2BP3 were increased in HGSOC (Figure 1E). These results suggest that there were widespread m⁶A modification disorders in OC.

IGF2BP2 expression is increased in OC and associated with survival and the malignant behavior of OC patients

Specifically, we analyzed the 5 dysregulated m⁶A-regulating genes. We found that IGF2BP2 mRNA expression changed most significantly in OC (Fold change = 8.817) (Figure 1E). Then, we analyzed the expression of IGF2BP2 by using Gene Expression Profiling Interactive Analysis (GEPIA) website and online UALCAN tool. The results showed that both the mRNA and protein expression IGF2BP2 was highly expressed in ovarian cancer tissues (Figure 1F-G). And the OC patients with high IGF2BP2 expression showed shorter overall survival (OS) and progression free survival (PFS) as shown by Kaplan-Meier Plotter [Ovarian] (Figure S1D-E).

To validate the dysregulation of IGF2BP2 in ovarian cancer, we first analyzed the IGF2BP2 mRNA expression in 45 ovarian cancer tissues and 15 normal ovarian tissues by qRT-PCR, which showed that IGF2BP2 mRNA was significantly increased in ovarian cancer tissues (Figure 1H). Then we analyzed the protein expression of IGF2BP2 in 46 ovarian cancer patients by immunohistochemical (IHC). The results confirmed that the level of IGF2BP2 was significantly higher in OC than that in normal oviduct epithelial and adjacent tissues and was further upregulated in patients at FIGO stage III and IV versus FIGO stage I and II (Figure 1I-J). Moreover, the IGF2BP2 level was positively correlated with FIGO stage, pathological grade, lymph node metastasis and peritoneal metastasis, and patients with higher IGF2BP2 had shorter overall survival and recurrence free survival (Table 1, Figure 1K). Taken together, these results suggest that the m⁶A reader IGF2BP2 is highly expressed in OC and is associated with poor prognosis of OC patients.

Table 1

Correlation between IGF2BP2 level and clinicopathological features of ovarian cancer (OC)

Characteristics	The IHC score of IGF2BP2		Difference (P-value)
Characteristics	High	Low	Difference (P-value)
Age (years)
<54	12	11
³54	11	12	0.768
FIGO stage
I-II	8	18
III-IV	15	5	0.003
Histopathologic cell type
Low grade	1	6
High grade	22	17	0.044
Lymph node metastasis
Yes	9	3
No	14	20	0.04
Peritoneal implantation
Yes	17	5
No	6	18	P < 0.001
Ki67(%)
<50	9	11
³50	14	12	0.552
CA125
£35	3	8
>35	20	15	0.084

In vitro, the mRNA and protein expression of IGF2BP2 were also increased in ovarian cancer cell lines (SKOV3, A2780, OVCAR3 and ES2) as compared with normal ovarian epithelial cell line (IOSE80, Figure S1F-G). And the cell proliferation, migration and invasion capacity were significantly enhanced in IGF2BP2-overexpressing A2780 and SKOV3 cells, while weakened in IGF2BP2-silenced A2780 and SKOV3 cells (Figure S2).

IGF2BP2 promotes OC growth and metastasis in vivo

Then the effects of IGF2BP2 on ovarian cancer were further explored in vivo. As shown in Figure 2A, the tumor growth rate was significantly faster in IGF2BP2-overexpressing group than that in the control group. After 4 weeks, the subcutaneous tumors were resected and weighted, the results showed that tumors derived from the IGF2BP2-overexpressing group were substantially larger than those from control group and the average tumor weight was markedly increased in IGF2BP2-overexpressing group (Figure 2B-C). Hematoxylin and Eosin staining of the subcutaneous tumor tissue showed that the tumor cells in both groups had hyperchromatic nuclei and disorganized interstitial structure (Figure 2D), which was consistent with the histological characteristics of tumor cells, indicating that the xenograft model was successfully established. IHC analysis verified that IGF2BP2 was overexpressed in the IGF2BP2-overexpressing group, and the proliferation index was increased as shown by Ki-67 staining (Figure 2D-E). Next, an orthotopic model was used to examine whether IGF2BP2 could affect peritoneal metastasis of OC cells. The orthotopic tumor weight and volume both increased significantly in IGF2BP2-overexpressing group. Besides, the liver metastasis rate, spleen metastasis rate and abdominal cavity (except for liver and spleen) metastasis rate of IGF2BP2-overexpressing and control orthotopic tumor was 83.33% and 100%, 50% and 33.3%, 100% and 33.3%, respectively. And the average metastasis site in liver, spleen and abdominal cavity (except for liver and spleen) is 44.3 and 62, 1.16 and 1, 6.67 (distributed in peritoneum, intestinal epithelium and fallopian tube) 0.33 (all in peritoneum), respectively (Figure 2F-H). Collectively, these results demonstrate the oncogenic role of IGF2BP2 in OC in vivo.

Identification of IGF2BP2 targets by MeRIP-seq, IGF2BP2 RIP-seq and RNA-seq

To investigate the underlying mechanisms of IGF2BP2 in OC development, we first performed RNA Immunoprecipitation sequencing (RIP-Seq) with IGF2BP2 specific antibody in IGF2BP2-overexpressing SKOV3 cells. The results identified 2703 candidate binding partner of IGF2BP2, among which 2599 were mRNAs (Figure 3A-B). RNA-seq of IGF2BP2-overexpressing SKOV3 cells and control cells identified 372 upregulated genes and 1287 downregulated genes (Figure 3C), while m⁶A-RIP-seq of SKOV3 cells identified 3815 genes with m⁶A peaks (Figure 3D). The intersection from the above sequencing results and HGSOC-MeRIP-seq results revealed that there were 154 mRNAs with m⁶A modification were bound to IGF2BP2. Among them, only the IGF2BP2 mRNA expression was increased, 23 mRNAs were downregulated, while other 130 genes were unchanged (Figure 3E). As an m⁶A reader, IGF2BP2 usually enhances RNA stability so that increasing the total amount of RNA[15]. Considering the reported function of IGF2BP2, we speculated that IGF2BP2 may not function as a classical RNA stabilizer in OC cells.

To verify our guess, we first verified the influence of IGF2BP2 on mRNA and protein level of c-Myc, a classical downstream target of IGF2BP2[15, 30, 36-38]. Although with m⁶A modification and could interact with IGF2BP2 in both SKOV3 and A2780 cells (Figure S3A-B), the expression of c-Myc mRNA did not change when IGF2BP2 expression was altered and the RNA stability of c-Myc was similar in IGF2BP2-overexpressing and control cells (Figure S3C-D), indicating that IGF2BP2 may enhance the aggressive behavior of ovarian cancer cells through other mechanism.

It has been reported that m⁶A reader YTHDF1 and YTHDF3 can interact with m⁶A modified RNA and regulate their translation, without affecting the RNA abundance of its targets[22, 39]. Considering that 130 of the 154 mRNAs that contain m⁶A sites and bind with IGF2BP2 were unchanged (Figure 3E). Therefore, we speculated whether IGF2BP2 could promote the translation of target mRNAs without affecting RNA stability. Cytoskeleton-associated protein 2-like (CKAP2L), Zinc Finger Protein 20 (ZNF20), MET Proto-Oncogene (MET) and ERCC Excision Repair 6 Like (ERCC6L) were chosen as candidate target RNAs due to their high IGF2BP2 affinity and m⁶A peaks. As a classical target of IGF2BP2, c-Myc was also included as a candidate target RNA. We examined the protein level of the five candidate genes in IGF2BP2-overexpressing and deficient cells. Results showed that only the protein abundance of CKAP2L was significantly upregulated upon IGF2BP2-overexpressing and reduced after knockdown of IGF2BP2, while c-Myc protein level was decreased upon IGF2BP2-knockdown and unchanged upon IGF2BP2-overexpressing (Figure 3F-G). Consistent with the sequencing results, CKAP2L mRNA expression was not changed in both IGF2BP2-overexpressing cells and silencing cells (Figure 3H). Subsequently, m⁶A-RIP-qPCR and IGF2BP2-RIP-qPCR demonstrated that CKAP2L mRNA contain m⁶A modification and could interact with IGF2BP2 (Figure 3I-J). In addition, combined immunofluorescence and fluorescence in situ hybridization (FISH) confirmed the co-localization of IGF2BP2 protein and CKAP2L mRNA (Figure 3K). Finally, the stability of CKAP2L mRNA was not increased in IGF2BP2-overexpressing ovarian cancer cells (Figure 3L), indicating IGF2BP2 may target CKAP2L through RNA stability independent manner.

IGF2BP2 regulates the translation of CKAP2L in a m⁶A dependent manner without affecting its protein stability

The total protein level can be increased by two ways: increased protein synthesis or decreased protein degradation[40-42]. We first treated IGF2BP2-overexpressing and control SKOV3 cells with the protein synthesis inhibitor Cycloheximide. Western blotting showed that overexpression of IGF2BP2 had no significant effect on the protein stability of CKAP2L, excluding the possibility that IGF2BP2 inhibited the degradation of CKAP2L protein (Figure 4A). Then, we analyzed new protein synthesis of CKAP2L after overexpression of IGF2BP2. Compared to control cells, IGF2BP2-overexpressing cells displayed an increased L-homopropargylglycine signal, indicating that IGF2BP2 could promote the new protein synthesis(Figure 4B). To verify whether the effect was m⁶A dependent, we first used Sequence-based RNA Adenosine methylation Site Predictor (SRAMP), a powerful mammalian m⁶A site predictor and has successfully predicted the m⁶A sites of multiple mRNAs[43-46]. It indicated that CKAP2L mRNA had a high abundance of m⁶A modifications. Combined with the m⁶A peak of HGSOC MeRIP-seq, we selected 4 loci with the highest confidence to construct the CKAP2L-m⁶A-MT vector (Figure 4C). Western blotting revealed that m⁶A modification site mutation could reduce the protein level of CKAP2L, suggesting that IGF2BP2 may regulated the protein synthesize of CKAP2L in a m⁶A dependent manner (Figure 4D). On the other side, the KH3-4 di-domain is indispensable for the m⁶A recognition and binding of IGF2BP2 while KH1-2 plays an accessory role[15, 47-49]. After introducing two point-mutations in KH3-4 di-domain of IGF2BP2 (Figure 4E), RIP-qPCR by using the antibody against IGF2BP2 showed that the mutations in KH3-4 di-domain significantly reduced the interaction between IGF2BP2 and CKAP2L mRNA (Figure 4F). Simultaneously, Western blotting showed that only IGF2BP2 wide-type vector could promote the translation of CKAP2L (Figure 4G). These data indicate that m⁶A binding domain is essential for IGF2BP2 to bind CKAP2L and regulate its protein level.

CKAP2L knockdown attenuates the oncogenic role of IGF2BP2

CKAP2L locates on chromosome 2, encodes a protein that is vital for cell division in neural progenitor cells and plays an oncogenic role in glioblastoma and lung adenocarcinoma[50, 51]. Recently, Garofalo M et al. found that CKAP2L promotes the progression of non-small cell lung cancer by regulating transcriptional elongation[52]. However, the role of CKAP2L has not been reported in OC. First, we examined the RNA expression of CKAP2L by GEPIA, which showed that the RNA expression of CKAP2L was significantly upregulated in OC samples (Figure 5A). Meanwhile, CKAP2L mRNA expression was negatively correlated with overall survival of OC patients (Figure 5B). Besides, CKAP2L expression was positively associated the expression of IGF2BP2 (Figure 5C). IHC analysis of CKAP2L in the subcutaneous xenograft tumor indicated that CKAP2L was increased in the IGF2BP2-overexpressing group (Figure 5D-E).

Besides, silencing of CKAP2L decreased the proliferation, invasion and migration capacity of ovarian cancer cells and reversed the oncogenic effect of IGF2BP2 on cell proliferation, migration and invasion (Figure 5F-J). These results suggest that CKAP2L mRNA is a crucial downstream target of IGF2BP2 in promoting ovarian cancer progression.

By using the orthotopic tumor model, we demonstrated that overexpression of IGF2BP2 significantly promoted the metastasis of ovarian cancer cells, especially the abdominal cavity (except the liver and spleen) metastasis, which is frequently happened in ovarian cancer patients. This is also consistent with the result that IGF2BP2 expression is positively associated with the FIGO stage, lymph node metastasis and peritoneal implantation of ovarian cancer patients. The biology of ovarian cancer differs from that of hematogenously metastasizing tumors because ovarian cancer cells primarily disseminate within the peritoneal cavity and are superficially invasive[53]. Therefore, in advanced stages peritoneal metastases is often present. However, due to absence of symptoms of EOC in early stages, approximately 80% of patients present with advanced stages[54]. The five-year relative survival for OC is only 49% and the chance of recurrent disease in advanced-stage OC is approximately 75%. Moreover, in relapsed patients, peritoneal recurrences are frequently found, causing ominous symptoms such as ascites and bowel ileus, ultimately leading to death[55]. Consequently, we hypothesized that IGF2BP2 may serve as an important therapeutic target to inhibit ovarian cancer metastasis or recurrence.

Before identified as a m⁶A reader, studies have reported that IGF2BP2 could promote the translation of its downstream target RNA. For example, Dai et al. found that IGF2BP2 could promote translation of downstream mRNAs through internal ribosomal entry sites rather than recruitment of eIF-4E in rhabdomyosarcoma cells[56]. Li et al. found that IGF2BP2 promoted the level of proteins such as c-Myc without increasing their mRNA expression in skeletal muscle cells[57]. More recently, Regué et al. showed that IGF2BP2 bound to the m⁶A modification sites of PDX1 mRNA, promoted PDX1 mRNA translation without affecting its mRNA expression and enhanced PDX1 polypeptide stability via the IGF2-AKT-GSK3b pathway in pancreatic β-cells[25]. These papers support our conclusion that IGF2BP2 can promote RNA translation without affecting the stability of the target RNA by binding to the m⁶A sites of the downstream target RNA.

In our study, CKAP2L was the major target of IGF2BP2 in regulating ovarian cancer progression, and we noticed that the protein level of c-Myc was decreased upon IGF2BP2-knockdown, but unchanged upon IGF2BP2-overexpressing, indicating that c-Myc was also a target of IGF2BP2 in ovarian cancer under some conditions. Besides, whether there are other target RNAs of IGF2BP2 and the mechanism through which IGF2BP2 promotes downstream RNA translation after binding to the m⁶A modification sites need to be further investigated.

However, MeRIP-seq showed that IGF2BP2 mRNA was modified by m⁶A in the CDS and 3’UTR region, and its m⁶A modification in 3’UTR was significantly increased in HGSOC tissues. We hypothesized that the increased m⁶A modifications recruited more m⁶A readers, which improved IGF2BP2 mRNA stability and promotes translation, ultimately leading to increased IGF2BP2 mRNA and protein level. However, small interfering RNA mediated knocking down of METTL3/14 and ALKBH5/FTO did not affect IGF2BP2 protein level (data not shown). As reported before, the IGF2BP2 expression was regulated through different mechanisms, such as miRNA mediated degradation[58], lncRNA mediated RNA stability[59] and promoter mediated transcription[60]. The specific mechanism that mediated the increased m⁶A modification and expression of IGF2BP2 needs to be further explored in the future.

In summary, IGF2BP2 is a highly expressed and oncogenic gene in OC cells. The expression of IGF2BP2 was positively correlated with the FIGO stage, pathological grade, lymph node metastasis and peritoneal metastasis of ovarian cancer. This suggests that IGF2BP2 is a promising new therapeutic target for OC. The role of IGF2BP2 in OC is different from that of other tumors, which provides a new theoretical basis to reveal the complex regulatory network of tumors.

Human Tissue samples

Ovarian cancer tissues and normal ovarian tissues were collected from the Women’s Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital). All samples were confirmed by another pathologist, rapidly frozen with liquid nitrogen and stored at -80°C. All of the patients signed informed consent forms. This study of patient specimens was approved by the Ethical Committee of Women’s Hospital of Nanjing Medical University. Clinical and pathological information for ovarian cancer patients is shown in the Supplementary table 1.

Cell culture

Human ovarian epithelial cancer cell line A2780 and OVCAR3 were purchased from Shanghai Mingjin Biology Co., Ltd.; human ovarian epithelial cancer cell line SKOV3 and human ovarian clear cell cancer cell line ES-2 were purchased from National Collection of Authenticated Cell Cultures; and human normal ovarian epithelial cell line IOSE80 was donated by Ms. Li Jing of the General Hospital of the Eastern Theater Command. SKOV3 was cultured in McCoy’s 5A medium (KeyGEN, China) supplemented with 10% FBS (Gibco, USA). OVCAR3 was cultured in RPMI-1640 medium (KeyGEN, China) supplemented with 20% FBS (Gibco, USA). A2780, ES-2, and IOSE80 were cultured in DMEM medium (KeyGEN, China) supplemented with 10% FBS (Gibco, USA). All the cells were supplemented with 1% Pen/Strep (Gibco, USA) and maintained in 5% CO₂cell culture incubator at 37℃.

Plasmids and siRNA

The CDS of IGF2BP2 was linked to the CMV promoter in pEZ-M98 vector to construct the IGF2BP2-overexpressing plasmid. IGF2BP2 KH3-4 mutant plasmid was constructed by mutating GxxG to GDDG in the IGF2BP2 KH3-4 domains. The CDS and 3’UTR of CKAP2L was linked to the CMV promoter in pVAX1 vector to construct the CKAP2L m⁶A wild type plasmid. CKAP2L m⁶A mutant plasmid was constructed by mutating A to G at positions 1260, 2369, 3332 and 3473 on CKAP2L mRNA. All plasmids were purchased from Nanjing Genebay Biotech Co., Ltd.

Small interfering RNAs (siRNAs) targeting IGF2BP2 and CKAP2L were synthesized by Guangzhou RiboBio Co., Ltd. The related sequences of siRNAs were as follows: si-IGF2BP2-1: 5’-CATGCCGCATGATTCTTGA-3’, si-IGF2BP2-2: 5’-GAACGAACTGCAGAACTTA-3’, si-CKAP2L: 5’-GGTGTACCTTCTAATGAAA-3’. The plasmids and siRNAs were transfected using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.

Stable cell line establishment

The IGF2BP2-overexpressing and control lentiviral were constructed and packaged by Nanjing Genebay Biotech Co., Ltd. Briefly, the IGF2BP2-overexpressing and control vector were constructed in the PLVX vector, and the vector as well as the assistant plasmid pSPAX2 and pMD2G were transfected into HEK293T cells to package the lentiviral particles. The lentiviral particles were then harvest and purified. The titer of IGF2BP2-overexpressing lentivirus and control lentivirus were 1.85 × 10⁸ TU/ml and 2.99 × 10⁸ TU/ml, respectively. SKOV3 and A2780 cells at 30% density were infected with IGF2BP2-overexpressing lentivirus and control lentivirus. About 72 hours after infection, puromycin (P8833, Sigma) was added to the culture medium to select the infected cells.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells and tissues using GeneJET RNA Purification Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instruction. For qRT-PCR, RNA was reverse transcribed to cDNA by using HiScript III RT SuperMix for qPCR Kit (Vazyme, China). qRT-PCR was subsequently conducted using an Applied Biosystems ViiA™ 7 DX machine (Thermo Fisher Scientific, USA). The primers used were shown in Supplementary table 2.

Methylated RNA immunoprecipitation sequencing

Total RNAs were extracted from the OC tissues or cells. An m⁶A-specific antibody (Sigma-Aldrich, no ABE572) was used to immunoprecipitate RNA. m⁶A RNA-seq service was provided by Shanghai Biotechnology Corporation (Shanghai, China). Briefly, m⁶A RNA immunoprecipitation was performed with the GenSeqTM m⁶A-MeRIP Kit (GenSeq Inc, Malaysia) following the manufacturer’s instructions. Both the input samples without immunoprecipitation and the m⁶A IP samples were used for RNA-seq library generation. The library quality was evaluated with Bioptic Qsep100 Analyzer (Bioptic lnc, China). Library sequencing was performed on illumina NovaSeq instrument with 150bp paired-end reads.

RNA immunoprecipitation sequencing and RIP-qPCR

RIP assays were performed using the Magna RIP RNA-binding Protein Immunoprecipitation Kit (17-700, Millipore) according to the manufacturer’s instructions. Briefly, cells were washed twice with PBS, collected and then the pellet was resuspended in complete RIP lysis buffer. Magnetic beads coated with 5 μg of antibody of IGF2BP2 (11601-1-AP, Proteintech) or negative control antibody (17–700, Millipore) were incubated with prepared cell lysates overnight at 4 °C, respectively. Then, the RNA-protein complexes were washed 6 times with wash buffer and incubated with proteinase K digestion buffer. RNA was finally extracted by phenol-chloroform RNA extraction methods. For qRT-PCR, RNA was reverse transcribed to cDNA by using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). For sequencing, cDNA libraries were produced by VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina® (Vazyme, NR603) and sequenced on Illumina HiSeq X Ten platform.

High-throughput RNA sequencing (RNA-seq)

Total RNA was isolated from IGF2BP2-overexpressing SKOV3 cells and control cells using TRIzol Reagent (Life technologies, USA). The sequencing libraries were generated using the TruSeq Stranded Total RNA Library Prep Kit from Illumina according to the manufacturer’s instructions. The samples were paired-end sequenced with a read length of 100 bp on an Illumina HiSeq 2500 sequencer. The process of sequencing was controlled by Illumina Data collection software. The library construction and sequencing were performed at Shanghai Biotechnology Corporation (Shanghai, China).

Western blotting

Cells were washed twice with PBS and lysed with RIPA (Servicebio, China) and 1% PMSF (Beyotime, China) on ice for 30 minutes. The lyses was collected in a centrifuge tube and centrifuged at 12000 rpm, 4℃ for 30min. Proteins were fractionated by SDS-PAGE, transferred onto PVDF membranes, blocked in 5% nonfat milk and then blotted with specific antibodies. Antibodies used were as follows: anti-β-actin (1:500, Santa Cruz), anti-IGF2BP2 (1:1500, Cell Signaling Technology), anti-CKAP2L (1:1000, Proteintech), anti-ERCC6L (1:1000, Proteintech), anti-ZNF20 (1:1000, Signalway Antibody), anti-MET (1:1000, Proteintech), anti-c-Myc (1:1000, Proteintech) , Goat anti-Mouse IgG-HRP (1:5000, Biosharp), Goat anti-Rabbit IgG-HRP (1:5000, Biosharp).

Hematoxylin and eosin staining and Immunohistochemistry

Human OC, fallopian tube and nude mice xenograft tumor tissues paraffin embedded sections were deparaffinized and rehydrated in succession. Sections were stained with hematoxylin and eosin. Immunohistochemistry was performed by using Anti-mouse/rabbit Immunohistochemistry Detection Kit (Proteintech, PK10006). Antibodies used were as follows: anti-IGF2BP2 (1:200, Proteintech), anti-CKAP2L (1:200, Proteintech), anti-Ki-67 (1:4000, Proteintech). Cells were viewed and photographed with a microscope. The score for staining was assessed on the staining intensity and the percentage of positive cells identified by Image J Immunohistochemistry Profiler. The intensity of the staining cell was graded as 0 (negative), 1 (low positive), 2 (postive), 3 (high positive). The final score was determined by multiplying the score for staining intensity with the percentage of positive cells.

Cell proliferation assay

For colony formation assay, about 2 × 10³cells were seeded into each well of six well plates and the medium were refreshed every 3 days. After 7 days, colonies were fixed with 4% paraformaldehyde (Biosharp, China) for 15min, stained with 0.1% crystal violet (Biosharp, China) for 15min and washed with ddH₂O.

Transwell migration and invasion assays

The migration and invasion assays were conducted using the transwell (Corning, USA) according to the protocol described before. 5 × 10⁴cells were seeded in the upper chambers with 200μL serum-free medium. 0.6 ml culture media with 20% FBS were added to the lower chamber. For invasion assay, 60μl Matrigel (1:7, Corning, USA) was prepared in the upper chamber. After 24~72h incubation, cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and washed with ddH₂O. Gently wipe off the remaining Matrigel and cells on the upper chamber of the Transwell, migrated or invaded cells were imaged and counted under a microscope.

Subcutaneous and orthotopic ovarian xenograft tumor studies

All mice used in this study were purchased from GemPharmatech (Nanjing, China). Female nude mice, aged 6 weeks (weighing approximately 20 g), were housed and cared for at the animal center of Nanjing Agricultural University.

To establish subcutaneous xenograft tumor model, 14 healthy female Balb/c nude mice were chosen and assigned to two groups: control group (injected with control SKOV3 cells) and ov-IGF2BP2 group (injected with IGF2BP2-overexpressing SKOV3 cells). 9×10⁶ cells suspended in 100ul PBS were subcutaneous injected into the right armpit of each mouse. Tumor volume was measured every 3 days. 3 weeks later, all mice were sacrificed and tumor weight was measured.

To establish orthotopic xenograft tumor model, 12 healthy female Balb/c nude mice were chosen and assigned to two groups. Nude mice were anesthetized by isoflurane inhalation, and after disinfecting the skin with iodophor, a 5 mm incision was made in the skin and abdominal wall, parallel and ventral to the spine, midway and between the last rib and the iliac crest. After pulling out the ovary, the cell suspensions (10 mL) containing 1 ´ 10⁶ IGF2BP2-overexpressing or control SKOV3 cells, were inoculated inserting the needle at the junction between the bursa and the fat pad. Ovary was put back in place, and if no bleeding was noted, the incision on the muscle layer and body wall was closed separately.

Fluorescence in situ hybridization (FISH) and Immunofluorescence

The location of CKAP2L mRNA was detected by FISH according to the instruction of Fluorescent In Situ Hybridization Kit (C10910, RiboBio). First, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Then, cells were incubated with prehybridization solution followed by hybridization solution containing 0.5 μM probe and washed with washing solution. Finally, nucleus was stained with DAPI.

The location of IGF2BP2 protein was detected by Immunofluorescence. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% TritonX-100, blocked in 5% goat serum and incubated with IGF2BP2 antibody (ab128175, Abcam) at 4℃ overnight. Then, cells were washed with PBS, incubated with Alexa Fluor Plus 647 goat anti-mouse IgG and stained with DAPI.

RNA and Protein stability assays

To evaluate RNA stability, medium of IGF2BP2-overexpressing SKOV3 cells and control cells were added with 5 ug/ml Actinomycin D (A9415, Sigma). Cells were collected at 0 min, 30 min, 60 min and 90 min to detect c-Myc mRNA expression, and collected at 0 h, 2 h, 4 h and 8 h to detect CKAP2L mRNA expression. RT-qPCR assay was used to detect mRNA expression in both groups.

To evaluate protein stability, medium of IGF2BP2-overexpressing SKOV3 cells and control cells were added with 20 ug/ml Cycloheximide (M4879, AbMole). The control group received constant volume of DMSO (Sigma-Aldrich, USA). Cells were collected at the specified time (0h, 4h, 8h, 12h). Western blotting assay was used to detect the level of CKAP2L protein in both groups.

Nascent protein synthesis assay

Nascent protein synthesis was detected by using Click-iT L-homopropargylglycine Alexa Fluor Protein Synthesis Assay Kits (C10428, Invitrogen) according to the manufacturer’s recommendations. Briefly, cells were cultured in L-methionine-free medium with L-homopropargylglycine at 37℃ for 30 min. Then, cells were washed once with PBS, fixed by 3.7% formaldehyde in PBS followed by a permeabilization step using 0.5% Triton X-100 for 20 min. Subsequently, Click-iT reaction cocktail mix was added with incubation for 30 min protected from light and washed with Click-iT reaction rinse buffer. Finally, nucleus was stained with high-content screening Nuclear Mask Blue Stain working solution for another 30 min and proceeded to imaging and analysis.

Data and software availability

Gene Expression Profiling Interactive Analysis (GEPIA) was used to analyze the gene expression in TCGA and GTEx databases. The University of Alabama at Birmingham CANcer data analysis Portal (UALCAN) was used to analyze the protein level in Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. The m⁶A sites of CKAP2L mRNA were predicted online using the SRAMP website.

Statistical Analysis

All the analyses were performed by SPSS (Statistical Package for the Social Sciences) version 26.0 (Chicago, USA). Unless otherwise noted, the data are expressed as mean ± SD. The significance of difference was evaluated using Student’s t test. The Kaplan–Meier method and the Gehan-Breslow-Wilcoxon test were applied to estimate overall survival. The correlation between IGF2BP2 levels and clinical characteristics of OC tissues was analyzed by chi-square test. Two-way ANOVA was used to analyze the difference in the growth curves of subcutaneous xenografts in nude mice. P values < 0.05 were considered significant. β-Actin was used as the loading control. The relative expression level was normalized to β-Actin.

OC: Ovarian cancer, m⁶A: N6-methyladenosine, IGF2BP2: insulin-like growth factor 2 mRNA-binding protein 2, MeRIP-seq: Methylated RNA Immunoprecipitation Sequencing, HGSOC: high-grade serous ovarian cancer, CDS: coding sequence, OS: overall survival, PFS: progression free survival, IHC: immunohistochemical, RIP-seq: RNA Immunoprecipitation sequencing, CKAP2L: Cytoskeleton-associated protein 2-like, ZNF20: Zinc Finger Protein 20, MET: MET Proto-Oncogene, ERCC6L: ERCC Excision Repair 6 Like, FISH: fluorescence in situ hybridization, SRAMP: Sequence-based RNA Adenosine methylation Site Predictor, qRT-PCR: RNA extraction and quantitative real-time PCR, RNA-seq: High-throughput RNA sequencing, GEPIA: Gene Expression Profiling Interactive Analysis, TCGA: The Cancer Genome Atlas, GTEx: The Genotype-Tissue Expression, UALCAN: The University of Alabama at Birmingham CANcer data analysis Portal, CPTAC: Clinical Proteomic Tumor Analysis Consortium

Author’s Contribution Acknowledgements

J. X. and X.M. J. conceived, designed, supervised the study and revised the manuscript. Y. Q. S., performed the experiment, analyzed the data and wrote the manuscript. Y. S. and X. Y. X. performed the experiment and analyzed the data. Z. G., X. Y. C. and X. C. contributed to bioinformatic analysis and animal experiments. L.L. G. provided clinical samples and conceptual advise. All authors read and approved the final submitted manuscript.

Conflict of interests

The authors declare that there are no conflicts of interest.

Acknowlegements

This work was supported by Jiangsu Provincial Key Research and Development Program (No. BE2020753 to J.X. and No. BE2019621 to X.M. J.) and “333 Project” of Jiangsu Province (J. X. and X. M. J.).

Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.
Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022;72(1):7-33.
Lheureux S, Braunstein M, Oza AM. Epithelial ovarian cancer: Evolution of management in the era of precision medicine. CA Cancer J Clin. 2019;69(4):280-304.
Lheureux S, Gourley C, Vergote I, Oza AM. Epithelial ovarian cancer. The Lancet. 2019;393(10177):1240-53.
Esteller M. The necessity of a human epigenome project. Carcinogenesis. 2006;27(6):1121-5.
Eckhardt F, Beck S, Gut I, Berlin K. Future potential of the Human Epigenome Project. Expert review of molecular diagnostics. 2004;4(5):609-18.
Desrosiers R, Friderici K, Rottman F. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Natl Acad Sci U S A. 1974;71(10):3971-5.
Zaccara S, Ries RJ, Jaffrey SR. Reading, writing and erasing mRNA methylation. Nat Rev Mol Cell Biol. 2019;20(10):608-24.
Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 2012;149(7):1635-46.
Zhang H, Shi X, Huang T, Zhao X, Chen W, Gu N, et al. Dynamic landscape and evolution of m6A methylation in human. Nucleic Acids Res. 2020;48(11):6251-64.
Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014;10(2):93-5.
Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011;7(12):885-7.
Zheng G, Dahl J, Niu Y, Fedorcsak P, Huang C, Li C, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol cell. 2013;49(1):18-29.
Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014;505(7481):117-20.
Huang H, Weng H, Sun W, Qin X, Shi H, Wu H, et al. Recognition of RNA N(6)-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat Cell Biol. 2018;20(3):285-95.
Alarcon CR, Goodarzi H, Lee H, Liu X, Tavazoie S, Tavazoie SF. HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events. Cell. 2015;162(6):1299-308.
Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature. 2015;518(7540):560-4.
Bokar J, Shambaugh M, Polayes D, Matera A, Rottman F. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA (New York, NY). 1997;3(11):1233-47.
Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, Sun BF, et al. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol Cell. 2016;61(4):507-19.
Roundtree IA, Luo GZ, Zhang Z, Wang X, Zhou T, Cui Y, et al. YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs. Elife. 2017;6.
Shi H, Wang X, Lu Z, Zhao BS, Ma H, Hsu PJ, et al. YTHDF3 facilitates translation and decay of N(6)-methyladenosine-modified RNA. Cell Res. 2017;27(3):315-28.
Liu T, Wei Q, Jin J, Luo Q, Liu Y, Yang Y, et al. The m6A reader YTHDF1 promotes ovarian cancer progression via augmenting EIF3C translation. Nucleic Acids Res. 2020;48(7):3816-31.
Li J, Xie H, Ying Y, Chen H, Yan H, He L, et al. YTHDF2 mediates the mRNA degradation of the tumor suppressors to induce AKT phosphorylation in N6-methyladenosine-dependent way in prostate cancer. Mol Cancer. 2020;19(1):152.
Xu X, Yu Y, Zong K, Lv P, Gu Y. Up-regulation of IGF2BP2 by multiple mechanisms in pancreatic cancer promotes cancer proliferation by activating the PI3K/Akt signaling pathway. J Exp Clin Cancer Res. 2019;38(1):497.
Regue L, Zhao L, Ji F, Wang H, Avruch J, Dai N. RNA m6A reader IMP2/IGF2BP2 promotes pancreatic beta-cell proliferation and insulin secretion by enhancing PDX1 expression. Mol Metab. 2021;48:101209.
Wang M, Yang Y, Yang J, Yang J, Han S. circ_KIAA1429 accelerates hepatocellular carcinoma advancement through the mechanism of mA-YTHDF3-Zeb1. Life Sci. 2020;257:118082.
Chen Y, Peng C, Chen J, Chen D, Yang B, He B, et al. WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1. Mol Cancer. 2019;18(1):127.
Shen C, Xuan B, Yan T, Ma Y, Xu P, Tian X, et al. m(6)A-dependent glycolysis enhances colorectal cancer progression. Mol Cancer. 2020;19(1):72.
Wang Q, Chen C, Ding Q, Zhao Y, Wang Z, Chen J, et al. METTL3-mediated m(6)A modification of HDGF mRNA promotes gastric cancer progression and has prognostic significance. Gut. 2020;69(7):1193-205.
Hu C, Liu T, Han C, Xuan Y, Jiang D, Sun Y, et al. HPV E6/E7 promotes aerobic glycolysis in cervical cancer by regulating IGF2BP2 to stabilize m(6)A-MYC expression. Int J Biol Sci. 2022;18(2):507-21.
Li T, Hu PS, Zuo Z, Lin JF, Li X, Wu QN, et al. METTL3 facilitates tumor progression via an m(6)A-IGF2BP2-dependent mechanism in colorectal carcinoma. Mol Cancer. 2019;18(1):112.
Pu J, Wang J, Qin Z, Wang A, Zhang Y, Wu X, et al. IGF2BP2 Promotes Liver Cancer Growth Through an m6A-FEN1-Dependent Mechanism. Front Oncol. 2020;10:578816.
Sa R, Liang R, Qiu X, He Z, Liu Z, Chen L. Targeting IGF2BP2 Promotes Differentiation of Radioiodine Refractory Papillary Thyroid Cancer via Destabilizing RUNX2 mRNA. Cancers (Basel). 2022;14(5).
Han L, Lei G, Chen Z, Zhang Y, Huang C, Chen W. IGF2BP2 Regulates MALAT1 by Serving as an N6-Methyladenosine Reader to Promote NSCLC Proliferation. Front Mol Biosci. 2021;8:780089.
Guo J, Zheng J, Zhang H, Tong J. RNA m6A methylation regulators in ovarian cancer. Cancer cell international. 2021;21(1):609.
Wang Y, Lu JH, Wu QN, Jin Y, Wang DS, Chen YX, et al. LncRNA LINRIS stabilizes IGF2BP2 and promotes the aerobic glycolysis in colorectal cancer. Mol Cancer. 2019;18(1):174.
Ye M, Dong S, Hou H, Zhang T, Shen M. Oncogenic Role of Long Noncoding RNAMALAT1 in Thyroid Cancer Progression through Regulation of the miR-204/IGF2BP2/m6A-MYC Signaling. Mol Ther Nucleic Acids. 2021;23:1-12.
Li J, Gao X, Zhang Z, Lai Y, Lin X, Lin B, et al. CircCD44 plays oncogenic roles in triple-negative breast cancer by modulating the miR-502-5p/KRAS and IGF2BP2/Myc axes. Mol Cancer. 2021;20(1):138.
Chang G, Shi L, Ye Y, Shi H, Zeng L, Tiwary S, et al. YTHDF3 Induces the Translation of mA-Enriched Gene Transcripts to Promote Breast Cancer Brain Metastasis. Cancer cell. 2020;38(6):857-71.e7.
Alber AB, Suter DM. Dynamics of protein synthesis and degradation through the cell cycle. Cell Cycle. 2019;18(8):784-94.
Hanna J, Guerra-Moreno A, Ang J, Micoogullari Y. Protein Degradation and the Pathologic Basis of Disease. Am J Pathol. 2019;189(1):94-103.
Rothman S. How is the balance between protein synthesis and degradation achieved? Theor Biol Med Model. 2010;7:25.
Zhou Y, Zeng P, Li YH, Zhang Z, Cui Q. SRAMP: prediction of mammalian N6-methyladenosine (m6A) sites based on sequence-derived features. Nucleic Acids Res. 2016;44(10):e91.
Zhao Q, Zhao Y, Hu W, Zhang Y, Wu X, Lu J, et al. m6A RNA modification modulates PI3K/Akt/mTOR signal pathway in Gastrointestinal Cancer. Theranostics. 2020;10(21):9528-43.
Yao F, Zhao C, Zhong F, Qin T, Wen F, Li M, et al. m(6)A Modification of lncRNA NEAT1 Regulates Chronic Myelocytic Leukemia Progression miR-766-5p/CDKN1A Axis. Front Oncol. 2021;11:679634.
Ai Y, Liu S, Luo H, Wu S, Wei H, Tang Z, et al. METTL3 Intensifies the Progress of Oral Squamous Cell Carcinoma via Modulating the m6A Amount of PRMT5 and PD-L1. J Immunol Res. 2021;2021:6149558.
Hollingworth D, Candel AM, Nicastro G, Martin SR, Briata P, Gherzi R, et al. KH domains with impaired nucleic acid binding as a tool for functional analysis. Nucleic Acids Res. 2012;40(14):6873-86.
Nielsen F, Nielsen J, Kristensen M, Koch G, Christiansen J. Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domains. J Cell Sci. 2002;115:2087-97.
Doyle G, Betz N, Leeds P, Fleisig A, Prokipcak R, Ross J. The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins. Nucleic Acids Res. 1998;26(22):5036-44.
Li YF, Tsai WC, Chou CH, Huang LC, Huang SM, Hueng DY, et al. CKAP2L Knockdown Exerts Antitumor Effects by Increasing miR-4496 in Glioblastoma Cell Lines. Int J Mol Sci. 2020;22(1).
Xiong G, Li L, Chen X, Song S, Zhao Y, Cai W, et al. Up-regulation of CKAP2L expression promotes lung adenocarcinoma invasion and is associated with poor prognosis. Onco Targets Ther. 2019;12:1171-80.
Monteverde T, Sahoo S, La Montagna M, Magee P, Shi L, Lee D, et al. CKAP2L Promotes Non-Small Cell Lung Cancer Progression through Regulation of Transcription Elongation. Cancer Res. 2021;81(7):1719-31.
Lengyel E. Ovarian cancer development and metastasis. The American journal of pathology. 2010;177(3):1053-64.
Radu M, Prădatu A, Duică F, Micu R, Creţoiu S, Suciu N, et al. Ovarian Cancer: Biomarkers and Targeted Therapy. Biomedicines. 2021;9(6).
Rakina M, Kazakova A, Villert A, Kolomiets L, Larionova I. Spheroid Formation and Peritoneal Metastasis in Ovarian Cancer: The Role of Stromal and Immune Components. International journal of molecular sciences. 2022;23(11).
Dai N, Rapley J, Angel M, Yanik MF, Blower MD, Avruch J. mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry. Genes Dev. 2011;25(11):1159-72.
Li Z, Gilbert JA, Zhang Y, Zhang M, Qiu Q, Ramanujan K, et al. An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis. Dev Cell. 2012;23(6):1176-88.
Ma Y, Shi B, Guo J, Liu J, Yang X, Xin R, et al. microRNA-320b suppresses HNF4G and IGF2BP2 expression to inhibit angiogenesis and tumor growth of lung cancer. Carcinogenesis. 2021;42(5):762-71.
Wang Y, Lu J, Wu Q, Jin Y, Wang D, Chen Y, et al. LncRNA LINRIS stabilizes IGF2BP2 and promotes the aerobic glycolysis in colorectal cancer. Molecular cancer. 2019;18(1):174.
Bai J, Yokomizo-Nakano T, Kubota S, Sun Y, Kanai A, Iimori M, et al. Overexpression of Hmga2 activates Igf2bp2 and remodels transcriptional program of Tet2-deficient stem cells in myeloid transformation. Oncogene. 2021;40(8):1531-41.

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IGF2BP2 promotes ovarian cancer growth and metastasis via upregulating CKAP2L protein expression in a m6A-dependent manner

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