RBM15B promotes hepatocellular carcinoma progression via IGF2BP1-mediated ITSN2 mRNA stabilization.

Introduction
Globally, hepatocellular carcinoma (HCC) is a major cause of cancer-related death and a leading cause of morbidity and mortality in patients with chronic liver disease and cirrhosis. HCC is the third most common cause for cancer-related death worldwide in 2022 (Sung et al. 2021). HCC is usually diagnosed at an advanced stage due to asymptomatic early stages, lack of specific symptoms, underlying liver disease such as cirrhosis caused by chronic hepatitis B or C infection, and the aggressive nature of HCC. Limited strategies are practicable for patients with advanced HCC, and individuals often miss the best opportunities for therapy. High postoperative recurrence and metastasis rates also result in poor outcomes. Five-year recurrence of postresection rate approaches 70%, with 2/3 of recurrences occurring within 2 years due to intrahepatic spread (Kulik and El-Serag 2019). These grim statistics demonstrate the severity and aggressiveness of HCC. Understanding its molecular pathogenesis and elucidating key oncogenic events would contribute greatly to the understanding of HCC occurrence and development and help to develop effective therapeutic strategies to improve clinical prognosis.
RNA modifications refer to the chemical alterations made to RNA after they have been transcribed from DNA. These modifications play crucial roles in various ways of RNA metabolism and function, including mRNA stability, translation efficiency, splicing, and RNA-protein interactions. Common RNA modifications are N6-methyladenosine (m6A), 5-methylcytosine (m5C), Ribose modifications 2’-O-methylation (2’-OMe) m6A, N1-methyladenosine (m1A), etc. According to MODOMICS (MODification of RNA: Dynamics and OMICS, a database that collects information about RNA modification pathways, enzymes, and genes involved in RNA modification) reported that the RNA of all living organisms could be post-transcriptionally modified through more than 150 different chemical modifications by the end of 2021 (Boccaletto et al. 2022). Among these modifications, N6-methyladenosine (m6A), where a methyl group is added to the nitrogen atom at the sixth position of the adenine base, is the most common in eukaryotes, at a frequency of 0.15–0.6% of all adenosines (Meyer et al. 2012). RNA m6A plays an important regulatory role in many physiological processes including embryogenesis (Mendel et al. 2018), circadian regulation (Zhong et al. 2018), DNA damage (Xiang et al. 2017), stress response (Zhou et al. 2015), and cell reprogramming (Aguilo et al. 2015). m6A is a post-transcriptional RNA modification that adds a methyl group to the 6th N of adenosine. It is catalyzed by the multi-component RNA methyltransferase complex (MTC), RNA demethylase, and m6A read protein (referred to as a “writer,” “eraser” and “reader,” respectively). Dynamic and reversible post-transcriptional modifications determine the destiny of target RNA by controlling different aspects of their behavior, including target RNA export, splicing, translation, and degradation (Roundtree et al. 2017). MTC, which catalyzes methyl group transfer, consists of a set of components, including methyltransferase-like 3 and 14 (METTL3 and METTL14), Wilms tumor 1-associated protein (WTAP), Vir-Like m6A methyltransferase associated (VIRMA), RNA-binding motif protein 15 (RBM15), and its homologous analog RNA-binding motif protein 15 B (RBM15B, also called HUMAGCGB, OTT3). Conversely, AlkB homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO) act as m6A erasers that remove N6-methyladenosine, thus maintaining the balance. Family members of the YT521-B homology and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) are identifiers of N6-methyladenosine (Gutschner et al. 2014). High-throughput m6A sequencing illustrated that N6-methyladenosine acts on numerous RNA in eukaryotes, especially at the 3′ untranslated regions (UTRs) and the end of the CDS region, including non-coding RNA (Dominissini et al. 2012). m6A participates in a series of pathological processes, including tumor initiation, progression, metastasis, cancer stem cell multipotency, and stemness.
RBM15B is an essential regulator of m6A and modulates diverse processes (Hiriart et al. 2005), such as selective splicing of mRNA and Xist RNA-mediated X chromosome inactivation (Patil et al. 2016). As a component of the WMM (WTAP, METTL3, and METTL14) complex, RBM15B mediates RNA N6-methyladenosine and plays an important role in mRNA splicing and degradation. RBM15B participates in m6A and WMM complex recruitment by binding to target RNA. We investigated the specific phenotype of RBM15B in HCC. Patients with HCC present with highly overexpressed RBM15B, which predicts a poor outcome. Furthermore, in vitro, and in vivo experiments confirmed the tumor-promoting role of RBM15B, which enhanced the propagation and invasion ability of HCC cells. We identify intersectin2 (ITSN2, also known as SH3D1B, SH3P18, SWA, or SWAP) as a potential downstream candidate of RBM15B. IGF2BP1 recognizes ITNS2 and mediates the stabilization of ITSN2 mRNA, which promotes HCC initiation and progression. At that time, Tan et al. reported that RBM15B promoted HCC proliferation and metastasis, and Sorafenib resistance by promoting TRAM2 (Tan et al. 2022). They conducted RNA immunoprecipitation-sequencing (RIP-seq) analysis and used a public dataset to do gene set enrichment analysis (GSEA). The intersection of RIP-seq and GSE screened out TRAM2 significantly affected by RBM15B. m6A dot blot analysis showed that RBM15B knockdown remarkably decreased the m6A level of HCC, while overexpression of RBM15B induced the opposite effect. We believe that RBM15B may regulate downstream genes through more than one pathway to promote liver cancer progression. The phenotypes of the two independent studies were highly consistent, which confirmed the role and status of RBM15B in the pathogenesis of liver cancer. We applied m6A-seq to further study the mechanism of RBM15B in liver cancer from the aspect of m6A modification.

Materials and methods

Patients and specimens
We collected tumor tissues and the corresponding para-cancerous tissues from 20 patients with HCC (Cohort 1) during surgery at our hospital. Fresh cancer specimens (200 mg each) were promptly frozen in liquid nitrogen for RNA and protein detection. We conducted tissue microarrays (TMA) for 56 HCC and para-cancerous tissues (Cohort 2) of patients with complete clinical and pathological data. The experimental protocols were approved by the Ethics Review Committees of our hospital and were performed in accordance with the Declaration of Helsinki (reference number:2019-421). Written informed consent was obtained from each individual according to the ethics committee’s terms.
Further details on the materials and methods are presented in Supplementary File 1.

Cell culture
Human HCC cell lines MHCC97H and HCCLM3 were procured from the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in DMEM containing 10% FBS, 50 µg/mL streptomycin, and 100 units/mL penicillin in a humidified incubator at 37 °C and 5% CO2. Short tandem repeat (STR) fingerprinting certificates are attainable upon demand. All the cells used in this study were confirmed to be mycoplasma-free.

Animal studies
To avoid the effects of physiological variability related to the estrous cycle in female mice, 4-week-old specific pathogen-free male nude mice were used for subcutaneous tumor transplantation. Mice are anesthetized with isoflurane, once the mouse is unconscious and not moving, we inject all 200 µL of the tumor/Matrigel mixture into the flank of the mouse. Observe mice weekly for the presence of tumor growth. Finally, mice were humanely euthanized using CO2 asphyxiation followed by cervical dislocation. The mice were maintained under specific pathogen-free conditions. The animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital, Zhejiang University School of Medicine (Animal Experimental Ethical Inspection of the First Affiliated Hospital, Zhejiang University School of Medicine, reference number:2021 − 1380).
Further details of euthanasia are presented in Supplementary File 4.

Statistical analysis
Statistical analyses were performed using the GraphPad Prism 8.0 software (GraphPad software, Inc., San Diego, CA, USA). All experiments were independently performed at least three times. Measured data are presented as mean ± standard deviation (SD). Quantitative data were analyzed using one-way analysis of variance (ANOVA) or two-tailed Student’s t-test, with statistical significance set at p < 0.05.

Results

RBM15B is upregulated in HCC and predicts poor prognosis
As CT et al. reported, The Cancer Genome Atlas (TCGA) database showed that RBM15B mRNA levels were remarkably elevated in tumor tissues compared with those in normal tissues (Tan et al. 2022). Furthermore, RBM15B mRNA levels increased with increasing tumor grade and lymph node metastasis stage (Tan et al. 2022). We examined RBM15B mRNA levels in Cohort 1 by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The paired t-test demonstrated that RBM15B mRNA levels were upregulated in tumors compared with those in para-cancerous tissues (Fig. 1A). Data from CPTAC (Clinical Proteomic Tumor Analysis Consortium) showed that RBM15B protein levels were also prominently elevated in primary HCC tumor compared with normal tissue (Fig. 1B). Western blot analysis of cohort 1 showed that RBM15B was highly expressed in tumor tissues (Fig. 1C). We investigated the relationship between RBM15B levels and clinical features in paired HCC MTA samples of cohort 2 and found that higher RBM15B levels were correlated with decreased tumor differentiation, increased microvascular infiltration, and increased Alpha-Fetoprotein (AFP) levels (Table 1). Immunohistochemistry (IHC) analysis of RBM15B in cohort 2 showed that tumor tissues had higher RBM15B protein expression than para-cancerous tissues (Fig. 1D). Kaplan–Meier analysis revealed that patients with high levels of RBM15B had shorter overall survival (OS) and progress-free survival (PFS) rates (Fig. 1E and F). These findings indicated that RBM15B was highly expressed in HCC and that patients with high RBM15B levels had worse outcomes than those with low RBM15B levels.

IHC scores were independently evaluated by two proficient pathologists. A semi-quantitative method was used to assess the staining intensity and percentage of positive cells. The staining intensity score was defined as follows:0 (no staining, negative), 1 (light yellow, weak), 2 (dark yellow, medium), and 3 (brown, yellow, strong). The percentage of positive cells was grouped as follows:0 (< 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%). The intensity score was multiplied by the positive cell score to yield the total score:0–7 was defined as low expression and ≥ 8 was regarded as high expression. Some cases were excluded from statistical analysis due to missing clinical data. A X2 test was used to test the differences between the two variables. Statistical significance: **p < 0.01.

RBM15B strengthens tumor migration and invasion ability in vitro
RBM15B was depleted and confirmed by RT-qPCR and western blot in MHCC97H and HCCLM3 cells, respectively. Both siRBM15B#1 and siRBM15B#2 showed excellent silencing efficiencies (Fig. 2A and B). We performed a Transwell assay and found that the deficiency of RBM15B restrained both the migration and invasion ability of MHCC97H cells. The histograms in Fig. 2C and D showed the number of penetrated cells on the right. Similar results were observed in the RBM15B-knockdown HCCLM3 cells (Fig. 2E and F). We performed a gain-of-function experiment and confirmed the overexpression of RBM15B using RT-qPCR and western blotting (Fig. 2G and H). Overexpression of RBM15B significantly enhanced the mobility and invasion capacity of MHCC97H and HCCLM3 cells (Fig. 2I and J). In summary, RBM15B promoted invading abilities of HCC cells, and the lack of RBM15B impaired the invasive abilities of HCC cells.

RBM15B facilitates HCC growth
To verify the proliferative phenotype of RBM15B, cell counting kit (CCK)-8 and colony formation assays were performed. The CCK-8 assay demonstrated that depletion of RBM15B inhibited the proliferation of MHCC97H and HCCLM3 cells (Fig. 3A and B). A colony formation assay showed that the depletion of RBM15B inhibited the colony-forming ability of HCC cells (Fig. 3C and D). To further clarify the role of RBM15B in cell proliferation, gain-of-function experiments were performed. CCK-8 assay showed that overexpression of RBM15B strongly accelerated cell propagation (Fig. 3E and F). Moreover, the colony-forming assay showed that upregulated RBM15B levels enhanced the colony-forming ability of both MHCC97H and HCCLM3 cells (Fig. 3G and H). To verify these effects in vivo, lentivirus (LV)-shRBM15B or LV-shNC was transfected into MHCC97H and HCCLM3 cells. Stable shRBM15B knockdown efficiency in MHCC97H and HCCLM3 cells was confirmed by western blot analysis (Fig. 3I), which were subcutaneously implanted into 4-week-old male nude mice. The LV-shRBM15B group yielded smaller tumors than the LV-shNC group (Fig. 3J–K). A remarkable decrease in tumor volume (1/2 × [length × width2]) was observed in the LV-shRBM15B group compared with that in the shNC group (Fig. 3L-M). IHC of RBM15B and Ki67 was performed for MHCC97H subcutaneous tumors. The RBM15B expression in the shRBM15B group was lower than that in the shNC group (Fig. 3N). Ki67 expression was lower in the shRBM15B group than that in the shNC group (Fig. 3O). In conclusion, RBM15B facilitated cancer propagation and depletion of RBM15B greatly inhibited tumor growth in vitro and in vivo.

ITSN2 is a potential downstream target of RBM15B in HCC
To determine the exact molecular mechanism underlying the tumor-promoting phenotype of RBM15B, methylated (m6A) RNA immunoprecipitation sequencing (meRIP-seq) and RNA-seq were performed to evaluate the m6A peaks and transcriptional alterations between shNC and shRBM15B in MHCC97H cells. Differential m6A peaks were determined at p < 0.05, and log fold change (FC) ≤ − 1. Differential gene expression was determined at p < 0.05, and log|FC| ≥ 1. FPKM values of RBM15B in shNC were 11.69, 6.99, 9.52, while FPKM values in shRBM15B were 0.84, 0.97, and 1.24, respectively, the FC of shRBM15B decreased by 3.72 times compared with that of shNC, indicating sufficient knockdown efficiency. To screen transcripts with significantly downregulated m6A peaks, we determined the intersection of meRIP-seq and RNA-seq data in a Venn plot, as indicated by the hypo-down and hypo-up sections in the density map (Fig. 4A). Consequently, 38 transcripts were identified in the present study. Transcripts with FPKM < 0.05 were excluded due to extremely low expression. Among these, there were two novel transcripts, one lincRNA, one antisense RNAs, and one pseudogene. We focused on the analysis of the mRNA transcripts (Fig. 4B). Theoretically, RBM15B can be combined with target transcripts to regulate m6A peaks and alter transcript expression. First, the possible candidates were verified by RT-qPCR. Among the 24 potential candidates, ITSN2 was the unique alternative whose levels declined consistently upon depletion of RBM15B with two separate ITSN2 siRNAs in MHCC97H and HCCLM3 cells (Fig. 4C-F). Intersectin 2 (ITSN2) plays a role in varies cellular processes, including endocytosis, adaptive immuneresponse, cell signaling and vesical transport, etc. ITSN2 mRNA is dramatically decreased after RBM15B knockdown in both cell lines and both siRNA sets, indicates stronger reproducibility of the results. Phosphodiesterase 4D Interacting Protein (PDE4DIP) serves to anchor phosphodiesterase 4D to the Golgi/centrosome region of the cell. PDE4DIP is upregulated both in NHCC97H and HCCLM3, but not two siRNA sets, indicates the chance of off-target and low specificity.Furthermore, RBM15B deficiency in MHCC97H and HCCLM3 cells induced a uniform decrease in the ITSN2 protein levels (Fig. 4G and H). Accordingly, we hypothesize that ITSN2 is a promising candidate target for RBM15B.

RBM15B promotes ITSN2 mRNA stabilization via IGF2BP1
As RBM15B is a member of the MTC family, we hypothesized that RBM15B acts on the downstream target ITSN2 in an m6A-dependant manner. We determined whether depletion of RBM15B would downregulate global m6A levels in HCC by m6A dot blots. The results showed that global m6A peaks were reduced upon inhibition of RBM15B in both MHCC97H and HCCLM3 cells (Fig. 5A and B). Potential m6A sites of ITSN2 were estimated using RBM v2.0, which is a comprehensive database to integrate epi-transcriptome sequencing data for exploring post-transcriptionally modifications of RNAs, as well as their relationships with microRNA binding events. As expected, the predicted m6A sites were predominantly distributed at the 3’ UTR and CDS near the termination codon, as shown in Supplementary File 2. Primers targeting these predicted m6A sites were subjected to methylated RNA immunoprecipitation (meRIP) analysis, followed by RT-qPCR. Fragments of ITSN2 were strongly reinforced in the m6A-IP groups compared to those in the IgG-IP groups. More importantly, compared to those in the shNC group, fewer fragments of m6A-modified ITSN2 transcripts were observed in the shRBM15B group, as determined by an assay with primers assigned to the four predicted m6A sites located at the 3′ UTR and end of the CDS (Fig. 5C and D). To further clarify the essential mechanism of m6A, we produced ITSN2 luciferase plasmids, including 288 bps of 3’ UTR and 126 bps of the CDS near the termination codon, or mutant (Mut) one, in which the four m6A sites were mutated. For the mutated one, the four m6A sites verified by meRIP were converted into cytosine (C) to eliminate the impact of m6A, whereas these four m6A sites were preserved in the wild type (WT) plasmid (Supplementary File 3). As expected, the luciferase activity of cells transfected with the ITSN2-WT plasmid was attenuated upon the elimination of RBM15B, whereas that of the ITSN2-Mut plasmid appeared to be insusceptible (Fig. 5E and F). The effect of m6A writers depends on their readers; therefore, we determined specific reader(s) for RBM15B. As common m6A readers, the IGF2BP family IGF2BP1, IGF2BP2, and IGF2BP3 are involved in the regulation of mRNA stabilization. Hence, we depleted IGF2BP1, IGF2BP2, and IGF2BP3 levels with the respective siRNAs and found that ITSN2 levels were distinctly decreased upon silencing of IGF2BP1 both in MHCC97H and HCCLM3 cells (Fig. 5G and H), but not with silencing of IGF2BP2 or IGF2BP3 (Fig. 5I-L). Western blotting analysis also indicated that ITSN2 levels decreased following IGF2BP1 depletion (Fig. 5M). RNA-binding protein immunoprecipitation (RIP) and RT-qPCR were performed to confirm the binding affinity of IGF2BP1 to ITSN2 mRNA. ITSN2 mRNA levels were evidently enriched by IGF2BP1 antibody compared to IgG antibody treatment (Fig. 5N and O). An RNA decay assay showed that the degradation of ITSN2 mRNA was strengthened upon reduction of RBM15B levels, thus reducing the levels of ITSN2 mRNA (Fig. 5P and Q). Therefore, we concluded that RBM15B facilitated the overall modification of m6A levels in HCC, especially at the 3′ UTR and CDS near the stop codon of ITSN2 mRNA. These results confirmed the importance of the RBM15B-IGF2BP1-ITSN2 regulation axis in HCC; specifically, RBM15B promoted the installation of m6A at specific sites of ITSN2 mRNA and, upon transfer to the cytoplasm, IGF2BP1 recognized the modified m6A and subsequently bonded to it to protect ITSN2 mRNA from degradation.

ITSN2 plays a vital role in promoting HCC
ITSN2 mRNA was prominently fortified in HCC tissues compared to that in normal liver tissues, according to TCGA (Fig. 6A). Moreover, ITSN2 mRNA levels increased with increasing tumor grade (Fig. 6B) and lymph node stage (Fig. 6C). We evaluated ITSN2 mRNA and protein levels in cohort 1 and found that tumor tissues had higher ITSN2 mRNA levels than paired para-cancerous tissues (Fig. 6D). Western blotting analysis also indicated that tumor tissues had enhanced protein expression of ITSN2 compared with para-cancerous tissues (Fig. 6E). Kaplan–Meier analysis revealed that individuals with higher expression of ITSN2 had worse OS (p = 0.046) than those with lower expression of ITSN2 (Fig. 6F). We performed TMA in cohort 2 to determine ITSN2 expression via IHC and found that tumor tissues had higher expression of ITSN2 in the cytoplasm (Fig. 6G). We explored the ITSN2 phenotype in HCC in vitro. CCK-8 and colony formation assays showed that exhaustion of ITSN2 levels inhibited the proliferation of MHCC97H and HCCLM3 cells (Fig. 6H–L). Transwell assays revealed that inhibition of ITSN2 expression decreased the migration and invasion abilities of MHCC97H (Fig. 6M and N) and HCCLM3 cells (Fig. 6O and P). Therefore, ITSN2 expedited HCC proliferation and invasion, and patients with higher levels of ITSN2 were predicted to have a worse clinical prognosis.

Depletion of ITSN2 abolishes tumor-promoting phenotype of RBM15B in HCC
Rescue experiments were performed by overexpressing RBM15B, followed by ITSN2 depletion. Western blot analysis verified the overexpression of RBM15B and the silencing of ITSN2 (Fig. 7A). CCK-8 and colony formation assays showed that ITSN2 silencing impaired the propagation-promoting effects of RBM15B overexpression (Fig. 7B and C), and ITSN2 exhaustion reversed the enhanced colony-forming ability associated with RBM15B overexpression (Fig. 7D, and 7E, respectively). Transwell assays indicated that ITSN2 silencing reversed the invasion-promoting effect of RBM15B upregulation (Fig. 7F–I). These findings suggest that enhanced ITSN2 levels may contribute to HCC development through the RBM15B-ITSN2 regulatory axis. In summary, ITSN2 depletion reversed the tumor-promoting effects of RBM15B upregulation.

Combined high expression of RBM15B and ITSN2 predicts an inferior prognosis in HCC
To assess the relationship between RBM15B and ITSN2, IHC for RBM15B and ITSN2 was performed using the TMA of cohort 2. IHC showed that RBM15B expression was positively correlated with ITSN2 expression (Fig. 8A) as shown in the histogram in Fig. 8B, showing high and low expression ratios of RBM15B and ITSN2, respectively. We observed a positive correlation between RBM15B and ITSN2 expression in the TCGA database (Fig. 8C). In summary, RBM15B expression was positively correlated with ITSN2 expression, and high co-expression of these two molecules predicted an inferior outcome in patients with HCC.

Discussion
In recent years, substantial and invertible chemical modifications of RNA have become a novel dimension of epigenetic regulation. m6A is the most common chemical modification in eukaryotes (Roundtree et al. 2017). In RNA m6A, a methyl group is deposited at the 6th N of adenosine. Studies have reported the profound impact of m6A in human diseases, especially malignant cancers (Yan et al. 2021). Regarding m6A writers, the effects and possible mechanisms of METTL3 (Yankova et al. 2021), METTL14 (Weng et al. 2018), and WTAP (Chen et al. 2019) have been extensively studied in various tumors. While the core m6A writers METTL3 and METTL14 have been extensively characterized in HCC, the roles of ancillary factors like RBM15B remain less defined. Our study addresses this gap by identifying RBM15B as a functionally significant writer that is upregulated in HCC and associated with aggressive clinicopathological features. This position RBM15B alongside established writers in the m6A epi-transcriptome of HCC.In this study, we found that both RBM15B mRNA and protein levels were increased in tumors. TMAs-containing samples from 56 HCC patients showed that RBM15B levels increased in tumors, which was correlated with a higher level of AFP, decreased tumor differentiation, and increased tumor microvascular invasion ability in HCC. Furthermore, we confirmed that RBM15B. overexpression significantly promotes HCC cell proliferation and invasion.In conclusion, the present study confirms that RBM15B is an important molecule that drives the growth and metastasis of HCC.
ITSNs are an evolutionarily conserved adaptor protein family involved in endocytic membrane traffic (Tsyba et al. 2011; Predescu et al. 2003). ITSN2 is widely expressed in multiple tissues (Pucharcos et al. 2001; Seifert et al. 2007). This protein family is also involved in clathrin-mediated endocytosis (Henne et al. 2010). ITSN2 is suggested to be involved in the regulation of Cdc42 activity (Burbage et al. 2018). It was reported that ITSN2 functions synergistically with WASp and Cdc42 in occupancy-induced T-cell receptor (TCR) endocytosis (McGavin et al. 2001). Mu et al. demonstrated that METTL3 deposited m6A on ITSN2 and promoted its stabilization to induce meiosis in oocytes (Mu et al. 2021). Locard-Paulet et al. revealed that ITSN2 controls T-cell effectors by reducing TCR levels upon antigenic agitation (Locard-Paulet et al. 2020). Our work unveils a previously unrecognized oncogenic axis in HCC: RBM15B-mediated m6A modification stabilizes ITSN2 mRNA. This finding is particularly innovative as it links an m6A writer to the regulation of ITSN2, a gene primarily studied in endocytosis and immune cell regulation, revealing a non-canonical, pro-tumorigenic function in the liver. This novel RBM15B-ITSN2 axis expands the mechanistic understanding of m6A-driven cancer progression. This aligns with previous studies that highlight the role of m6A modifications in regulating oncogenic transcripts and underscores the potential of ITSN2 as a therapeutic target in HCC. Moreover, ITSN2 silencing reversed the tumor-promoting effect of RBM15B overexpression. ITSN2 expression was positively correlated with RBM15B expression, and higher levels of ITSN2 predicted worse prognosis in patients with HCC. Notably, the downstream regulatory axis of ITSN2 has not yet been studied but will be investigated in a future study.
Writers and erasers determine the abundance and location of methylation, whereas readers perform m6A-related functions. m6A readers, including the YTH domain-containing family protein (YTHDF) (Du et al. 2016), YTH domain-containing protein (YTHDC) (Xiao et al. 2016), and IGF2BPs families (Yin et al. 2022), act by recognizing m6A sites on RNA and mediating the metabolism of the target RNA. YTHDF2 accelerates mRNA decay by recruiting RNA decay factors (Chai et al. 2021). YTHDC1 regulates mRNA splicing by recruiting pre-mRNA splicing elements into the binding regions of the target mRNA (Li et al. 2022). In contrast to YTHDF2, members of the IGF2BP family, including IGF2BP1 (Xue et al. 2021), IGF2BP2 (Yin et al. 2022), and IGF2BP3 (Wan et al. 2022), protect m6A-modified mRNA from degradation in the cytoplasm. Among these, IGF2BP1 is the most explicit and well-defined reader that mediates the stabilization of m6A-modified RNA. Zhang et al. (2021) verified that IGF2BP1 combined specifically with the 3′ UTR of LDHA in colorectal cancer, enhanced the stabilization of LDHA transcripts and promoted cancer progression. Consistent with the established role of IGF2BP1 as an m6A “reader” that enhances mRNA stability, our data confirm its critical function in this axis. The depletion of IGF2BP1 led to a reduction in ITSN2 transcripts, suggesting that RBM15B-dependent m6A marks on ITSN2 mRNA are likely recognized and stabilized by IGF2BP1. This aligns with the canonical mechanism observed in other cancers, such as the stabilization of LDHA by IGF2BP1 in colorectal cancer (Zhang et al. 2021), and validates the functional integrity of the m6A writer-reader axis in our model. Although we investigated the function of IGF2BP1 in RBM15B-mediated m6A in HCC, the effects of other readers, such as HuR and YTHDC, remain largely unclear. Follow-up studies are needed to identify other m6A reader candidates.
Although this study is one of the first to demonstrate that RBM15B facilitates HCC progression through the m6A-dependent stabilization of ITSN2 mRNA, revealing a novel axis in HCC pathology, our work has some limitations. MeRIP-seq and RNA-seq data were used to screen for target molecules with differentially expressed m6A peaks and transcripts.A key limitation of our screening approach, which relied on MeRIP-seq and RNA-seq, is its focus on transcript abundance. It potentially overlooks m6A-mediated regulation at the level of translation efficiency, a rapidly emerging dimension of m6A biology. Consequently, we might have missed critical RBM15B targets whose protein levels, rather than mRNA levels, are modulated by m6A. Future employment of techniques like Ribo-seq (Meyer and Jaffrey, 2017; Ingolia et al. 2009) will be crucial to paint a complete picture of RBM15B’s regulatory network and could uncover additional oncogenic pathways driven by translational control. Techniques such as Ribo-seq, which analyzes ribosome occupancy, would provide additional insights into the translational regulation mediated by m6A modifications (Meyer and Jaffrey, 2017; Ingolia et al. 2009). To narrow down the potential candidate targets, we overlapped the meRIP-seq and RNA-seq data using a Venn plot, which we acknowledge may be insufficient to identify appropriate candidates and could have omitted some important molecules in HCC pathology that deserve further investigation. Second, to avoid physiological variability linked to sex hormones in female mice, male mice were solely used for subcutaneous transplantation. The effect on sex hormones in HCC tumorigenesis and progression requires further investigation. Third, further studies should explore the role of RBM15B in tumor immunity. We constructed a mouse liver carcinoma in situ model to explore the expression profiles of RBM15B in tumor-infiltrating lymphocytes and the potential immune mechanisms, which will be explored in future studies. Finally, although RBM15B’s primary role in our study involves m6A-dependent RNA regulation, it may also have m6A-independent functions that contribute to HCC progression. These functions could involve protein-protein interactions or alternative RNA-binding activities, which remain poorly understood. Investigating these mechanisms could uncover additional pathways through which RBM15B drives oncogenesis (Xing and Chen 2018; Hentze et al. 2018). Notwithstanding the future work required to address these limitations, our findings possess considerable translational potential.From a clinical translation perspective, our findings hold significant promise. The strong correlation of both RBM15B and ITSN2 with poor prognosis in HCC patients positions them as potential novel prognostic biomarkers. More importantly, the elucidated RBM15B-ITSN2-IGF2BP1 axis reveals a network of potential therapeutic targets. Given the burgeoning development of small-molecule inhibitors against m6A writers and readers, targeting this specific axis could offer a new strategic avenue for a subset of HCC patients with high RBM15B/ITSN2 expression. Future studies validating this axis in larger patient cohorts and developing targeted interventions are warranted to translate these mechanistic insights into clinical benefits.

Conclusions
This study highlights the transcriptomic regulation of the RBM15B-IGF2BP1-ITSN2 axis in HCC. RBM15B contributes to the installation and m6A modification of ITSN2 mRNA, which is subsequently stabilized by IGF2BP1. Enhanced ITSN2 expression favors HCC cell propagation and metastasis. This study offers new insights into the roles and epigenetic mechanisms of RBM15B in HCC, and suggests a potential strategy for the clinical management of HCC.

Supplementary Information
Below is the link to the electronic supplementary material.