RBM15B Promotes Hepatocellular Carcinoma Progression via IGF2BP1-Mediated ITSN2 mRNA Stabilization

doi:10.21203/rs.3.rs-3806743/v1

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RBM15B Promotes Hepatocellular Carcinoma Progression via IGF2BP1-Mediated ITSN2 mRNA Stabilization

https://doi.org/10.21203/rs.3.rs-3806743/v1

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Background: N⁶-methyladenosine (m⁶A), the most abundant form of RNA chemical methylation, is aberrantly expressed in hepatocellular carcinoma (HCC), where it affects multiple biological processes including targeted RNA splicing, transport, degradation, stabilization, and translation.

Method: The RNA-binding motif protein 15 B (RBM15B) is a pivotal coordinator of m⁶A activation, such as alternative splicing of mRNA. We investigated the biological effects and potential mechanisms of action of RBM15B in HCC using TCGA database, in-vitro and in vivo assays, and Merip-seq.

Results: High levels of RBM15B, which predicts a poor prognosis, have been identified in patients with HCC. RBM15B promotes HCC propagation and invasion in vitro and in vivo. RBM15B regulates the m⁶A of intersectin2 (ITSN2) mRNA via insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1)-facilitated stabilization of ITSN2 mRNA. ITSN2 depletion rescued the tumor-promoting phenotype induced by RBM15B overexpression.

Conclusion: In summary, RBM15B-regulated m⁶A in ITSN2 facilitates HCC progression via IGF2BP1-guided stabilization of ITSN2 mRNA. Our study illustrates the importance of the RBM15B-IGF2BP1-ITSN2 regulatory axis based on m⁶A activity and provides new insights into epi-transcriptomic maladjustments of initiation and metastasis in HCC.

IGF2BP1

ITSN2

N6-methyladenosine

RBM15B

According to the World Health Organization, hepatocellular carcinoma (HCC) is the fifth most common cancer and second most common cause of cancer-related deaths worldwide [1]. As HCC is usually diagnosed at an advanced stage, limited strategies are practicable, and HCC patients often miss the best opportunities for therapy. High postoperative recurrence and metastasis rates also result in poor outcomes. Less than 20% of patients with HCC survive for more than one year after diagnosis [2]. 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.

By the end of 2021, MODOMICS reported that the RNA of all living organisms could be post-transcriptionally modified through more than 150 different chemical modifications [3]. Among these modifications, N6-methyladenosine (m⁶A) is the most common in eukaryotes [4]. RNA m⁶A plays an important regulatory role in many physiological processes including embryogenesis [5], circadian regulation [6], DNA damage [7], stress response [8], and cell reprogramming [9]. 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 [10]. 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 [11]. 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 [12]. 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 [13], such as selective splicing of mRNA and Xist RNA-mediated X chromosome inactivation [14]. 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 [14]. RBM15B participates in m⁶A and WMM complex recruitment by binding to target RNA. However, the precise phenotype of RBM15B and its molecular mechanisms of action in HCC remain unclear.

In this study, 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 identifies intersectin2 (ITSN2, also known as SH3D1B, SH3P18, SWA, or SWAP) as a potential downstream candidate of RBM15B. IGF2BP1 recognizes ITNS2 and mediates stabilization of ITSN2 mRNA, which promotes HCC initiation and progression. In summary, our study elucidates an epigenetic mechanism contributing to HCC pathogenesis and metastasis and identifies RBM15B as a biological candidate marker with diagnostic and therapeutic potential in HCC.

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 − 1421). Written informed consent was obtained from each individual according to the ethics committees’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.

RBM15B is upregulated in HCC and predicts poor prognosis.

Firstly, we evaluated the mRNA levels of RBM15B in human HCC using The Cancer Genome Atlas (TCGA) database. RBM15B mRNA levels were remarkably elevated in tumor tissues compared with those in normal tissues (Fig. 1A). Furthermore, RBM15B mRNA levels increased with increasing tumor grade and lymph node metastasis stage (Fig. 1B and 1C). 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. 1D). Data from TCGA database showed that RBM15B protein levels were also prominently expressed in HCC tumor tissues (Fig. 1E). Western blot analysis of cohort 1 showed that RBM15B was highly expressed in tumor tissues (Fig. 1F). 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 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. 1G). Kaplan–Meier analysis revealed that patients with high levels of RBM15B had shorter overall survival (OS) and disease-free survival (DFS) rates (Fig. 1H and 1I). These findings indicate that RBM15B is highly expressed in HCC and that patients with high RBM15B levels have worse outcomes than those with low RBM15B levels.

Table 1

Clinical characteristics of the 56 patients with HCC in Cohort 2
Features		RBM15B-low	RBM15B-high	p-value
Gender	Male	19	2	0.696
	Female	29	6
Age	< 65	14	7	0.1077
	≥ 65	30	5
AFP	< 20 µg/L	10	9	0.0032^**
	≥ 20 µg/L	4	28
HbsAg	Negative	5	30	0.2977
	Positive	6	15
Differentiation	Low	14	13	0.0023^**
	High	2	19
Microvascular infiltration	Absent	17	4	0.0021^**
	Present	13	22
Recurrence	No	10	11	0.1411
	Yes	8	23

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 2B). 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 2D showed the number of penetrated cells on the right. Similar results were observed in the RBM15B-knockdown HCCLM3 cells (Fig. 2E and 2F). We performed a gain-of-function experiment and confirmed the overexpression of RBM15B using RT-qPCR and western blotting (Fig. 2G and 2H). Overexpression of RBM15B significantly enhanced the mobility and invasion capacity of MHCC97H and HCCLM3 cells (Fig. 2I and 2J). In summary, RBM15B promotes the invading abilities of HCC cells, and lack of RBM15B impairs 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 3B). A colony formation assay showed that the depletion of RBM15B inhibited the colony-forming ability of HCC cells (Fig. 3C and 3D). 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 3F). Moreover, the colony-forming assay showed that upregulated RBM15B levels enhanced the colony-forming ability of both MHCC97H and HCCLM3 cells (Fig. 3G and 3H). 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.S2A), which were subcutaneously implanted into 4-week-old male nude mice. The LV-shRBM15B group yielded smaller tumors than the LV-shNC group (Fig. 3I-L). A remarkable decrease in both tumor volume (1 / 2×[length × width²]) and weight was observed in the LV-shRBM15B group compared with that in the shNC group (Fig. 3M-P). 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. S2B). Ki67 expression was lower in the shRBM15B group than that in the shNC group (Fig. 3Q). In conclusion, RBM15B facilitates cancer propagation and depletion of RBM15B greatly inhibits 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 were determined at p < 0.05, and log|FC| ≥ 1. 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 only unique alternative whose levels declined consistently upon depletion of RBM15B with two separate ITSN2 siRNAs in MHCC97H and HCCLM3 cells (Fig. 4C-F). Furthermore, RBM15B deficiency in MHCC97H and HCCLM3 cells induced a uniform decrease in the ITSN2 protein levels (Fig. 4G and 4H). 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 5B). Potential m⁶A sites of ITSN2 were estimated using RBM v2.0. 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 5D). 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 5F). 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 5H), 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 5O). 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 5Q). Therefore, we conclude that RBM15B facilitates the overall modification of m⁶A levels in HCC, especially at the 3′ UTR and CDS near the stop codon of ITSN2 mRNA. These results confirm the importance of the RBM15B-IGF2BP1-ITSN2 regulation axis in HCC; specifically, RBM15B promotes the installation of m⁶A at specific sites of ITSN2 mRNA and, upon transfer to the cytoplasm, IGF2BP1 recognizes the modified m⁶A and subsequently binds 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 6N) and HCCLM3 cells (Fig. 6O and 6P). Therefore, ITSN2 expedites HCC proliferation and invasion, and patients with higher levels of ITSN2 are 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. S1A). CCK-8 and colony formation assays showed that ITSN2 silencing impaired the propagation-promoting effects of RBM15B overexpression (Fig. S1B and S1C), and ITSN2 exhaustion reversed the enhanced colony-forming ability associated with RBM15B overexpression (Fig. S1D, and S1E, respectively). Transwell assays indicated that ITSN2 silencing reversed the invasion-promoting effect of RBM15B upregulation (Fig. S1F-I). These findings suggest that enhanced ITSN2 levels may contribute to HCC development through the RBM15B-ITSN2 regulatory axis. In summary, ITSN2 depletion reverses 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. S2C) as show in the histogram in Fig. S2D, 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. S2E). In summary, RBM15B expression is positively correlated with ITSN2 expression, and high co-expression of these two molecules predicts an inferior outcome in patients with HCC.

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 [10]. In RNA m⁶A, a methyl group is installed at the 6th N of adenosine. Studies have reported the profound impact of m⁶A in human diseases, especially malignant cancers [15]. Regarding m⁶A writers, the effects and possible mechanisms of METTL3 [16], METTL14 [17] and WTAP [18] have been extensively studied in various tumors. However, the role of RBM15B in HCC is unclear. 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 enhanced the propagation and invasion abilities of HCC cells. In conclusion, the present study confirms that RBM15B is an important molecule that drives growth and metastasis of HCC.

ITSNs are an evolutionary conserved adaptor protein family involved in endocytic membrane traffic [19, 20]. ITSN2 is widely expressed in multiple tissues [21, 22]. This protein family is also involved in clathrin-mediated endocytosis [23]. ITSN2 is suggested to be involved in the regulation of Cdc42 activity [24]. It was reported that ITSN2 functions synergistically with WASp and Cdc42 in occupancy-induced T-cell receptor (TCR) endocytosis [25]. Mu et al. demonstrated that METTL3 deposited m⁶A on ITSN2 and promoted its stabilization to induce meiosis in oocytes [26]. Locard-Paulet et al. revealed that ITSN2 controls T-cell effectors by reducing TCR levels upon antigenic agitation [27]. We found that ITSN2 levels were markedly higher in tumors than in paired para-cancerous tissues, and the downregulation of ITSN2 expression impaired the proliferation and invasion abilities of HCC cells. 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)[28], YTH domain-containing protein (YTHDC) [29], and IGF2BPs families [30], act by recognizing m⁶A sites on RNA and mediating the metabolism of the target RNA. YTHDF2 accelerates mRNA decay by recruiting RNA decay factors [31]. YTHDC1 regulates mRNA splicing by recruiting pre-mRNA splicing elements into the binding regions of the target mRNA [32]. In contrast to YTHDF2, members of the IGF2BP family, including IGF2BP1 [33], IGF2BP2 [30], and IGF2BP3 [34], 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. [35] verified that IGF2BP1 combined specifically with the 3′ UTR of LDHA in colorectal cancer, enhanced the stabilization of LDHA transcripts and promoted cancer progression. In our study, we also found that ITSN2 transcript number was decreased by IGF2BP1 depletion. 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 is among the first studies to demonstrate that RBM15B facilitates HCC progression via the m⁶A-dependent stabilization of ITSN2 mRNA, 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. However, m⁶A may not affect mRNA expression but may influence translation efficiency, which cannot be determined by RNA-seq. 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, further studies on the m⁶A-independent functions of RBM15B are needed.

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.

Declaration of interest statement

The authors report that there are no competing interests to declare.

Ethics approval and consent to participate

We complied with all relevant ethical regulations for working with patient resources and experimental animals (reference number:2021 − 1380). The animal experiments were carried out in compliance with the ARRIVE guidelines. The clinical study was approved by the Institutional Review Board at The First Affiliated Hospital, Zhejiang University School of Medicine (reference number:2019 − 1421) and Affiliated Hangzhou First People's Hospital (reference number:2021 − 302). Informed consent was obtained from all the patients.

Funding

This work was funded by Medicine and Health Science and Technology Plan Projects of Zhejiang Province under Grant 2022KY245; Research Project of Jinan Micro-ecological Bio-medicine Shandong Laboratory under Grant JNL-2022039C; Zhejiang Provincial Natural Science Foundation of China under Grant LZ22H080002; and Research Unit Project of Chinese Academy of Medical Sciences under Grant 2019-I2M-5-030.

Author Contribution

Yongfang Xu,Wei Chen and Mengqi Xiong performed the in vivo, in vitro, and clinical sample experiments; Chengjie Yao performed statistical analysis and animal experiments; Yunhao Chen guided the meRIP experiment and modified the draft; Da Man aided in bioinformatics analysis; Xiujin Ye,Haiyang Xie and Changku Jia designed this experiment and revised the first draft to the final draft.

Acknowledgements

The authors thank technician Danjing Guo for the technical guidance with IHC. We also thank Dr. Rong Su for her help with flow cytometry experiments.

Availability of data and materials

The datasets used and/or analyzed in this study are available from the corresponding author upon request.

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No competing interests reported.

image7.png
Fig. S1 Silencing of ITSN2 reversed the tumor-promoting role of RBM15B. (A) Rescue assay by overexpression of RBM15B and/or depletion of ITSN2 verified by western blotting in MHCC97H (upper chart) and HCCLM3 (lower chart) cells. (B-C) CCK-8 assay upon ITSN2 knockdown and RBM15B overexpression. (D-E) Colony forming assay after ITSN2 knockdown and RBM15B overexpression, with charts showing colonies of indicated groups. (F-I) Transwell migration and invasion assay, with histograms (H and I) showing the numbers of penetrated cells on the right. Data are presented as the mean ± SD. ***p < 0.001, ****p < 0.0001.
image8.png
Fig. S2 (A) shRBM15B knockdown efficiency verified by western blotting. (B) Representative IHC images of RBM15B in the subcutaneous tumor. (C) Representative IHC images of RBM15B and ITSN2 (scale bar, 50 μm; magnification, 200× and 400×). (D) Histograms of RBM15B and ITSN2 levels determined by IHC. (E) Correlation analysis of RBM15B and ITSN2 based on TCGA database. Data are presented as the mean ± SD. ****p < 0.0001.
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RBM15B Promotes Hepatocellular Carcinoma Progression via IGF2BP1-Mediated ITSN2 mRNA Stabilization

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