RBM23 Drives Hepatocellular Carcinoma by Activating NF-κB Signaling Pathway

Purpose Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and angiogenesis has been proven to be significantly involved in its progression. However, the molecular mechanism underlying HCC angiogenesis has not been well researched. In this study, RNA Binding Motif Protein 23 (RBM23) was identified as a novel proangiogenic factor in HCC cell lines and tissues. Materials and Methods Firstly, we analyzed the correlation of clinical specimens. In HCC tissues, the levels of RBM23 and microvessel density (MVD) showed a strong positive correlation. Furthermore, data from related cytology experiments showed that the knockdown of RBM23 expression in HCC cells significantly inhibited the tube formation by the human vascular endothelial cells in vitro. The mechanism of this phenomenon was found to be through increasing the mRNA of p65 and enhanced the nuclear accumulation of p65. Consequently, RBM23 activated the NF-κB signaling pathway and promoted expression of the proangiogenic cytokines selectively. Results and Conclusion. In summary, this study revealed that RBM23 promotes the angiogenesis properties of HCC via the NF-κB signaling pathway. It may, therefore, be a potential therapeutic target for the treatment of hepatocellular carcinoma.


Introduction
Hepatocellular carcinoma (HCC) is the most common primary malignancy of liver cancer with a high global prevalence [1]. Owing to its high potential for vascular invasion, metastasis, and postoperative recurrence, most HCC patients die from a locally advanced or metastatic disease in a relatively short time [2]. However, the mechanisms responsible for HCC progression and metastasis are not clear at the moment [3]. Therefore, it is pending for us to identify new driving factors to deepen our understanding of the occurrence and development of this disease.
Angiogenesis, as we all know, is a hallmark of various malignancies, which is usually induced early during the multistage development of invasive cancers when we studied both in animal models and in humans [4]. HCC is a hypervascular malignancy accompanied by a high level of neovascularization, which ensures sufficient nutrition for the tumor and makes HCC cells prone to early metastasis through the blood tract [5]. The early blood tract metastasis not only makes a large number of patients lose the chance of radical surgery but also leads to a poor prognosis [6]. So far, only sorafenib has been approved by FDA for clinical treatment [7], but its effect on patients with advanced hepatocellular carcinoma is limited, and drug resistance is very easy to develop and accompanied by the emergence of some related complications [8], which brings great troubles for clinical use of liver cancer patients. This urged us to investigate further to discover new driving factors of HCC angiogenesis [9]. Based on this, we launched relevant research and initially identified RBM23 as a factor that promotes microvascular proliferation in hepatocellular carcinoma.
Patients with HCC often develop from chronic hepatitis and chronic cirrhosis [10]. During this development, a large number of inflammatory factors make the NF-κB signaling pathway abnormally activated [11]. As is known to all, nuclear factor κB (NF-κB) is an important and wellresearched transcription factor which regulates genes associated with a variety of cellular functions, such as cell proliferation [12], survival [13], angiogenesis [14], and cancer metastasis [15]. The dominant cellular NF-κB is a heterodimer of p50 and p65 subunits, which are normally sequestered in the cytoplasm of nonstimulated cells. The p65 subunits are normally phosphorylated, then translocated into the nucleus once cells are exposed to extracellular stimuli [16]. In various diseases, the NF-κB signaling pathway often has abnormal activation, resulting in the transcription of a large number of cancer-promoting genes, including cytokines that promote angiogenesis [17]. Therefore, to improve the prognosis of cancer, the NF-κB signaling pathway activation must be terminated at the appropriate time. Thus, discovery of the molecule involved in controlling the aberrant activation of the NF-κB signaling pathway is a potential target for therapeutic strategies against cancer.
In this study, we identified RBM23, which has not been researched before, as a novel promoter of HCC angiogenesis. We first analyzed the level of RBM23 in fresh tissue samples collected, which is positively correlated with microvessel density (MVD) in HCC tissues (indicated by CD31-positive cells). The results were further verified in the tissue microarray of hepatocellular carcinoma. Then, we analyzed the content of RBM23 in several HCC cell lines and established stable cell lines based on it, and various molecular and cytological experiments were used to demonstrate the promotion of HCC angiogenesis by RBM23 both in in vitro and in vivo assays. Mechanically, RBM23 improved the activity of the NF-κB signaling pathway to produce more proangiogenesis cytokines by increasing the expression of RelA/p65.

Patients and Tissue
Specimens. This study was conducted on 32 paraffin-embedded hepatocellular carcinoma (HCC) samples and corresponding noncancerous tissues, which were purchased from Servicebio (Wuhan, China); this process was approved by the Ethics Committee of the Affiliated Hospital of Nantong University, and relevant experiments were carried out. Additionally, 12 HCC specimens and matched adjacent noncancerous tissues from the Affiliated Hospital of Nantong University were frozen and stored in liquid nitrogen until further use. (The samples are well preserved in liquid nitrogen. The patients are all newly diagnosed hepatocellular carcinoma patients and have not received any form of anticancer treatment before surgery.) Prior consent from patients was obtained, and all the experimental procedures were approved by the Institutional Research Ethics Committee.

RBM23 Promotes Tumor Angiogenesis In Vitro.
Through the above experiments, we found that there is a large clinical correlation between RBM23 and microvessel density (MVD), which can affect the complete tubular structures formed by HUVECs. Then, the functional role of RBM23 in angiogenesis was also determined through the investigation of in vitro HUVEC growth and migration by cell counting kit-8 assays and wound healing assays. As it was showed, the migration of HUVECs was inhibited in conditioned medium from HepG2-sh-RBM23 cells and promoted in the conditioned medium from Huh-7-ov-RBM23 cells compared with the corresponding controls (Figure 2(a)). Similar results were found in the cell counting kit-8 assays (Figure 2(b)). Moreover, to reveal the effect of conditioned medium from Huh-7-ov-RBM23 and corresponding control cells on angiogenesis in vivo, we mixed the cell supernatants with Matrigel and injected the mixture into a subcutaneous location and took out the plugs 5 days later (Figure 2(c)). On examining the mRNA level of IL-6, IL-8, and VEGF, which are the main cytokine that promotes angiogenesis, we found that RBM23 mainly affected IL8 and had a relatively small effect on IL-6 and VEGF (Figure 2(d)).

RBM23 Activates NF-κB Activity and Promotes the Expression of Its Target Proangiogenesis Genes.
To explore the specific mechanism by which RBM23 affects angiogenesis, we first examined the key molecules of related signaling pathways, including HIF-1a, NF-kappaB, STAT3, and AP1 pathways (the cells of each group were cultured in a hypoxic environment with 1% oxygen for 16 hours, and then, the expression of HIF-1a was detected). Western blotting assays revealed that RBM23 mainly activated the NF-κB signaling pathway selectively (Figure 3(a)). Then, we investigated the potential mechanisms of the NF-κB signaling pathway controlled by RBM23. No significant change in the expression of other important molecules in this signaling pathway was observed between the modified RBM23 expression and respective control cells. However, NF-κB p65 and its phosphorylated (serine-536) levels decreased in HepG2sh-RBM23 and increased in Huh-7-ov-RBM23 compared with the corresponding control cells (Figure 3(b)). Moreover, we verified that RBM23 could affect the NF-kappaB pathway using immunofluorescence (Figure 3(c)). Using cell fractionation and western blot, we found that both the cytoplasmic p65 and nuclear p65 were significantly increased upon RBM23 overexpression in Huh-7 cells and significantly decreased in RBM23 knockdown in HepG2 Huh-7-ov-vector Huh-7-ov-RBM23 HepG2-sh-vector HepG2-sh-RBM23-1 HepG2-sh-RBM23-2 (a)  (Figure 3(d) and Figure S1C). These results indicated that RBM23 affected the activity of the NF-κB signaling pathway by affecting the content of RelA/p65. The ELISA results also showed that the use of RelA/p65 short hairpin RNA or signaling pathway inhibitors (Bay11-7082, IMD-0354) could rescue the increase in IL-8 caused by overexpression of RBM23 in Huh-7 cells (Figure 3(e)). This further showed that RBM23 promoted tumor angiogenesis by activating the NF-κB signaling pathway.
3.4. In Vivo RBM23 Promotes Angiogenesis in Cancer. The above experimental results showed that RBM23 can affect the NF-κB signaling pathway, and then, we used the above tissue chip and select the same site to analyze the content of p-p65 (ser-536), which is found to be highly expressed in cancer tissues compared to the matched normal tissues and has a good correlation with RBM23 (Figure 4(a)). To investigate the role of RBM23 in tumorigenesis in vivo, Huh-7-ov-RBM23 and corresponding control cells were injected subcutaneously into nude mice, and the tumors were removed after six weeks (Figure 4(b)). The microvessel density was evaluated by CD31 immunohistochemical staining, and the tumor proliferation was evaluated by Ki-67 immunohistochemical staining. The results obtained proved that RBM23 could promote tumor neovascularization in vivo (Figure 4(c)).

Discussion
In this study, we found that RBM23 was highly expressed in hepatocellular carcinoma (HCC) and correlated with tumor microvessel density (as indicated by CD31-positive cells), suggesting that RBM23 may be involved in the formation of microvessels in HCC and promote the proliferation and distant metastasis of HCC. Angiogenesis promotes rapid progress and early metastasis in hepatocellular carcinoma (HCC) [18]. This brings challenges to clinical treatment because a large number of patients lose the opportunity of surgery due to the early metastasis, which not only brings great challenges to the radical cure of hepatocellular carcinoma but also creates huge economic and social pressure for the society and individual patients [19]. In the course of clinical treatment, it can be found that a large number of patients have lost the chance of radical surgery due to the early metastasis of the intrahepatic portal vein system or the metastasis of distant organs through the bloodstream [20]. Nonetheless, it has also brought new ideas for treatment such as targeted therapy for tumor neovascularization. Sorafenib, a small inhibitor of several tyrosine protein kinases, contains vascular endothelial growth factor receptor, plateletderived growth factor receptor, and Raf. Although sorafenib has been recently approved by the FDA for the treatment of advanced HCC [21] and showed improved survival rates in these patients, it still has huge risks and challenges [22]. At the same time, sorafenib has been the only first-line drug for advanced hepatocellular carcinoma for many years [23]. Therefore, it is urgent to identify new therapeutic targets to develop a treatment for HCC treatment. In this study, we identified RBM23 as a new molecule that promotes tumor angiogenesis, which provides a new perspective for us to deepen our understanding of angiogenesis in hepatocellular carcinoma.

BioMed Research International
Through further studies, we found that RBM23 promotes angiogenesis by promoting the expression and secretion of IL-8. From a mechanical point of view, RBM23 promotes IL-8 transcription by increasing the expression of RelA/p65 and then the activity of the NF-κB signaling pathway.
The NF-κB signaling pathway, a critically vital signaling pathway in tumorigenesis, regulates numerous downstream cancer-promoting genes, including some proangiogenesis cytokines, such as IL-6, IL-8, and VEGF [24]. Recently, researchers demonstrated that ubiquitination-and phosphorylation-mediated signaling transductions critically control the activation of the NF-κB signaling pathway [25]; among them, IkBa-mediated cytoplasmic accumulation of the NF-κB p65/p50 complex has been considered a key mechanism of terminating the NF-κB signaling pathway [26], for the reason that deterring RelA/p65, the most important molecule in this signaling pathway, would completely block the activity of the NF-κB signaling pathway [27]. For instance, Wang and colleagues reported in gastric cancer that CHIP interacted with RelA/p65 and promoted its ubiquitination and degradation via a proteasome terminating NF-κB signaling pathway activity and inhibiting angiogenesis induced by IL-8 [28]. Besides, Liu et al. found in hepatocellular carcinoma that PROX1 elevated the level of RelA/p65 and stabilized RelA/p65 by recruiting ubiquitin-specific protease 7 (USP7). This prevented RelA/p65 from degradation thereby enhancing the activity of the signaling pathway [29].
However, it is not yet known whether there is a new regulatory mechanism for RelA/p65. In this study, RBM23 did not affect the activation and expression of IkBa in our study; unexpectedly, we found that RBM23 could improve the level of RelA/p65 mRNA, thereby increasing the activity of the NF-κB signaling pathway to promote the angiogenesis of hepatocellular carcinoma.
In summary, this study demonstrates that RBM23 regulates the activity of the NF-κB signaling pathway in HCC via directly targeting RelA/p65; these data revealed the RBM23-RelA/p65-NF-κB axis as a mechanism of promoting the angiogenesis in hepatocellular carcinoma. At the same time, knocking down RBM23 inhibited HCC cell malignant phenotypes and, importantly, suppressed HCC angiogenesis by inhibiting NF-κB activity through reducing the expression of RelA/p65 and downregulation of the proangiogenic cytokine IL-8. We propose that RBM23 may serve as a promising prognostic marker for hepatocellular carcinoma, and knocking down CHIP may be a novel strategy for antiangiogenesis therapy for human HCC.

Conclusions
In this study, we identified RBM23, a molecule that was not researched before, as a novel proangiogenic factor in hepatocellular carcinoma (HCC), which facilitates the angiogenesis of hepatocellular carcinoma (HCC) via modulating the NF-κB signaling pathway, and through analysis of the clinical samples of multiple cohorts and performing in vivo experiments, we believe that RBM23 can be a new target for HCC treatments.

Data Availability
The statistics data used to support the findings of this study are included within the supplementary information file.