Upregulation of RBM24 Exacerbates Bladder Cancer Progression by Formatting Runx1t1/TCF4/miR-625-5p Feedback Loop

Background: RNA-binding motif protein 24 (RBM24) acts as a multifunctional determinant of cell fate, proliferation, apoptosis, and differentiation during development through regulation of pre-mRNA splicing and mRNA stability. It is also implicated in carcinogenesis, but the functions of RBM24 in bladder cancer (BC) remains unclear. Methods: Cell viability was examined by colony forming and MTT assays. Real-time quantitative PCR (RT-qPCR) and western blot analysis were used to detect the protein and mRNA levels. Co-immunoprecipitation (CoIP) and proximity ligation assay (PLA) were used to determine the protein-protein interaction. Chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), and oligo pull-down assays were used to verify DNA/RNA–protein interactions. Luciferase assay analysis was used to detect effects on transcription factor activity. Results: In the present study, we revealed that RBM24 was upregulated in BC tissues. Importantly, we found that higher level of RBM24 was correlated with poor prognosis in BC patients. Overexpression of RBM24 promoted while depletion of RBM24 inhibited BC cell proliferation in vivo and in vitro. Mechanically, RBM24 positively regulated Runx1t1 expression in BC cells by binding to and enhancing Runx1t1 mRNA stability. Runx1t1 in turn promoted RBM24 expression by interacting with TCF4. Furthermore, Runx1t1 in turn promoted RBM24 expression by interacting with the transcription factor TCF4 and depressing transcription of miR-625-5p, which directly targets and normally suppresses RBM24 expression. RBM24-regulated BC cells proliferation was moderated via the Runx1t1/TCF4/miR-625-5p feedback loop. Conclusions: In summary, these results indicate that a RBM24/Runx1t1/TCF4/miR-625-5p positive feedback loop plays a key role in BC oncogenesis. Disruption of this pathway may be a potential therapeutic strategy for BC treatment. interaction between Runx1t1 and TCF4 that suppresses miR-625-5p in transcriptional level, which play as negative regulator of RBM24 by directly targeting RBM24. Taken together, our results indicate that RBM24, Runx1t1, TCF4, and miR-625-5p form a positive feedback loop that can drive the proliferation of BC cells. The RBM24/Runx1t1/TCF4/miR-625-5p pathway may be a potential therapeutic target for BC treatment.


Background
Bladder cancer (BC) is one of the most common malignancies in the urinary system with an estimated 450,000 new cases per year worldwide [1]. Among these cases, approximately 70% of new cases are non-muscle invasive (NMIBC) while 30% are muscle invasive (MIBC) [2]. Surgery, chemotherapy, and radiation therapy are the mainly treatments for BC patients [3]. However, the 5-year survival rate of high-risk patients is still very low, and the current main treatment methods cannot prevent the recurrence or progression of these BC patients [4]. One important reason is the poor understanding of the mechanisms underlying BC development and progression.
Therefrom, it is necessary for a better knowledge of the molecular basis of BC and exploration of innovative therapeutic strategies.
RNA-binding motif protein 24 (RBM24) is a multifunctional protein involved in regulation of pre-mRNA splicing, mRNA stability, and translation, and through these functions it acts as a critical determinant of cell fate and differentiation [5]. It was once believed that RBM24 is preferentially expressed in cardiac and skeletal muscle tissues and primarily serves to regulate embryonic heart development [6,7]. However, recent studies have demonstrated that RBM24 also regulates cancer progression [8]. Hua et al found that RBM24 inhibited the progression of nasopharyngeal carcinoma by upregulating miR-25, which in turn downregulates MALAT1 [9].
Using immunoprecipitation coupled to reverse transcription and microarray analysis (RIP-ChIP), Yu et al demonstrated that RBM24 is a multi-tasking RNA-binding protein (RBP) capable of regulating the stability and expression of multiple bound targets [10]. These RBPs may function as suppressors or facilitators of disease depending on the speci c upstream regulators and downstream effectors (targets) [3]. However, the role RBM24 in BC is still unclear.
Runt-related transcription factor 1 (Runx1t1), is a member of Eight-Twenty-One (ETO) family proteins [11], Run1t1 was rst identi ed through its involvement in a t (8;21) translocation associated with acute myeloid leukemia (AML) [12]. Subsequent studies reported that Runx1t1 acts as a transcriptional co-repressor by interacting with DNA-bound transcription factors and recruiting other proteins to facilitate transcriptional repression [13]. A recent study found upregulated expression of Runx1t1 in cord blood-derived endothelial colony-forming cells [14]. In accord with functions in vascular endothelial development, a Runx1t1 de cient mouse showed reduced angiogenesis [15]. however, Runx1t1 was also reported to suppress colorectal cancer through regulation of cell proliferation and chemotherapeutic drug resistance [16]. and to upregulate the cell cycle genes Cdk4 and Cdk6 by recruiting a histone deacetylase (HDAC)-containing nuclear co-repressor complex [17,18]. But the role of Runx1t1 in BC remains still unclear.
In the present study, we found that the higher RBM24 and Runx1t1 levels in BC tissue which were correlated with poor patient survival in BC patients. Moreover, overexpression of RBM24 upregulated Runx1t1 by stabilizing Runx1t1 mRNA and concomitantly accelerated BC proliferation. We identi ed a protein-protein interaction between Runx1t1 and TCF4 that suppresses miR-625-5p in transcriptional level, which play as negative regulator of RBM24 by directly targeting RBM24. Taken together, our results indicate that RBM24, Runx1t1, TCF4, and miR-625-5p form a positive feedback loop that can drive the proliferation of BC cells. The RBM24/Runx1t1/TCF4/miR-625-5p pathway may be a potential therapeutic target for BC treatment.

Results
RBM24 is upregulated in BC tissues and contributes to poor prognosis.
Our previous study has revealed that RBM5 is downregulated in BC tissue [3]. In contrast, mRNA and protein levels of RBM24 were frequently increased in BC tissue (T) samples compared to normal bladder tissues (N) as revealed by RT-qPCR and western blot analysis of 62 BC specimens ( Fig. 1A and B). Similar results were obtained by immuno uorescence staining with a RBM24-speci c antibody in a cohort of 161 BC specimens ( Fig. 1C and D), and correlation analysis showed that the mRNA level of RBM24 was signi cantly associated with tumor size and stage, but not with other clinicopathologic factors such as age, sex, and tumor grade (Table 1). Additionally, the TCGA database also revealed that higher RBM24 mRNA levels in BC patients were associated with poor overall survival (P = 0.00215, Fig. 1E). Together, these clinical data suggest that upregulation of RBM24 may be a critical event driving BC progression.  To investigate speci c functions of RBM24 in BC, we rst compared protein levels between a normal bladder cell   line (SV-HUC-1) and a series of BC cell lines (UM-UC-3, 253J, T24, and J82). RBM24 was signi cantly elevated in two tumor cell lines (UM-UC-3 and 253J) but downregulated in one (J82 cells) compared to the normal bladder cells ( Fig. 2A). However, mRNA expression of RBM24 was upregulated only in the 253J cell line and decreased in J82 cell line (Fig. 2B). Therefore, these two cell lines were selected for subsequent loss-and gain-of-function experiments. In light of the correlation between RBM24 expression and tumor stage, we speculated that RBM24 may be involved in cell proliferation. Indeed, RBM24 knockdown in 253J cells using a speci c shRNA decreased while overexpression in J82 cells by transfection with a pWPI-RBM24 vector increased RBM24 expression compared to a shRNA control vector and empty overexpression vector, respectively (Fig. 2C). In addition, RBM24 knockdown in 253J cells suppressed expression of the cell proliferation marker CDK4 while overexpression in J82 cells increased CDK4 expression (Fig. 2C). Further, MTT and colony formation assays revealed that overexpression of RBM24 promoted J82 cell proliferation, while RBM24 knockdown restrained the proliferation of 253J cells ( Fig. 2D and E). Together, these data suggest that RBM24 promotes BC cell proliferation.

Runx1t1 mediates the RBM24-induced cell proliferation
Previous studies have reported that RBM24 functions as a regulator in multiple genes linked to cell proliferation, fate, differentiation, and apoptosis [10]. To investigate the molecular mechanisms underlying regulation of BC cell proliferation by RBM24, we examined the expression levels of candidate effectors selected according to previous studies [10]. Among 6 candidate genes, Runx1t1 was signi cantly downregulated in RBM24-depleted 253J cells and upregulated in RBM24-overexpressing J82 cells as revealed by western blot (Fig. 3A). Runx1t1 mRNA levels were also signi cantly upregulated in BC tissue samples compared to normal bladder tissues as measured by RT-qPCR ( Fig. 3B), as were Runx1t1 protein levels according to western blot and immuno uorescence staining ( Fig. 3C and 3D). Moreover, human clinical data from the TCGA database of Oncolnc revealed that higher Runx1t1 expression was associated with poor prognosis (P = 0.0064, Fig. 3E). Additionally, correlation analysis showed that RBM24 mRNA expression was positively correlated with Runx1t1 mRNA expression in BC tissue (Fig. 3F).
To examine whether Runx1t1 contributes to RBM24-regulated BC cell proliferation, we performed a rescue experiment. As shown in Fig. 3G, 253J cell growth was reduced markedly following knockdown of RBM24 and Runx1t1 together compared to knockdown of either gene alone. Conversely, the enhanced proliferation observed in J82 cells overexpressing RBM24 was abolished by co-transfection with shRunx1t1 as evidenced by colony formation assay (Fig. 3H). Together, these data suggest that Runx1t1 participates in RBM24-mediated regulation of BC cell proliferation.
RBM24 promotes Runx1t1 expression by stabilizing its mRNA Because RBM24 and Runx1t1 were positively correlated in BC, we then investigated whether RBM24 regulates Runx1t1 expression in BC cells and the underlying mechanisms. RT-qPCR results showed that overexpression of RBM24 upregulated mRNA expression level of Runx1t1 while knockdown of RBM24 downregulated the mRNA expression level of Runx1t1 in BC cells (Fig. 1A). As previous studies have reported that RBM24 enhances RNA stability [10,19], we investigated if elevated RBM24 prolongs Runx1t1 mRNA expression following blockade of new transcription using actinomycin D (ActD). Indeed, RBM24 knockdown in 253J cells accelerated whereas overexpression in J82 cells prolonged Runx1t1 mRNA expression, consistent with RBM24-mediated enhancement of Runx1t1 mRNA stability (Fig. 4B). To con rm this notion, we examined whether RBM24 binds to Runx1t1 mRNA using in vitro RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays. Coimmunoprecipitation con rmed that a RBM24 antibody effectively pulled down endogenous RBM24 protein (Fig. 4C), and PCR analysis of RIP products revealed the presence of STAT3 and Runx1t1 mRNA 3'-untranslated regions (3′UTRs), but not the 3′UTR of GAPDH (Fig. 4D). Consistent with this nding, a biotinylated Runx1t1 mRNA-3′UTR and positive control STAT3-mRNA probe pulled down RBM24 protein, while a GAPDH mRNA-3′UTR probe did not (Fig. 4E). These results suggest that RBM24 can bind to the Runx1t1 mRNA 3′UTR, thereby enhancing mRNA stability.
We then used western blot to determine whether Runx1t1 regulates the expression levels of genes associated with cell proliferation. Depletion of Runx1t1 in 253J cells reduced expression of the proliferation marker gene CDK4 while overexpression of Runx1t1 in J82 cells increased CDK4 protein level ( Fig. 4F). While RBM24 protein expression was positive regulated by Runx1t1 protein expression, neither depletion of Runx1t1 in 253J cells nor overexpression in J82 cells altered RBM24 mRNA levels ( Fig. 4G).
Runx1t1 interacts with transcription factor TCF4 to mediate RBM24 upregulation and cell proliferation Runx1t1 functions as a transcription cofactor by interacting with various partner proteins [20,21]. To identify the partner(s) involved in RMB24 regulation, we performed co-immunoprecipitation coupled with mass spectrometry (CoIP-MS). Eight proteins were upregulated in RBM24-overexpressing BC cells, including the transcription factor TCF4 (Fig. 5A), and reciprocal immunoprecipitation showed a strong interaction between Runx1t1 and TCF4 in BC cells (Fig. 5B). Also, an in situ proximity ligation assay (PLA) con rmed direct binding between Runx1t1 and TCF4 ( Fig. 5C). In addition, TCF4 mRNA levels were signi cantly upregulated in BC tissues compared to normal bladder tissues (Fig. 5D) and positively correlated with Runx1t1 mRNA (Fig. 5E). Survival analysis from the TCGA database showed that higher expression of TCF4 was associated with poor prognosis (Fig. 5F).
To investigate whether TCF4 participates in a RBM24/Runx1t1 axis to regulate BC cell proliferation, we performed rescue experiments. Co-transfection of 253J cells with shTCF4 and shRunx1t1 inhibited expression of RBM24 and CDK4 to a greater extent that knockdown of Runx1t1 alone (Fig. 5G). Transfection of J82 cells with shTCF4 also sharply reduced RBM24 and CDK4 expression levels, and this reduction was reversed by cotransfection with Runx1t1 overexpression vector (Fig. 5H). Knockdown of TCF4 and Runx1t1 also decreased the proliferation rate of 253J cells to a greater extent than knockdown of either protein alone. Conversely, overexpression of Runx1t1 promoted J82 cell growth, an effect abolished by shTCF4 co-transfection (Fig. 5I).
Since Runx1t1 alone did not regulate RBM24 transcription (Fig. 4G), we examined whether TCF4 regulates RBM24 transcription. However, neither Runx1t1 nor TCF4 regulated RBM24 mRNA expression (Fig. 5J). Together, these data suggest that Runx1t1 interacts with TCF4 to promote RBM24 protein expression rather than gene transcription. miR-625-5p mediates Runx1t1/TCF4-regulated proliferation by direct targeting in BC cells Regulation of RMB24 protein expression by Runx1t1/TCF4 without in uencing RBM24 mRNA level suggests that Runx1t1/TCF4 regulates RBM24 at the posttranscriptional level. MicroRNAs (miRNAs) are critical regulators of gene expression at the posttranscriptional level, so we conducted bioinformatics analyses using TargetScan, miRanda, and RNA22 to predicted miRNA sequences targeting the RBM24 3'-UTR and then used a T7 RNA transcriptase to generate a RBM24 3'-UTR containing biotin-labeled uracil (Fig. 6A). After transfection, complementary miRNAs were extracted by pull-down assay and the expression levels of 9 candidates compared by RT-PCR. The result showed that miR-149-3p, miR-216a-5p, miR-625-5p, miR-449a, and miR-578, were enriched by the RBM24 3'-UTR pull-down assay (Fig. 6B). Next, we assessed miRNA expression levels after up-and downregulation of Runx1t1 and TCF4 expression to identify the miRNAs involved in Runx1t1/TCF4-mediated regulation of RBM24 expression. As shown in Fig. 6C, only miR-625-5p was regulated by both Runx1t1 and TCF4.
To investigate whether the Runx1t1/TCF4 complex regulates RBM24 expression by directly depressing miR-625-5p transcription, we rst predicted the potential TCF element within the 2-kb 5'-promoter region of miR-625-5p using Ensembl and PROMO 3.0 websites, which identi ed three potential TCF elements (Fig. 6J). ChIP analysis con rmed that Runx1t1/TCF4 bound predominantly to the region − 522 to − 532 bp upstream of the transcription start site within the miR-625-5p promoter (Fig. 6K), and a luciferase assay yielded similar results (Fig. 6L). Together, these ndings indicate that Runx1t1/TCF4 directly inhibits the transcription of miR-625-5p, which in turn disinhibits RBM24 expression.
Disruption of the RBM24/Runx1t1/TCF4/miR-625-5p axis inhibits BC xenograft growth in vivo Finally, we examined whether RBM24 and Runx1t1 regulate BC cell growth in vivo using a xenograft model. Injection of 253J cells with stable knockdown of RBM24 and Runx1t1 yielded smaller tumors in nude mice than injection of sham-transfected 253J cells. Furthermore, the tumor volume was much smaller in mice implanted with RMB25/Runx11 double knockdown cells compared to single knockdown cells (Figs. 7A and B). These ndings were mirrored by mean tumor wet weights following excision (Fig. 7C). Western blot analysis of extracted tumor tissue also demonstrated that silencing of either Runx1t1 or RBM24 signi cantly downregulated Runx1t1, RBM24, and CDK4 compared to tumors derived from control cells, and this downregulation was further enhanced by simultaneous knockdown of both Runx1t1 and RBM24 (Fig. 7D).These ndings suggest RBM24/Runx1t1/TCF4/miR-625-5p axis inhibits cell proliferation in BC cells in vivo. Figure 8 shows our proposed model illustrating the role of the RBM24/Runx1t1/TCF4/miR-625-5p feedback loop in BC.

Discussion
In this study, we identi ed a RBM24/Runx1t1/TCF4/miR-625-5p feedback loop that drives BC oncogenesis. First, RBM24 expression was signi cantly higher in BC tissues than corresponding normal tissues, and elevated RBM24 expression was correlated with poor prognosis. Second, RBM24 overexpression promoted BC cell proliferation in vivo and in vitro, and enhanced Runx1t1 protein expression by increasing Runx1t1 mRNA stability. Third, Runx1t1 interacted with the transcription factor TCF4, and this Runx1t1-TCF4 complex positively regulated RBM24 protein expression by suppressing expression of the RBM24 negative regulator miR-625-5p, resulting in formation of a positive feedback loop driving elevated RBM24 expression and BC cell proliferation.
These ndings suggest that the RBM24/Runx1t1/TCF4/miR-625-5p axis is a critical promoter of BC initiation and progression.
RBPs in uence the structure and interactions of target RNAs, thereby in uencing RNA biogenesis, stability, function, transport, and subcellular localization [22]. Many RBPs are expressed in a tissue-speci c manner to drive developmental processes [23]. For instance, RBM24 is highly expressed in heart and muscle tissues [7], and regulates cardiac embryonic stem cell differentiation by a splicing-mediated mechanism [24]. In addition, RBM24 was reported to suppress nasopharyngeal cancer progression [9]. In the current study, however, RBM24 accelerated BC cell proliferation both in vivo and in vitro. This discrepancy may be explained by differences in upstream and downstream regulatory factors [25]. Consistent with this accelerated proliferation, high levels of RBM24 promoted the expression of Runx1t1 and correlated with poor prognosis in BC patients.
Transcription factor 4 (TCF4) is a member of helix-loop-helix (bHLH) transcription factor family that recognize and bind the Ephrussi box (E-Box) DNA element (5′-ACANNTGT-3′) [26]. TCF4 regulates chromatin remolding and transcription by recruiting histone acetyltransferases (HATs) such as p300 [27]. Numerous studies have also implicated TCF4 in cancer progression. Jagrut et al reported that TCF4 signaling was upregulated in colon cancer stem cells and promoted growth and self-renewal [28]. The expression of TCF4 is associated with breast cancer chemoresistance [29]. Additionally, high expression of TCF4 was an independent adverse prognostic factor in acute myeloid leukemia [30]. However, the expression and function of TCF4 in BC is largely unknown. In the present study, we found that TCF4 was upregulated in BC tissues and that high expression was predictive of poor prognosis. We demonstrated that interaction with Runx1t1 and subsequent downregulation of miR-625-5p, resulting in RBM24 upregulation, is one mechanism underlying the oncogenic effect of TCF4.

Conclusion
The present study demonstrates that upregulation of RBM24 enhances BC cell proliferation and leads to poor BC prognosis by initiating a Runx1t1/TCF4/miR-625-5p feedback loop. These ndings highlight the RBM24/Runx1t1/TCF4/miR-625-5p axis as a potential therapeutic target for BC treatment.

Clinical samples
Human primary BC tissues and the corresponding normal bladder tissues were collected from the BC patients who were admitted to the Department of Urology of the Second Hospital of Hebei Medical University from July  FAST system (Life Technologies) with primers listed in Table 2. Relative transcript expression levels were normalized to GAPDH and calculated using the 2 −ΔΔCt formula as previously described (29).

Proximity ligation assay
The proximity ligation assay (PLA) was performed as described previously [31]. Brie y, 253J cells were seeded into 6 well chamber slides and cultured for 24 hours. Then 4% paraformaldehyde were used to xate the sliders.
anti-Runx1t1 and anti-TCF4 were used to stain the slides. Rabbit PLUS and Mouse MINUS Duolink in situ proximity ligation assay (PLA) kits were used to detect the interaction between the two proteins following the manufacturer's protocols. Fluorescence was detected using a laser scanning confocal microscope.

Western blot analysis
Western blot analysis was carried on as described previously [32]. Vector construction and luciferase reporter assay The 3' untranslated region (UTR) sequences of RBM24 containing wild-type forms of the miR-625-5p target site were inserted into the Xho1 and EcoR1 digested-psiCHECK Vector (Promega Corp). 2 kb miR-625 promoter sequence was obtained by PCR with primer and inserted into the Mlu1 and Xho1 digested-pGL3-basic vector (Promega Corp., Madison, WI, USA). Luciferase assay was performed as described previously (32). In brief, 293A cells were seeded into a 24-well plate, RBM24 reporter construct (wild-type or mutant) or the empty reporter vector was co-transfected with miR-625-5p mimic and pRL-TK, or co-transfected with mimic control and pRL-TK, or 293A cells were co-transfected with pGL3-miR-luc vector or oeRBM24 or shTCF4 for 24 h. Luciferase activity was measured by Dual-Glo Luciferase Assay System (Promega, Madison, WI) with a Flash and Glow (LB955, Berthold Technologies) reader. The speci c target activity was expressed as the relative activity ratio of re y luciferase to Renilla luciferase.

Xenograft animal model
Xenograft model was performed as described previously [3,32,33]. Male BALB/c nude mice at 4-6 weeks of age

Morphometry and histology
Human bladder cancer and normal bladder tissues were xed with formalin solution and then processed for routine embedding in para n. Ten consecutive 5-µm-thick sections were prepared for hematoxylin and eosin staining. The cross-section Images were acquired using a Leica microscope (Leica DM6000B, Switzerland) and digitized with LAS V.4.4 (Leica).

RNA immunoprecipitation (RIP) assays
Chromatin immunoprecipitation (ChIP) assay The chromatin immunoprecipitation (ChIP) assay was performed as described previously [3]. In brief, 253J cells were treated with 1% formaldehyde for 10 min to cross-link proteins with DNA. The cross-linked chromatin was then prepared and sonicated to an average size of 400-600 bp. The samples were diluted 10-fold and then precleared with protein A-agarose/salmon sperm DNA for 30 min at 4 °C. The DNA fragments were immunoprecipitated overnight at 4 °C with anti-Runx1t1, or anti-TCF4 or anti-IgG (as negative control) antibodies. After cross-linking reversal, TCF4 and Runx1t1 occupancy on the miR-625 promoter was examined. Results were determined by qRT-PCR. ChIP primer sequences were summarized in Table 2       Kaplan-Meier analysis was used to analyze the overall survival of BC patients with low or high TCF4 level from TCGA database. P = 0.0014. G and H, Western blot analysis examined the TCF4, Runx1t1, CDK4 and RBM24 expression in 253J cells transfected with shTCF4 and shRunx1t1 either alone or together (G), or J82 cells transfected with oeRunx1t1 and shTCF4 either alone or together (H). Button panel shown densitometric analysis in three independent experiments. *P < 0.05, **P < 0.01vs. corresponding control. (I) Cells were prepared as (G and H), cell viability was measured by MTT assay. *P < 0.05 vs. corresponding control. (J) Cells were prepared as (G and H), RT-qPCR was used to detect the expression of RBM24 mRNA. Figure 6 miR-625-5p mediates Runx1t1/TCF4-regulated proliferation by direct targeting in BC cells . (A) Venn diagram displaying potential microRNAs associated with RBM24 3'UTR sequence from three online target-prediction programs. (B) Biotin-labeled RBM24 3'-UTR RNA was transfected into J82 cells followed by a biotin pull-down assay using Streptavidin-coupled Dynabeads. The miRNAs were extracted from the sedimented beads, and the relative levels of 9 candidate miRNAs were detected by RT-qPCR. *P< 0.05, **P < 0.01 vs. control probe. (C) J82 cell was transfected with oeRunx1t1 or shTCF4 or co-transfected with both vectors together. RT-qPCR was used to detect the expression of 5 miRNAs. *P < 0.05, **P < 0.01 vs. their corresponding controls. (D) J82 cell was cotransfected with RBM24 3'UTR and indicated miRNAs mimic. Luciferase reporter assays was showed miR-625-5p and miR-449a reduced the RBM24 3'UTR luciferase activity. *P < 0.05, **P