Background
Bladder cancer is the fourth most common malignant tumor and the 13
th leading cause of death in cancer patients worldwide [
1,
2]. In addition, bladder cancer is the most common cancer in the urinary system in China [
3]. Approximately 90% of bladder cancers are uroepithelial cancers. According to the depth of invasion, BCa can be classified into nonmuscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) [
4]. A total of 19.8%-54% of high-risk NMIBC patients will progress to MIBC [
5]. The overall 5-year survival rate of MIBC is approximately 60%-70%. About 10% of new-onset MIBC is metastasized, and the 5-year survival rate is 5%-30%. Localized MIBC is feasible for radical cystectomy and pelvic lymph node dissection, but approximately half of these patients will have distant metastasis and recurrence after surgery [
1,
6]. Therefore, more in-depth investigations into the tumorigenesis and progression of bladder cancer are of great significance.
With the updating and advancement of high-throughput sequencing and molecular biological technologies, noncoding RNAs have exerted emerging roles in various physiological and pathological processes. CircRNA is a profound mystical kind of long noncoding RNA. Most circRNAs consist of exons from existing protein-coding genes through noncanonical back-splicing [
7]. Because of their covalent loop structure, circRNAs can tolerate degradation by exonucleases and are more stable than linear RNAs [
8]. Emerging literature has reported that circRNAs regulate the occurrence and development of multiple human diseases, especially cancers [
9‐
11]. To date, it has been elucidated that circRNAs can serve as microRNA sponges or decoys [
12,
13], modulate the expression levels of specific proteins [
14,
15], and serve as scaffolds accelerating interactions between proteins by colocalization [
16,
17]. Our previous studies have indicated that circRNAs participate in bladder cancer progression by acting as miRNA sponges [
18,
19]. However, more thorough research on circRNAs modulating the tumorigenesis and development of bladder cancer is of great significance.
The Hippo pathway is a highly conserved modulating system controlling organ development, regeneration of tissue, cell proliferation and immune regulation [
20]. Upstream signaling transmission of the Hippo pathway is mainly mediated by phosphorylation. Phosphorylated TAZ/YAP is retained in the cytoplasm and eventually degraded. LATS1, a core factor in the kinase cascade of the Hippo pathway, plays a vital role during this process. LATS1 phosphorylates TAZ/YAP, increasing the retention rate of TAZ/YAP in the cytoplasm and thus suppressing the transcriptional activity of TAZ/YAP [
21]. Notably, emerging evidence supports the aberrant status of the Hippo pathway in human cancer, promoting tumorigenesis, metastatic progression, and metabolic shift [
22‐
25].
Histone lactylation was newly discovered by Zhang et al. in 2019. This brand-new epigenetic modification relies on lactate produced by intracellular metabolism and regulates cell biological functions by activating downstream gene transcription and expression [
26]. Aerobic glycolysis, also termed the Warburg effect, is one of the most characteristic features of tumor cells and refers to the preferential production of energy through glycolysis rather than oxidative phosphorylation even under normoxia [
27]. Therefore, tumor cells will produce and accumulate more lactate than normal cells, which makes the functions of histone lactylation worth exploring in tumors. However, the specific roles of histone lactylation in human bladder cancer remain unclear.
Here, we discovered, for the first time, that circXRN2 activates the Hippo pathway by stabilizing LATS1, which in turn suppresses H3K18 lactylation-driven tumor progression in human bladder cancer. Our findings provide an original molecular mechanism of circRNA and expand our understanding of the Hippo pathway and histone lactylation in bladder cancer.
Materials and methods
Ethical approval
The use of clinical samples with patient consent (Approval Number: IIT20220447B) and the xenograft tumorigenesis model and lung-metastatic model (Approval Number: 20221595) in this study were approved by the Ethical Committee, The First Affiliated Hospital, Zhejiang University School of Medicine, and the operations and experimental protocols were performed according to the laboratory guidelines of the NIH. Detailed information on the surgical samples is listed in Supplementary file
1.
RNA immunoprecipitation (RIP)
An RNA Immunoprecipitation Kit (Geneseed, Guangzhou) was used for the RIP assay according to the manufacturer’s instructions. In brief, 100 μL of cell lysates was set aside for input control. To obtain the antibody-bead complex, pretreated magnetic Protein A + G beads were coincubated with specific antibodies and IgG (5 μg) at 4 °C and rotated for 120 min at 10 rpm. After the reaction was completed, the supernatant was discarded on the magnetic frame. RNA complexes bound to beads were washed, and RNA was extracted for subsequent assays.
CUT&Tag
The CUT&Tag assay was performed using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina according to the manufacturer’s instructions. Briefly, prepared concanavalin A-coated magnetic beads (ConA beads) were added to resuspended cells and incubated at room temperature to bind cells. The nonionic detergent digitonin was used to permeate the cell membrane. Then, H3K18la antibody (PTM-1427RM, PTM BIO, Hangzhou, China), secondary antibody and Hyperactive pA-Tn5 Transposase were incubated with the cells that were bound by ConA beads. Therefore, the hyperactive pA-Tn5 transposase can exactly cut off the DNA fragments that were bound with the target protein. In addition, the cut DNA fragments can be ligated with P5 and P7 adaptors by Tn5 transposase, and the libraries were amplified by PCR with the P5 and P7 primers. The purified PCR products were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Finally, these libraries were sequenced on the Illumina NovaSeq6000 platform, and 150 bp paired-end reads were generated for the following analysis. The results are presented in Supplementary files
6 and
7.
RNA fluorescence in situ hybridization (FISH)
The Cy3-labeled circXRN2 probe was synthesized and obtained from RiboBio (Guangzhou, PR China). Later, a FISH Detection Kit (RiboBio, PR China) was employed according to the manual. Cell nuclei were stained with DAPI. The results were captured by a microscope (Olympus, Tokyo, Japan). The details of the probe used in this study are listed in Supplementary file
3.
Agilent Seahorse XFe24, a noninvasive and real-time analyzer of glycolysis in living cells, was used to evaluate the glycolytic rate of different cells in this research. The rate of glycolysis can be reflected by the extracellular acidification rate (ECAR). To eliminate the influence of mitochondrial respiration, the extracellular acidification caused by mitochondrial respiration is calculated through the oxygen consumption rate (OCR). Briefly, equal numbers of tumor cells (5 × 104) were seeded into 24-well plates. The next day, the cells were rinsed with Seahorse detection buffer. Then, the analyzer injected Rot/AA and 2-DG (2-deoxy-glucose) automatically. The glycolytic proton efflux rate (glycoPER), basal glycolysis rate and compensatory glycolysis rate can be calculated to reflect the real-time glycolytic status of cells by the analyzer.
Glycolytic process evaluation
To evaluate the status of glycolysis, 2-NBDG and Glucose Uptake Colorimetric Assay Kits (Biovision, USA) were used to determine glucose uptake ability, and the production of lactate was measured by lactate colorimetric assay kits (Biovision, USA). All procedures were performed according to the manual.
Cell lines and culture
All cell lines cultured in this study were obtained from the Chinese Academy of Sciences. In detail, RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin‒Streptomycin Solution (P/S) was prepared for culturing 5637, T24 and EJ; F-12K medium supplemented with 10% FBS and 1% P/S was prepared for culturing SV-HUC-1; MEM medium supplemented with 10% FBS and 1% P/S was prepared for TCCSUP; DMEM supplemented with 10% FBS and 1% P/S was prepared for UM-UC-3. The culture conditions were 37 °C and 5% CO2.
RNA extraction and quantitative real-time PCR analysis
Total RNA was extracted by NucleoZOL® RNA Isolation Reagent (MACHEREY–NAGEL, Germany). After reverse transcription by using the PrimeScript™ RT Reagent Kit (Perfect Real Time), polymerase chain reaction was conducted by an Applied Biosystems QUANT5 Studio PCR system. GAPDH was used as an endogenous control. The amplification conditions were as follows: a. denaturation at 95 °C for 10 s; b. annealing at 72 °C for 20 s; and c. extension at 60 °C for 20 s for 40 cycles. The Ct values were collated according to the results, and the 2
−ΔΔCt method was used to calculate the relative expression levels of target genes. The primers used in this study are listed in Supplementary file
3.
SiRNA, plasmid and lentivirus
SiRNAs were obtained from TranSheepBio (Shanghai, China). We transfected siRNAs into cells with Lipofectamine RNAiMAX (Invitrogen). To overexpress circXRN2 in bladder cancer cells, Genomeditech (Shanghai, China) designed and obtained cDNA containing full-length circXRN2 and embedded it into the pcD-ciR vector. Then, the plasmids were transfected into cells via Lipofectamine 3000 (Invitrogen). For lentivirus-related experiments, we treated cells with puromycin for one week to ensure the efficiency of transfection. The sequences of siRNAs in this study are listed in Supplementary file
3.
Chromatin immunoprecipitation (ChIP)
To investigate the interaction between H3K18la and the promoter region of LCN2, a SimpleChIP® Plus Enzymatic Chromatin IP Kit (CST, #9004, USA) was used according to the manufacturer’s procedure in our study.
Protein immunoprecipitation (IP)
The IP experiment was performed using the Absin Co-IP kit (Absin, ab955, China) according to the manufacturer’s instructions. Briefly, a total of 1 × 107 cells were collected and washed with PBS. After 15 min of lysis, the supernatant was incubated with specific antibodies at 4 °C for 12 h with rotation. The next day, 5 μL of Protein A and 5 μL of Protein G were added, incubated with rotation for 2 h at 4 °C, and then centrifuged at 12,000 × g for 1 min. Finally, the pellet was retained for later use.
CCK-8 assay
To evaluate cell viability, we employed Cell Counting Kit-8 (APEBIO, USA) according to the manufacturer’s instructions. Briefly, 1 × 104 pretreated cells were seeded into 96-well plates after counting, and the volume of medium added to each well was 100 μL. The next day, 10 μL CCK8 solution was added to each well, and the cells were incubated for 2 h in a 37 °C incubator. Finally, absorbance (450 nm) was measured by a SpectraMax i3x reader. Before relative cell viability was calculated, the values of the blank control well were subtracted.
A total of 2000 cells were cultured in 12-well plates for colony formation. After one week of incubation, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. The colonies were photographed and counted.
Transwell migratory experiment
To evaluate cell migratory ability, equal numbers (3 × 104) of T24 cells were seeded into the upper chamber and incubated overnight. Subsequently, 500 μL complete medium supplemented with 10% FBS was added into the lower chamber of the Transwell insert to promote cell migration. After another 24-h incubation, cells migrating through the membrane of Transwell inserts were stained with crystal violet and photographed by microscopy (100 ×).
Wound healing assay
We used an Ibidi culture insert (Ibidi, Germany) to evaluate the migratory ability of different cells. Briefly, bladder cancer cells were seeded into cultured plates according to the manufacturer’s protocol and incubated overnight. The next day, the inserts were removed, and serum-free medium was added. After 36 h, bright field images were acquired with a microscope. ImageJ was used to calculate the migratory rate.
Analysis of apoptosis
The apoptotic rate was determined by an Apoptosis Detection Kit (BD, USA). Annexin V-FITC binds to outward phosphatidylserine in the early stage of apoptosis. In addition, necrotic or late apoptotic cells can be stained by PI. Briefly, cells were harvested and stained with Annexin V-FITC and PI at room temperature. The apoptotic rate was determined and analyzed by BD FACS Calibur (BD, USA) and FlowJo software.
Western blot
RIPA buffer (Beyotime, Shanghai) was used to extract total protein from cells. Then, we measured the concentration of protein with a BCA assay kit (Beyotime, Shanghai). Furthermore, samples were separated by SDS‒PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Primary antibodies against specific genes were diluted in the NCM Universal Antibody Diluent (WB500D, NCM Biotech, China) and incubated with PVDF membranes overnight at 4 °C. The next day, the membranes were incubated with secondary antibodies at room temperature for 2 h. The chemiluminescence method was used to determine the relative expression levels of different genes. Antibody against H3K18la were purchased from PTM BIO (PTM-1406RM, Hangzhou, China).
Immunofluorescence assay
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X. Later, 2.5% BSA solution was used for blocking. After incubation with primary antibodies and secondary antibodies, images were captured by a microscope (Nikon, Japan). The nuclei were stained with DAPI.
Subcutaneous tumorigenesis model
T24 cells (6 × 106) were suspended in PBS and injected subcutaneously into nude mice (4 weeks old). The parameters of subcutaneous tumors were recorded for 7 days. Of note, the volume of the tumor was speculated by the formula below: tumor volume = 0.5 × length × width2 (mm3). After 35 days, tumors were harvested from sacrificed mice and weighed. We took care of the experimental animals in accordance with the guidelines of the Laboratory Animal Center, Zhejiang University.
T24 cells (5 × 106) were suspended in PBS and injected via the tail vein into 6-week-old nude mice. Fifty days later, the mice were sacrificed, and the lung tissues were dissected. Tissues were fixed for H&E staining. We took care of the experimental animals in accordance with the guidelines of the Laboratory Animal Center, Zhejiang University.
Statistical analysis
Differences between groups were analyzed by one-way or two-way ANOVA in SPSS software, and the data in this study are presented as the mean ± standard deviation (SD). A P value < 0.05 was regarded as statistically significant.
Discussion
In general, due to the huge amount of information in the genome and variable coding mechanisms, the third nucleotide of the codons in the exons often varies. However, the exons involved in the formation of the relevant circRNA are on the contrary, and their sequences are evolutionarily conserved, which suggests that circRNA plays an important noncoding role in eukaryotic cells [
38]. Existing studies have only clarified and excavated a small part of the biological functions of circRNAs, in which most studies have reported that they act as microRNA sponges or decoys [
12,
13]. For example, our previous studies demonstrated that circRIMS1 and circPPP1CB could serve as miRNA sponges to regulate bladder cancer progression [
18,
19]; circRHOBTB3 is an endogenous competitive RNA for miR-654-3p to suppress gastric cancer growth [
39]; the circNSD2/miR-199b-5p regulatory axis was confirmed to be involved in the progression of colorectal cancer [
40] and hsa_circ_0003258 regulates prostate cancer progression by binding to miR-653-5p and modulating IGF2BP3 expression [
41]. Nevertheless, current studies rarely investigate novel molecular mechanisms and regulatory models of circRNAs in human bladder cancer, and further studies are urgently needed.
First, our study demonstrated that circXRN2 could interact with the LATS1 protein and was dysregulated in bladder cancer cell lines and surgical samples. Meanwhile, circXRN2 suppressed cell proliferation and migration in vitro and in vivo. In addition, we validated that circXRN2 overexpression upregulated LATS1 protein levels by inhibiting its ubiquitylation and degradation.
Apart from functioning as endogenous competitive RNAs, circRNAs can also serve as scaffolding molecules that modulate protein‒protein interactions and the stability of proteins and then regulate various signaling pathways and biological processes. For example, by forming the circFOXO3/p21/CDK2 ternary complex, circFOXO3 enhances the interaction between p21 and CDK2 and reduces CDK2 phosphorylation activity [
17]. A similar effect is also found in circNFIX, which strengthens the interplay between Y-box binding protein 1 (YBX1) and NEDD4-like E3 ubiquitin ligase (Nedd4l), leading to YBX1 ubiquitination [
42]. A recent study showed that SPOP increases ubiquitination of the LATS1 protein and promotes its degradation in kidney cancer [
29]. SPOP, as a subunit of the Cullin-RING E3 ligase, exerts its biological functions by recognizing specific substrates for ubiquitination [
43]. In light of this evidence, we further demonstrated that circXRN2 interacts with the SPOP-binding degron on the LATS1 protein and prevents LATS1 from undergoing SPOP-mediated ubiquitination and degradation.
The Hippo signaling pathway consists of a kinase cascade and two executors (TAZ and YAP) [
31]. As research has progressed, increasing attention has been garnered on the regulation of tumorigenesis by mediating the Hippo pathway. For instance, the E3 ubiquitin ligase PARK2 promoted esophageal squamous carcinoma progression by inhibiting the Hippo/YAP signaling pathway [
44]. MYBL2 impairs the Hippo signaling pathway and regulates castration resistance and metastasis in prostate cancer [
45]. In addition, the Hippo pathway also participates in tumorigenesis in multiple human cancers [
46‐
48]. Considering that LATS1 is a key molecule in the kinase cascade of the Hippo pathway, we further demonstrated that circXRN2 activates the Hippo signaling pathway, thereby regulating biological functions in bladder cancer cells.
Histones are a type of protein that binds to DNA in eukaryotic nucleosomes and regulates DNA-templated processes. Usually, there are a large number of posttranslational modifications (PTMs) at the N-terminal tail, such as methylation, acetylation, and succinylation [
49]. In 2019, histone lactylation was discovered and reported for the first time by Zhang et al. [
26]. Histone lactylation is derived from lactate produced by cellular metabolism and modulates biological processes by promoting gene transcription. Different from normal cells, glycolysis, instead of aerobic oxidation, is preferred for tumor cells to acquire enough energy for maintaining their rapid growth and survival, even under well-oxygenated conditions. The above biological phenomenon is also termed the Warburg effect and is one of the metabolic characteristics in tumor cells [
50]. Therefore, tumor cells will produce and accumulate more lactate than normal cells, suggesting that histone lactylation in tumors is more likely to be aberrant and worthy of investigation. To date, emerging literature has shown that histone lactylation drives tumor progression in various types of human cancer. Numb/Parkin-mediated mitochondrial fitness governs the differentiation of prostate cancer and lung adenocarcinoma cells via regulation of histone lactylation [
51]. Meanwhile, H3K18la promotes METTL3-mediated m
6A modification to enhance the immunosuppressive effect of tumor-infiltrating myeloid cells [
52]. H3K18la can also modulate the expression of YTHDF2 to participate in the progression of ocular melanoma [
53]. Nevertheless, the exact roles of histone lactylation in human bladder cancer remain to be discovered. In the current research, we discovered the vital role and aberrant expression level of H3K18 lactylation in human bladder cancer tumorigenesis. Until now, it has been well known that histone lactylation and acetylation at lysine residues engage in a competitive relationship and serve as indicators for the levels of lactate and acetyl-CoA [
54]. The fate of cells, whether they lean toward malignancy or not, hinges on the outlet of pyruvate committed to lactate or acetyl-CoA generation as the end product of glycolysis. While increased acetyl-CoA synthesis propels the tricarboxylic acid (TCA) cycle toward the efficient utilization of glucose and the generation of ATP, tumor cells are distinguished by their heightened production of lactate, which fuels uncontrolled cell growth by facilitating excessive biomass production [
55]. Moreover, histone lactylation exhibits distinct temporal dynamics and exerts different effects on gene transcription when compared to histone acetylation. However, the investigation of histone lactylation, its interaction with histone acetylation, and other posttranslational modification events is still in its early stages. In forthcoming research, our focus will be on delving deeper into the underlying mechanisms governing these histone epigenetic marks and their phenotypic manifestations, thus broadening our comprehension of the treatment of human bladder cancer.
Notably, dysregulation of the Hippo pathway induces metabolic reprogramming in human cancers, including glucose metabolism, glutamine metabolism, fatty acid metabolism and other metabolites [
56]. A study reported that METTL3 regulates glycolysis by regulating m6A methylation of the key molecule of the Hippo pathway, LATS1, in breast cancer [
57]. Moreover, the HIF-1α/YAP signaling axis modulates glucose/iodine metabolism in papillary thyroid cancer progression [
58]. Xu and colleagues verified that LINC00941 enhances the Warburg effect of pancreatic cancer cells by modulating the Hippo pathway [
59]. In light of this evidence, our research also confirmed for the first time that circXRN2 suppressed H3K18 lactylation by activating the Hippo pathway and that H3K18 lactylation exerted its oncogenic functions by enhancing oncogene LCN2 expression. However, the underlying mechanism by which LCN2 promotes bladder cancer needs to be explored in the future.
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