NAT10 affects the progression of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating CCL2

doi:10.21203/rs.3.rs-4099955/v1

Download PDF

Research Article

NAT10 affects the progression of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating CCL2

https://doi.org/10.21203/rs.3.rs-4099955/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Intrahepatic cholangiocarcinoma (ICC) is a highly lethal hepatobiliary tumor and its incidence is on the rise. As a cancer of unknown primary causes, the pathogenesis and related biomarkers of ICC still needs to be investigated. N-acetyltransferase 10 (NAT10) is essential for cellular mRNA stability and tumor cell progression; however, the detailed mechanism underlying its role in ICC is unknown. Here, we examined the role of NAT10 in ICC and deeply investigated its effect on macrophage polarization. Tissue microarray (TMA) analysis shown that high expression of NAT10 was positively associated with poor clinicopathological manifestations of CCA. Silencing of NAT10 inhibited the proliferation of ICC cells in vitro and tumor growth in vivo, whereas NAT10 overexpression promoted ICC progression. Mechanistically, NAT10 binds to the C-C motif chemokine ligand 2 (CCL2) mRNA and elevates its protein levels, thereby promoting the proliferation of ICC cells and M2 polarization of macrophages. Molecular docking screening and the surface plasmon resonance (SPR) identified a natural product, berberine (BBR), which targeted CCL2 and thereby inhibited ICC progression and reduced M2 polarization of macrophages. In summary, NAT10 promotes ICC progression and M2 polarization of macrophages by increasing CCL2. BBR inhibits ICC progression by targeting CCL2 and is an attractive novel compound for targeted therapy.

NAT10

CCL2

intrahepatic cholangiocarcinoma

M2 polarization

berberine

Cholangiocarcinoma (CCA) is a highly aggressive malignancy that originates from the bile duct epithelium. CCA accounts for approximately 3% of all gastrointestinal malignancies and can be divided based on its anatomic location of origin into intrahepatic CCA (ICC), perihilar CCA, and distal CCA [1, 2]. ICC accounts for 10% of all primary liver cancers [1, 3, 4], and its incidence is increasing [5–7]. Surgical resection is the only possible curative treatment, and the 5-year overall survival rate is 15–40% [8]. However, up to two-thirds of patients will relapse after surgical removal [9]. Although gemcitabine combined with cisplatin is effective in patients who do not undergo surgery or require adjuvant chemotherapy, the median survival in these patients is only 11.8 months [10]. In addition, there is no targeted therapy for ICC. Therefore, uncovering the pathogenesis and identifying therapeutic targets is burning question for the treatment of ICC.

In recent years, the role of RNA acetylation, particularly ac4C modification, in tumors has begun to attract attention. N-acetyltransferase 10 (NAT10), a GCN55-related N-acetyltransferase, is the only writer of ac4C. It has an acetyltransferase domain and a lysine-rich carboxyl terminus. NAT10 plays critical role in mRNA stability and translation efficiency by modifying mRNA with ac4C [11], multiple studies have examined how NAT10 regulates tumor progression by modifying mRNA with ac4C [12–16]. However, its regulating pattern in ICC and the tumor microenvironment (TME) remains unclear. The TME is composed of all non-cancerous host cells and non-cellular components in the tumor and plays an important role in the occurrence and development of tumors. Macrophages are an important component of the TME. They are mainly divided into two states with opposite functions: M1 macrophages (pro-inflammatory, usually antitumor) and M2 macrophages (anti-inflammatory, pro-tumor) [17], which can be transformed into each other [18].The intracellular distribution of NAT10 is mainly located in the nucleolar region, which affects the progression of tumor cells by regulating other proteins or pathways. In the present study, the specific regulatory mechanisms of NAT10 in ICC and its relationship with TME will be uncovered.

NAT10 is upregulated in ICC

Surfing in The Cancer Genome Atlas (TCGA) database, we found NAT10 was significantly upregulated in CCA (P < 0.01) (Fig. 1A), as well as in a variety of other solid tumors of the digestive system in TCGA, including stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), and rectum adenocarcinoma (READ) (Fig. 1B). There was no difference in overall survival (OS) or disease-free survival (DFS) between CCA patients with high and low NAT10 expression. However, patients with high NAT10 expression had significantly poorer OS and DFS than those with low expression in most solid tumors (Fig. 1C, Supplementary Fig. 1). The possible reasons for this result are the high degree of malignancy of CCA, late stage when the patient diagnosed with no chance of surgery, short survival time, and small number of samples in the database.

The tissue microarray (TMA) of 90 patients with CCA and corresponding adjacent tissues were used to further verify the expression of NAT10 and its clinicopathological features. Data shown that higher NAT10 expression was observed in CCA tissues (Fig. 1D, E), and NAT10 expression was significantly correlated with tumor location, histological grade, and primary tumor stage (Supporting Table 1). qRT-PCR data shown that NAT10 was upregulated in CCA cells (RBE, HuCCT1, and TFK-1) compared to bile duct epithelial cells (HIBEpiC) (Fig. 1F).

NAT10 promotes the growth of ICC in vivo and in vitro

To investigate the functional role of NAT10 in ICC, we constructed three independent short hairpin RNA (shRNA) sequences (shNAT10#1, shNAT10#2, and shNAT10#3). ShNAT10#1 and shNAT10#2 were selected for subsequent experiments own to their silencing efficiency (Fig. 2A, B). In addition, we constructed NAT10-overexpressing cell lines and validated them at the mRNA and protein levels (Supplementary Fig. 2A, B). The CCK8 assay showed that knockdown of NAT10 significantly inhibited the proliferation of ICC cells, whereas overexpression of NAT10 increased the proliferation of ICC cells (Fig. 2C, Supplementary Fig. 2C). Live-cell imaging was used to photograph the cells every 2 h for 120 h, and the data were consistent with the CCK8 (Fig. 2D, E, Supplementary Fig. 2D, E). Colony formation assay was performed to determine the long-term effects of NAT10 on ICC cell proliferation. After 8–12 days, NAT10 knockdown significantly reduced colony formation (Fig. 2F), whereas NAT10 overexpression significantly increased colony formation (Supplementary Fig. 2F). To evaluate the effects of NAT10 on ICC in vivo, we constructed an animal xenograft model by injecting HuCCT1 cells subcutaneously into the left forelimbs of nude mice. Consistent with the in vitro results, the growth rate of NAT10-knockdown xenografts was slower than that of control xenografts (Fig. 2G-I). NAT10 also promotes ICC growth in vivo.

Subcutaneous tumor tissues from xenograft animal models were stained with hematoxylin and eosin (H&E) (Supplementary Fig. 2G), and the knockdown of NAT10 was confirmed by WB and IHC (Fig. 2J, Supplementary Fig. 2H). Data shown that NAT10 promotes ICC proliferation and may be an oncogene of ICC.

CCL2 is a downstream target of NAT10

To explore the role of NAT10 in the development of ICC and identify its downstream targets, we conducted ONT full-length transcriptome sequencing to examine changes after NAT10 knockdown. In RBE cells, 9 and 39 genes were consistently upregulated and downregulated, respectively, by two independent shRNAs (Fig. 3A). We observed good agreement between the two independent shRNAs for the downregulated genes. Gene ontology (GO) analysis showed that differentially expressed genes were involved in apoptosis, cell surface receptor signaling pathways, immune response, secretion, and gene concentration in the extracellular regions and space, suggesting that NAT10 may have an important impact on ICC biology (Fig. 3B). CCL2 was concerned as the most prominent candidate target gene. qRT-PCR and Western blot verified that NAT10 knockdown decreased the mRNA and protein levels of CCL2 (Fig. 3C, D). Additionally, CCL2 expression was significantly decreased in NAT10-knockdown tumors in vivo (Fig. 3E). The data indicate that NAT10 promotes CCL2 expression in ICC both in vitro and in vivo. Based on the acetyltransferase properties of NAT10, we further determine the regulatory mechanism of NAT10 on CCL2. RNA immunoprecipitation-quantitative PCR (RIP-qPCR) and coimmunoprecipitation (COIP) was conducted. The data showed that NAT10 exhibited strong binding and interaction with CCL2 mRNA. The two proteins did not interact (Fig. 3F and G). In summary, CCL2 is a downstream target directly regulated by NAT10.

CCL2 promotes ICC growth in vitro and in vivo

To verify the tumor-promoting function of CCL2, we established stable CCL2-knockdown cell lines, which were validated by western blot (Fig. 4A). Using CCK8 and live-cell imaging assays, we found that CCL2 knockdown significantly inhibited the proliferation of ICC cells (Fig. 4B-D). Additionally, CCL2 knockdown significantly inhibited colony formation (Fig. 4E). To confirm the role of CCL2 in ICC in vivo, we used a xenograft animal model wherein HuCCT1 cells were inoculated subcutaneously into the right forelimbs of nude mice. Consistent with the in vitro results, the growth rate of subcutaneous tumors in the CCL2-knockdown group was slower than that of tumors in the control group (Fig. 4F-H). Therefore, CCL2 promotes ICC growth in vivo. We stained the subcutaneous tumor tissues of xenograft animal models with H&E (Fig. 4I), and the knockout of CCL2 was confirmed by IHC (Fig. 4J).

We next infected NAT10-knockdown ICC cells with a lentivirus carrying CCL2 (Supplementary Fig. 3A) and performed CCK8 assays and live-cell imaging, which showed that CCL2 overexpression partially rescued the loss of proliferation observed in NAT10-knockdown ICC cells (Supplementary Fig. 3B-D). Taken together, CCL2 is a downstream target of NAT10 and plays a role in promoting ICC growth both in vivo and in vitro.

NAT10 polarizes macrophages toward the M2 type through its regulation of CCL2

To study whether ICC cells can cause macrophage polarization and the type of polarization, we cultured RAW264.7 mouse macrophages with conditioned medium from ICC cells and performed qRT-PCR to detect the iNOS and Arg-1 levels after 24 h. Compared with control cells, co-cultured RAW264.7 was polarized (Fig. 5A). We also used a transwell chamber to co-culture ICC and RAW264.7 cells and obtained similar but more significant results (Fig. 5B). We also assessed this by flow cytometry. There was no difference in CD86 expression, representing M1 macrophages, after co-culture; however, there was a significant increase in CD206 expression, representing M2 macrophages (Fig. 5C). These results indicate that ICC cells could polarize RAW264.7 cells toward the M2 type.

CCL2 can polarize macrophages toward the M2 type. Therefore, to assess whether the polarization of RAW264.7 cells to M2 type by ICC cells was dependent on NAT10, we first conducted immunofluorescence staining of the NAT10-knockdown tumors, which showed that the expression of CD86, which represents M1 macrophages in mice, was higher in the knockdown tumors than in the control tumors. By contrast, the expression of CD163, which represents M2 macrophages, was decreased (Fig. 5D). We then knocked down NAT10 in ICC cells and used WB and enzyme-linked immunosorbent assay (ELISA) to detect the levels of CCL2 in the ICC cells and cell supernatants. NAT10 knockdown reduced the expression of CCL2 in both the cells and cell supernatants (Fig. 5E, F).

Lentivirus-mediated gene silencing was used to knock down CCL2 in RBE and HuCCT1 cells (Fig. 5G). Cells were co-cultured with RAW264.7 cells, and the levels of CD86 and CD206 were assessed by flow cytometry. Although ICC cells could still polarize RAW264.7 cells toward the M2 type after CCL2 knockdown, the effect was significantly reduced (Fig. 5H). We also performed immunofluorescence staining of the CCL2-knockdown tumors, and the results were consistent with those of the NAT10-knockdown tumors. The expression of CD86, representing M1 macrophages, was increased in CCL2-knockdown tumors, whereas the expression of CD163, representing M2 macrophages, was decreased (Fig. 5I). These results suggest that ICC cells can polarize macrophages toward the M2 type and that NAT10 plays an important role through its regulation of CCL2.

Berberine can target binding and inhibit CCL2

Based on the anti-ICC function of NAT10 and CCL2, we tried to screen their natural targeted inhibitors. Fortunately, a natural product, berberine (BBR) was concerned, which presents high anti-inflammatory and anticancer activity. Molecular docking shown that the binding affinity of BBR with NAT10 and CCL2. was − 8.3 kcal/mol and − 6.3 kcal/mol, respectively (Fig. 6A). After treated with BBR, HuCCT1 cells were performed using RNA sequencing. There was no significant change in NAT10 expression after BBR treatment, but CCL2 was significantly decreased (Fig. 6B). The data suggested that BBR might exert antitumor effects on ICC by inhibiting CCL2. qRT-PCR and Western blot also present a significantly down-regulation of CCL2 but not NAT10 in mRNA and protein level (Fig. 6C, D). To further verify whether BBR could specifically bind to CCL2, we used surface plasmon resonance (SPR) assay to detect its affinity. The data showed a strong binding of BBR to CCL2, and the concentration gradient trend was significant. There was specific binding with an affinity of 423.4 µM (Fig. 6E, F).

Antitumor effects of BBR on ICC in vitro and in vivo

To demonstrate the effect of BBR on the proliferation of ICC cells, we treated RBE and HuCCT1 cells with different concentrations of BBR (0, 10, 20, 40, 80, 160, and 200 µM) and performed CCK8 assays. BBR significantly reduced the viability of both cell lines. This inhibition was time- and concentration-dependent (Fig. 7A). BBR had a stronger effect on HuCCT1 cells, showing a significant inhibitory effect at 48 hours at a very low dose. We then treated these two cell lines with BBR and conducted live-cell imaging. There were significantly fewer cells in the BBR condition than in the DMSO condition, which was consistent with the results of the CCK8 assay (Fig. 7B, C).

To deeply uncovered the inhibitory effect of BBR on the proliferation of ICC cells, RBE and HuCCT1 cells were treated with BBR for 48 h, and changes in the cell cycle were detected using flow cytometry. After treatment with BBR, the percentage of GO/G1 phase cells increased, whereas the percentage of cells in the S and G2/M phases significantly decreased (Supplementary Fig. 4A, B). We also used flow cytometry to assess apoptosis in RBE and HuCCT1 cells after 48 h of BBR treatment. BBR induced apoptosis in both cell lines (Supplementary Fig. 4C, D). Taken together, the data suggest that BBR exerts antitumor effects by blocking ICC cells in the G0/G1 phase and inducing apoptosis.

To further verified the effect of BBR on tumor growth in vivo, HuCCT1 cells were subcutaneously transplanted into nude mice. When tumors appeared, the mice were randomly divided into two groups, with six tumor-bearing mice per group, and BBR (50 mg/kg/d) or sterile water was administered orally. After 18 days of continuous administration of BBR, tumor size and weight were significantly lower than those in the control group (Fig. 7D-F). The tumor tissues were stained with H&E (Supplementary Fig. 4E), and IHC was used to detect the expression of NAT10 and CCL2. There was no significant difference in the expression of NAT10 between the two groups; however, the expression of CCL2 in the tumors of the BBR group was significantly lower than that in tumors in the control group (Supplementary Fig. 4F), which was consistent with the results of the in vitro experiments. Finally, immunofluorescence was used to detect macrophages in the tumors. Compared to the DMSO group, the expression of CD86, representing M1, was increased in the BBR group, whereas the expression of CD163, representing M2, was significantly decreased (Supplementary Fig. 4G), which was consistent with what we observed in NAT10- and CCL2-knockdown tumors. In summary, BBR inhibits the proliferation of ICC and affects the polarization of macrophages by specifically binding to CCL2, thus playing an antitumor role.

Post-transcriptional modification plays an important role in gene expression and function. At present, more than 100 kinds of RNA modifications have been discovered, of which the most common is RNA methylation modification, namely m6A modification. The role of m6A in ICC has been reported to regulate the proliferation, metastasis, immunity and tumor microenvironment of ICC [19–22]. In recent years, another acetylation modification, known as ac4C modification, has begun to attract attention. As the only writer of ac4C, NAT10 is not only involved in several crucial cellular processes [23–26], but also in the occurrence and development of tumors. More and more evidence shows that NAT10 plays an important role in the pathogenesis of multiple solid tumors [12, 14, 15, 27–30]. In this study, we observed a significant upregulation of NAT10 in CCA tissues. High expression of NAT10 was significantly correlated with the histological grade and primary tumor stage of CCA patients. In addition, we demonstrated that NAT10 promotes ICC cell proliferation and tumor growth in vivo. These findings suggest that NAT10 may serve as a potential biomarker for predicting the onset and progression of ICC and could potentially become a new therapeutic target.

The occurrence and development of tumors are not only related to tumor cells themselves, but are also affected by the TME. Macrophages are important components of the TME, infiltrated into the tumor by the recruitment of CCL2 in the TME, and then polarized into M2 macrophages by binding with CCL2 through the receptor CCR2 on the cell surface [31–33]. M2 macrophages induced by CCL2 also secrete CCL2, which on the one hand induces more macrophages to infiltrate into the tumor and polarize toward M2 [34, 35],and on the other hand can act on tumor cells to promote tumor progression [33, 36, 37]. In addition to secreting CCL2, M2 macrophages also release cytokines such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor-β(TGF-β) to promote tumor cell epithelial–mesenchymal transition (EMT), angiogenesis and extracellular matrix (ECM) remodeling [36, 38]. Tumor cells and macrophages promote each other's progression through CCL2 (Fig. 8). In the present study, we confirm that CCL2 is the downstream target of NAT10 in ICC. NAT10 promotes ICC cells proliferation and tumor growth in vivo through CCL2. At present, there is no report on whether NAT10 can affect the polarization of macrophages. We determined that CCL2 is the downstream target of NAT10, and CCL2 affects macrophage polarization. Further validation demonstrates that ICC can affect macrophage polarization through the regulation of CCL2 by NAT10. In vivo and in vitro experiments, NAT10 polarizes macrophages towards M2 type by regulating CCL2. NAT10 is an acetylase, which can acetylate not only protein [30] but also mRNA [11]. In order to further determine the potential regulatory mechanism of NAT10 on CCL2, we performed RIP-qPCR and COIP simultaneously. We found that NAT10 acts by binding to the mRNA of CCL2 in ICC, rather than its protein. In the present study, we not only confirmed the intracellular regulatory mechanism by which NAT10 promotes ICC progression, but also found the relationship between NAT10 and macrophage polarization. It is precisely because NAT10 and CCL2 have such an important role in ICC that they are likely to serve as potential diagnostic markers and therapeutic targets for ICC.

ICC has no specific targeted drugs. Berberine is a small isoquinoline alkaloid, which can be extracted from coptidis and hydrastis canadensis [39, 40]. Berberine has been found to have therapeutic effects on many diseases [40–45]. More and more studies have reported that berberine also has anti-tumor effects [46–51]. Our study demonstrates that as the downstream target of NAT10, CCL2, a chemokine, plays an important role in both tumor and inflammation. CCL2 also plays critical role in ICC cell proliferation and macrophage polarization. Our results suggest that berberine can specifically bind to CCL2 and thus play an anti-tumor role on ICC. Berberine primarily exerts its effects by inhibiting tumor cell proliferation and promoting apoptosis. For different tumor cells, berberine can block them at different stages of the cell cycle [52–56]. This study confirmed that berberine can arrest ICC cells in G0/G1 phase and promote cell apoptosis. In addition, immunofluorescence staining was performed on subcutaneous tumors of nude mice in the berberine treated group. The expression of CD86, representing M1 macrophages, was significantly up-regulated, while CD163, representing M2 macrophages, was significantly down-regulated. These findings were consistent with the results of CCL2 knockdown. Our results indicate that berberine can not only play an anti-tumor role on ICC cells through CCL2, but also regulate macrophage polarization and affect the TME by influencing the level of CCL2 secreted into the extracellular cell. At present, the research methods of targeted drugs are mainly large-scale screening through activity-based protein profiling (ABPP) and molecular docking [57, 58]. However, these methods are mainly based on molecular structure. In our study, we adopted a different approach, namely the functional consistency of the drug and the molecule. This discovery not only provides a potential targeted therapeutic drug for the treatment of ICC, but also offers a possible new method for the study of targeted drugs.

In summary, we found that NAT10 expression is upregulated in ICC and is associated with poor clinicopathological features in tumor patients. Mechanically, NAT10 promotes ICC progression through the regulation of CCL2 mRNA and causes macrophages to become M2-type. BBR inhibited the progression of ICC and macrophage M2-type polarization by targeting CCL2. These findings not only provide new potential diagnostic markers and therapeutic targets for ICC, but also provide possible targeted therapeutic strategies.

Cholangiocarcinoma (CCA) specimens and cell lines

The human intrahepatic CCA (ICC) cell lines RBE and HuCCT1 were purchased from Shanghai Fu Heng Biological (Shanghai, China) and cultured with RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 5% CO2 and 37°C. Macrophage RAW264.7 cells were purchased from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in special medium (CM-0190, Procell). The CCA tissue microarray (TMA) containing 90 CCA tissues and 31 interlobular bile duct tissues was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China).

Construction of stable knockdown and overexpressing cell lines

NAT10 overexpression and knockdown lentiviruses were purchased from Genechem Co., Ltd. (Shanghai, China), and CCL2 overexpression and knockdown lentiviruses were purchased from Genecarer Biotech Co., Ltd. (Xi ‘an, China). Transfection was performed according to the manufacturer instructions. Stable clones were screened with 2 µg/mL puromycin (HY-B1743A, MCE) for at least 1 week. Knockdown and overexpression were validated at the RNA and protein levels. The short hairpin RNAs used in this study are listed in Supporting Table 2.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using Trizol reagent (Life Technologies). cDNA synthesis was performed using PrimeScript RT Master Mix (RR036A, Takara). The obtained cDNA was used as a template for quantitative reverse transcription polymerase chain reaction using TB Green Premix Ex Taq II (RR820A; Takara). The relative RNA expression was normalized to GAPDH using the 2-ΔΔCt methods. Primers used in this study are listed in Supporting Table 2.

Western blotting

Cells were lysed in RIPA lysis buffer and centrifuged at 12 000 ×g at 4°C for 30 min. The supernatant was collected, and the protein concentration was determined using a bicinchoninic acid protein assay (abs9232, Absin). Proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking with non-fat milk, membranes were incubated overnight at 4°C with primary antibodies anti-NAT10 (1:2000, ab194297, Abcam) and anti-CCL2 (1:1000, ab214819, Abcam). After three washes with TBST, the membranes were incubated with secondary antibody for 1 h, and bands were visualized using ECL detection reagent. Protein band intensity was quantified using ImageJ software, and relative protein expression was normalized to GAPDH.

Cell proliferation, live-cell imaging, and colony formation assays

For the cell proliferation assays, 2 × 103 cells were inoculated into 96-well plates, and cell proliferation was measured for 5 consecutive days to evaluate cell proliferation activity using a Cell Counting Kit-8 (K1018, APExBIO). For live-cell imaging, 1 × 103 cells were inoculated into a 96-well plate for live-cell imaging using a Cytation 1 imaging system (Biotek). Images were taken every 2 h for 5 days. Finally, the total number of cells was counted to evaluate the cell proliferation. For colony formation assays, 800 and 500 knockdown and overexpression cells, respectively, were inoculated into 6-well plates. After 8–12 days, the cells were fixed with 4% paraformaldehyde, subjected to crystal violet staining, and photographed, and the colonies were counted.

Animal studies

All animal experiments were approved by the Ethics Committee of the First Hospital of Lanzhou University (approval no. LDYYLL-2024-38) and complied with the Code of Ethics for Animal Experiments. In vivo experiments were conducted in male BALB/c nude mice aged 4–5 weeks to analyze the effects of NAT10 and CCL2 on ICC. HuCCT1-shNC, HuCCT1-shNAT10#1, and HuCCT1-shCCL2 cells (1 × 107/100 µL per mouse, n = 6 per group) suspended in pre-cooled serum-free RPMIS-1640 culture solution were injected subcutaneously into the flanks of mice. Changes in subcutaneous tumor volume (V = 0.5 × L × W2) were closely monitored. At the end of the experiment, the mice were sacrificed by injection of excess pentobarbital sodium, and the tumors were removed and weighed. The tumors were resected for hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), immunofluorescence, and WB.

H&E staining, IHC, and immunofluorescence

Tissue samples were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E staining, IHC, and immunofluorescence. During IHC, the TMA and xenograft tumor tissues were successively dewaxed, hydrated, subjected to antigenic repair and serum blocking, and incubated at 4°C with primary antibody overnight. The primary antibodies used were anti-NAT10 (1:500, ab194297, Abcam) and anti-CCL2 (1:2000, 25542-1-AP, Proteintech). The slices were then incubated with the secondary antibody for 30 min and further incubated with DAB and hematoxylin. Finally, tissue sections were photographed and analyzed. The expression intensity of NAT10 was determined independently by two senior pathologists who were blinded to the clinicopathological data. The expression of NAT10 was expressed using the H-score: H-score = π(i + 1), where π is the percentage of positive cells and i is the staining intensity (0–3). The staining intensity was divided into four grades: 0, negative expression; 1, weak expression; 2, moderate expression; and 3, strong expression. The samples were classified as having low or high expression based on the median H-score.

For immunofluorescence, xenograft tumor sections underwent dewaxing, hydration, antigen repair, and incubation in 3% H2O2 at 37°C for 25 minutes to inhibit endogenous peroxidase. Next, the sections were incubated with 3% bovine serum albumin at room temperature for 30 min, and the primary and corresponding secondary antibodies were administered successively. The following antibodies were used: F4/80 (1:2000, 28463-1-AP, Proteintech), CD86 (1:2000, 13395-1-AP, Proteintech), and CD163 (1:2000, ab182422, Abcam).

Nanopore Technologies (ONT) full-length transcriptome sequencing

ONT full-length transcriptome sequencing was performed on NAT10-knockdown (shNAT10#1 and shNAT10#2) and control (shNC) RBE cells. The experiments were performed according to the standard protocol provided by Nanopore Technologies, including sample quality inspection, library construction, library quality inspection, and library sequencing. The DESeq R software package (1.10.1) was used to analyze the differential expression between the two conditions. A fold change ≥ 1.5 and P < 0.05 was defined as significantly differentially expressed. Gene ontology (GO) analysis and KEGG signaling pathway enrichment analyses were performed on the differentially expressed genes. GO enrichment analysis was performed using the GOseq R packet based on the Wallenius non-central hypergeometric distribution. KEGG pathway enrichment analysis of differentially expressed genes was performed using KOBAS software.

RNA immunoprecipitation quantitative polymerase chain reaction (RIP-qPCR)

HuCCT1 cells and the Imprint® RNA Immunoprecipitation Kit (SAB4200085, Merck Millipore) were used for this assay. HuCCT1 cells were transfected according to the manufacturer instructions, and samples were collected 48 h later. The sample and 5 µg anti-NAT10 antibody (ab194297, Abcam) were added to magnetic beads, and the samples were incubated at room temperature for 30 min. The beads were washed twice with washing buffer, and then RIP immunoprecipitation buffer was added. The immune complex was obtained by adding the cell lysate to the magnetic bead-antibody complex. Finally, the immune complex was separated using washing buffer and the RNA was used for quantitative polymerase chain reaction.

Coimmunoprecipitation (COIP) assay

HuCCT1 cells were lysed on ice with immunoprecipitation lysis buffer for 30 minutes and then centrifuged at 4°C at 14 000 rpm for 30 minutes to collect the supernatant. Anti-NAT10 (ab194297, Abcam) or control IgG was added, and the samples were incubated at 4°C overnight. Then, 30 µL of beads were added, and the sample was rotated overnight at 4°C. The beads were washed three times with cracking buffer, and the supernatant was collected for western blotting.

Enzyme-linked immunosorbent assay (ELISA)

The levels of CCL2 in the cell supernatants were determined using an ELISA kit (E-EL-H6005, Elabscience). First, a reference standard working solution was used to construct a standard curve. The cell supernatant was then analyzed according to the manufacturer’s instructions. After adding the substrate solution, the plate was incubated at 37°C for approximately 15 minutes, depending on the color development. Finally, the Stop Solution was added, and the optical density was measured at 450 nm on a microplate reader.

Molecular docking

The structure of BBR was downloaded from PubChem, and the 3D structures of NAT10 (PDB: 0000) and CCL2 (PDB: 1DOL) were obtained from the RCSB PDB. The protonation state of the small molecule was set at pH = 7.4, and the compound was extended to a 3D structure using Open Babel. The AutoDock tool (ADT3) was used to prepare the receptor proteins and ligands. The docking box was generated using the AutoGrid program, and molecular docking was performed using AutoDock Vina (1.2.0). The optimal combination conformation was selected to analyze the interactions. Finally, a protein-ligand interaction diagram was generated using the PyMOL software.

RNA sequencing after BBR treatment

Cells were treated with BBR for 48 h, and RNA was harvested. RNA sequencing was performed by Wuhan Ruixing Biotechnology Co., Ltd. (Wuhan, China). Illumina Novaseq 6000 was used for high-throughput sequencing in PE150 mode. The DESeq2R software package was used to analyze the differential expression between the two groups. Fold change ≥ 2 or ≤ 1/2 and P < 0.05 were the screening criteria. Differentially expressed genes were analyzed using GO (http://geneontology.org/) and KEGG (http://www.kegg.jp/).

Surface plasmon resonance (SPR)

SPR used a Biacore T20 (GE Healthcare) and a CM5 chip. After replacing the new CM5 chip, it was cleaned with NaOH, activated, protein-coupled, and sealed. Finally, the buffer and sample were run and an affinity test was performed. The LMW kinetics model was selected for sample injection.

Toxicity testing

BBR (HY-17577, MCE) was diluted to different concentrations, and its inhibition rate was determined using the CCK8 method. The concentration of a drug with an inhibition rate of 50% was identified as the IC50. Live-cell imaging was also used to test the toxicity of BBR.

Flow cytometry

ICC cells were treated with BBR for 48 h. Cell cycle (C1052, Beyotime) and apoptosis (E-CK-A211, Elabscience) kits were used to collect and stain the cells according to the manufacturer’s instructions. Flow cytometry was performed using an Agilent flow cytometer. To detect macrophage polarization, macrophages were co-cultured with ICC cells in a transwell chamber. After 24 hours, macrophages were collected and incubated with CD86 (12-0862-82, Invitrogen) and CD206 (17-2061-80, Invitrogen) antibodies at 4°C for 30 minutes. Macrophage polarization was analyzed using an Agilent flow cytometer.

Antitumor effect of BBR in vivo

HuCCT1 cells (1 × 107) were suspended in 100 µL serum-free RPMI-1640 medium and injected subcutaneously into the right forelimb of male BALB/c nude mice aged 4–5 weeks. The tumor size was closely monitored, and when the tumor size reached approximately 5 mm3, the mice were randomly divided into two groups of six mice each. BBR was administered orally (50 mg/kg/d), and sterile water was administered to the control group. Tumor volume and animal weight were measured every 3 days. Mice were sacrificed approximately 18 d after treatment, and the tumors were removed and weighed. The resected tumors were used for subsequent experiments.

Statistical analysis

Statistical analysis was performed using GraphPad Prime 8.0 and SPSS Statistics 20.0. All data were expressed as mean ± SD and were analyzed by Student’s t test or one-way ANOVA. The relationship between NAT10 expression and clinicopathological parameters was determined using the chi-square test. Statistical significance was set at P < 0.05.

Acknowledgments

The study protocol was approved by the Ethics Committee of the First Hospital of Lanzhou University (LDYYLL-2024-38) and complied with the code of ethics for animal experiments.

Author contributions

TC and WM designed the experiment. TC completed the experiment, data analysis and the writing of the manuscript draft. ZB, WM and JD analyzed and interpreted the data and reviewed the manuscript. YL participated in the bioinformatics analysis. ZB and WM supervised the study, provided funding and resources, and revised the manuscript. All authors reviewed and approved the final manuscript. TC and WM as guarantors are responsible for the overall content.

Funding

This study was supported by the National Natural Science Foundation of China (82060551; 82060666).

Data availability

Data are available on reasonable request.

Conflict of interest

The authors declare no competing interests.

Rizvi S, Gores GJ. Pathogenesis, Diagnosis, and Management of Cholangiocarcinoma. Gastroenterology. 2013;145:1215–29.
Rizvi S, Khan SA, Hallemeier CL, Kelley RK, Gores GJ. Cholangiocarcinoma — evolving concepts and therapeutic strategies. Nat Reviews Clin Oncol. 2017;15:95–111.
DeOliveira ML, Cunningham SC, Cameron JL, Kamangar F, Winter JM, Lillemoe KD, Choti MA, Yeo CJ, Schulick RD. Cholangiocarcinoma. Ann Surg. 2007;245:755–62.
Nakeeb A, Pitt HA, Sohn TA, Coleman J, Abrams RA, Piantadosi S, Hruban RH, Lillemoe KD, Yeo CJ. Cameron JL: Cholangiocarcinoma. Ann Surg. 1996;224:463–75.
Taylor-Robinson SD, Toledano MB, Arora S, Keegan TJ, Hargreaves S, Beck A, Khan SA, Elliott P, Thomas HC. Increase in mortality rates from intrahepatic cholangiocarcinoma in England and Wales 1968–1998. Gut. 2001;48:816–20.
Khan SA, Taylor-Robinson SD, Toledano MB, Beck A, Elliott P, Thomas HC. Changing international trends in mortality rates for liver, biliary and pancreatic tumours. J Hepatol. 2002;37:806–13.
Patel T. Worldwide trends in mortality from biliary tract malignancies. BMC Cancer. 2002;2:10.
Bridgewater J, Galle PR, Khan SA, Llovet JM, Park JW, Patel T, Pawlik TM, Gores GJ. Guidelines for the diagnosis and management of intrahepatic cholangiocarcinoma. J Hepatol. 2014;60:1268–89.
Spolverato G, Vitale A, Cucchetti A, Popescu I, Marques HP, Aldrighetti L, Gamblin TC, Maithel SK, Sandroussi C, Bauer TW et al. Can hepatic resection provide a long-term cure for patients with intrahepatic cholangiocarcinoma? Cancer 2015, 121:3998–4006.
Franssen S, Holster JJ, Jolissaint JS, Nooijen LE, Cercek A, D'Angelica MI, Homs MYV, Wei AC, Balachandran VP, Drebin JA, et al. Gemcitabine with Cisplatin Versus Hepatic Arterial Infusion Pump Chemotherapy for Liver-Confined Unresectable Intrahepatic Cholangiocarcinoma. Ann Surg Oncol. 2024;31:115–24.
Arango D, Sturgill D, Alhusaini N, Dillman AA, Sweet TJ, Hanson G, Hosogane M, Sinclair WR, Nanan KK, Mandler MD, et al. Acetylation of Cytidine in mRNA Promotes Translation Efficiency. Cell. 2018;175:1872–e18861824.
Li Z, Li D, Yang T, Yao C. NAT10 promotes the tumorigenesis and progression of laryngeal squamous cell carcinoma through ac4C modification of FOXM1 mRNA. Cancer Biol Ther. 2023;24:2274143.
Zhang X, Jiang G, Sun M, Zhou H, Miao Y, Liang M, Wang E, Zhang Y. Cytosolic THUMPD1 promotes breast cancer cells invasion and metastasis via the AKT-GSK3-Snail pathway. Oncotarget. 2017;8:13357–66.
Pan Z, Bao Y, Hu M, Zhu Y, Tan C, Fan L, Yu H, Wang A, Cui J, Sun G. Role of NAT10-mediated ac4C-modified HSP90AA1 RNA acetylation in ER stress-mediated metastasis and lenvatinib resistance in hepatocellular carcinoma. Cell Death Discovery. 2023;9:56.
Chen X, Hao Y, Liu Y, Zhong S, You Y, Ao K, Chong T, Luo X, Yin M, Ye M, et al. NAT10/ac4C/FOXP1 Promotes Malignant Progression and Facilitates Immunosuppression by Reprogramming Glycolytic Metabolism in Cervical Cancer. Adv Sci. 2023;10:e2302705.
Zhang Y, Jing Y, Wang Y, Tang J, Zhu X, Jin WL, Wang Y, Yuan W, Li X, Li X. NAT10 promotes gastric cancer metastasis via N4-acetylated COL5A1. Signal Transduct Target Ther. 2021;6:173.
Funes SC, Rios M, Escobar-Vera J, Kalergis AM. Implications of macrophage polarization in autoimmunity. Immunology. 2018;154:186–95.
Arneth B. Tumor Microenvironment. Medicina (Kaunas) 2019, 56.
Gao Y, Yu M, Liu Z, Liu Y, Kong Z, Zhu C, Qin X, Li Y, Tang L. m6A demethylase ALKBH5 maintains stemness of intrahepatic cholangiocarcinoma by sustaining BUB1B expression and cell proliferation. Translational Oncol. 2024;41:101858.
Xiang D, Gu M, Liu J, Dong W, Yang Z, Wang K, Fu J, Wang H. m6A RNA methylation-mediated upregulation of HLF promotes intrahepatic cholangiocarcinoma progression by regulating the FZD4/β-catenin signaling pathway. Cancer Lett. 2023;560:216144.
Zhou S, Yang K, Chen S, Lian G, Huang Y, Yao H, Zhao Y, Huang K, Yin D, Lin H, Li Y. CCL3 secreted by hepatocytes promotes the metastasis of intrahepatic cholangiocarcinoma by VIRMA-mediated N6-methyladenosine (m(6)A) modification. J Transl Med. 2023;21:43.
Qiu X, Yang S, Wang S, Wu J, Zheng B, Wang K, Shen S, Jeong S, Li Z, Zhu Y, et al. M(6)A Demethylase ALKBH5 Regulates PD-L1 Expression and Tumor Immunoenvironment in Intrahepatic Cholangiocarcinoma. Cancer Res. 2021;81:4778–93.
Liu H, Ling Y, Gong Y, Sun Y, Hou L, Zhang B. DNA damage induces N-acetyltransferase NAT10 gene expression through transcriptional activation. Mol Cell Biochem. 2007;300:249–58.
Chi YH, Haller K, Peloponese JM Jr., Jeang KT. Histone acetyltransferase hALP and nuclear membrane protein hsSUN1 function in de-condensation of mitotic chromosomes. J Biol Chem. 2007;282:27447–58.
Fu D, Collins K. Purification of human telomerase complexes identifies factors involved in telomerase biogenesis and telomere length regulation. Mol Cell. 2007;28:773–85.
Ito S, Horikawa S, Suzuki T, Kawauchi H, Tanaka Y, Suzuki T, Suzuki T. Human NAT10 is an ATP-dependent RNA acetyltransferase responsible for N4-acetylcytidine formation in 18 S ribosomal RNA (rRNA). J Biol Chem. 2014;289:35724–30.
Jin C, Wang T, Zhang D, Yang P, Zhang C, Peng W, Jin K, Wang L, Zhou J, Peng C, et al. Acetyltransferase NAT10 regulates the Wnt/β-catenin signaling pathway to promote colorectal cancer progression via ac4C acetylation of KIF23 mRNA. J Experimental Clin Cancer Res. 2022;41:345.
Feng Z, Li K, Qin K, Liang J, Shi M, Ma Y, Zhao S, Liang H, Han D, Shen B, et al. The LINC00623/NAT10 signaling axis promotes pancreatic cancer progression by remodeling ac4C modification of mRNA. J Hematol Oncol. 2022;15:112.
Wang G, Zhang M, Zhang Y, Xie Y, Zou J, Zhong J, Zheng Z, Zhou X, Zheng Y, Chen B, Liu C. NAT10-mediated mRNA N4‐acetylcytidine modification promotes bladder cancer progression. Clin Translational Med. 2022;12:e738.
Liu HY, Liu YY, Yang F, Zhang L, Zhang FL, Hu X, Shao ZM, Li DQ. Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer. Nucleic Acids Res. 2020;48:3638–56.
Petty AJ, Yang Y. Tumor-associated macrophages: implications in cancer immunotherapy. Immunotherapy. 2017;9:289–302.
Yang H, Zhang Q, Xu M, Wang L, Chen X, Feng Y, Li Y, Zhang X, Cui W, Jia X. CCL2-CCR2 axis recruits tumor associated macrophages to induce immune evasion through PD-1 signaling in esophageal carcinogenesis. Mol Cancer. 2020;19:41.
Li D, Ji H, Niu X, Yin L, Wang Y, Gu Y, Wang J, Zhou X, Zhang H, Zhang Q. Tumor-associated macrophages secrete CC‐chemokine ligand 2 and induce tamoxifen resistance by activating PI3K/Akt/mTOR in breast cancer. Cancer Sci. 2019;111:47–58.
Sierra-Filardi E, Nieto C, Domínguez-Soto A, Barroso R, Sánchez-Mateos P, Puig-Kroger A, López-Bravo M, Joven J, Ardavín C, Rodríguez-Fernández JL, et al. CCL2 shapes macrophage polarization by GM-CSF and M-CSF: identification of CCL2/CCR2-dependent gene expression profile. J Immunol. 2014;192:3858–67.
Chen Y, Song Y, Du W, Gong L, Chang H, Zou Z. Tumor-associated macrophages: an accomplice in solid tumor progression. J Biomed Sci. 2019;26:78.
Izumi K, Fang LY, Mizokami A, Namiki M, Li L, Lin WJ, Chang C. Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation. EMBO Mol Med. 2013;5:1383–401.
Qian BZ, Li J, Zhang H, Kitamura T, Zhang J, Campion LR, Kaiser EA, Snyder LA, Pollard JW. CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature. 2011;475:222–5.
Chanmee T, Ontong P, Konno K, Itano N. Tumor-associated macrophages as major players in the tumor microenvironment. Cancers (Basel). 2014;6:1670–90.
Chen XW, Di YM, Zhang J, Zhou ZW, Li CG, Zhou SF. Interaction of herbal compounds with biological targets: a case study with berberine. ScientificWorldJournal 2012, 2012:708292.
Tillhon M, Guamán Ortiz LM, Lombardi P, Scovassi AI. Berberine: new perspectives for old remedies. Biochem Pharmacol. 2012;84:1260–7.
Tang QL, Lai ML, Zhong YF, Wang AM, Su JK, Zhang MQ. Antinociceptive effect of berberine on visceral hypersensitivity in rats. World J Gastroenterol. 2013;19:4582–9.
Zhang M, Wang CM, Li J, Meng ZJ, Wei SN, Li J, Bucala R, Li YL, Chen L. Berberine protects against palmitate-induced endothelial dysfunction: involvements of upregulation of AMPK and eNOS and downregulation of NOX4. Mediators Inflamm. 2013;2013:260464.
Ma X, Yu X, Li R, Cui J, Yu H, Ren L, Jiang J, Zhang W, Wang L. Berberine-silybin salt achieves improved anti-nonalcoholic fatty liver disease effect through regulating lipid metabolism. J Ethnopharmacol. 2024;319:117238.
Heidarian E, Rafieian-Kopaei M, Khoshdel A, Bakhshesh M. Metabolic effects of berberine on liver phosphatidate phosphohydrolase in rats fed on high lipogenic diet: an additional mechanism for the hypolipidemic effects of berberine. Asian Pac J Trop Biomed. 2014;4:S429–435.
Ansari N, Khodagholi F. Natural products as promising drug candidates for the treatment of Alzheimer's disease: molecular mechanism aspect. Curr Neuropharmacol. 2013;11:414–29.
Chu SC, Yu CC, Hsu LS, Chen KS, Su MY, Chen PN. Berberine reverses epithelial-to-mesenchymal transition and inhibits metastasis and tumor-induced angiogenesis in human cervical cancer cells. Mol Pharmacol. 2014;86:609–23.
Choi MS, Oh JH, Kim SM, Jung HY, Yoo HS, Lee YM, Moon DC, Han SB, Hong JT. Berberine inhibits p53-dependent cell growth through induction of apoptosis of prostate cancer cells. Int J Oncol. 2009;34:1221–30.
Hwang JM, Kuo HC, Tseng TH, Liu JY, Chu CY. Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells. Arch Toxicol. 2006;80:62–73.
Piyanuch R, Sukhthankar M, Wandee G, Baek SJ. Berberine, a natural isoquinoline alkaloid, induces NAG-1 and ATF3 expression in human colorectal cancer cells. Cancer Lett. 2007;258:230–40.
Liu Y, Hua W, Li Y, Xian X, Zhao Z, Liu C, Zou J, Li J, Fang X, Zhu Y. Berberine suppresses colon cancer cell proliferation by inhibiting the SCAP/SREBP-1 signaling pathway-mediated lipogenesis. Biochem Pharmacol. 2020;174:113776.
Li G, Zhang C, Liang W, Zhang Y, Shen Y, Tian X. Berberine regulates the Notch1/PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells. Pharm Biol. 2021;59:21–30.
Xiao Y, Tian C, Huang T, Han B, Wang M, Ma H, Li Z, Ye X, Li X. 8-Cetylberberine inhibits growth of lung cancer in vitro and in vivo. Life Sci. 2018;192:259–69.
Su YH, Tang WC, Cheng YW, Sia P, Huang CC, Lee YC, Jiang HY, Wu MH, Lai IL, Lee JW, Lee KH. Targeting of multiple oncogenic signaling pathways by Hsp90 inhibitor alone or in combination with berberine for treatment of colorectal cancer. Biochim Biophys Acta. 2015;1853:2261–72.
Eo SH, Kim JH, Kim SJ. Induction of G₂/M Arrest by Berberine via Activation of PI3K/Akt and p38 in Human Chondrosarcoma Cell Line. Oncol Res. 2014;22:147–57.
Gao X, Wang J, Li M, Wang J, Lv J, Zhang L, Sun C, Ji J, Yang W, Zhao Z, Mao W. Berberine attenuates XRCC1-mediated base excision repair and sensitizes breast cancer cells to the chemotherapeutic drugs. J Cell Mol Med. 2019;23:6797–804.
Wu HL, Chuang TY, Al-Hendy A, Diamond MP, Azziz R, Chen YH. Berberine inhibits the proliferation of human uterine leiomyoma cells. Fertil Steril. 2015;103:1098–106.
Feng P, Chen D, Wang X, Li Y, Li Z, Li B, Zhang Y, Li W, Zhang J, Ye J, et al. Inhibition of the m(6)A reader IGF2BP2 as a strategy against T-cell acute lymphoblastic leukemia. Leukemia. 2022;36:2180–8.
Luo P, Zhang Q, Zhong TY, Chen JY, Zhang JZ, Tian Y, Zheng LH, Yang F, Dai LY, Zou C, et al. Celastrol mitigates inflammation in sepsis by inhibiting the PKM2-dependent Warburg effect. Mil Med Res. 2022;9:22.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

NAT10 affects the progression of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating CCL2

Status:

Version 1

Abstract

Figures

Introduction

Results

NAT10 is upregulated in ICC

NAT10 promotes the growth of ICC in vivo and in vitro

CCL2 is a downstream target of NAT10

CCL2 promotes ICC growth in vitro and in vivo

NAT10 polarizes macrophages toward the M2 type through its regulation of CCL2

Berberine can target binding and inhibit CCL2

Antitumor effects of BBR on ICC in vitro and in vivo

Discussion

Materials and methods

Cholangiocarcinoma (CCA) specimens and cell lines

Construction of stable knockdown and overexpressing cell lines

Quantitative real-time polymerase chain reaction (qRT-PCR)

Western blotting

Cell proliferation, live-cell imaging, and colony formation assays

Animal studies

H&E staining, IHC, and immunofluorescence

Nanopore Technologies (ONT) full-length transcriptome sequencing

RNA immunoprecipitation quantitative polymerase chain reaction (RIP-qPCR)

Coimmunoprecipitation (COIP) assay

Enzyme-linked immunosorbent assay (ELISA)

Molecular docking

RNA sequencing after BBR treatment

Surface plasmon resonance (SPR)

Toxicity testing

Flow cytometry

Antitumor effect of BBR in vivo

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1