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

NAT10 affects the progression of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating CCL2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article NAT10 affects the progression of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating CCL2 Teng Cai, Jianye Dai, Yanyan Lin, Zhongtian Bai, Wenbo Meng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4099955/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction 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. Results 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. 1 A), 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. 1 B). 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. 1 C, 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. 1 D, 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. 1 F). 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. 2 A, 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. 2 C, 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. 2 D, 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. 2 F), 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. 2 G-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. 2 J, 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. 3 A). 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. 3 B). 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. 3 C, D). Additionally, CCL2 expression was significantly decreased in NAT10-knockdown tumors in vivo (Fig. 3 E). 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. 3 F 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. 4 A). Using CCK8 and live-cell imaging assays, we found that CCL2 knockdown significantly inhibited the proliferation of ICC cells (Fig. 4 B-D). Additionally, CCL2 knockdown significantly inhibited colony formation (Fig. 4 E). 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. 4 F-H). Therefore, CCL2 promotes ICC growth in vivo. We stained the subcutaneous tumor tissues of xenograft animal models with H&E (Fig. 4 I), and the knockout of CCL2 was confirmed by IHC (Fig. 4 J). 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. 5 A). We also used a transwell chamber to co-culture ICC and RAW264.7 cells and obtained similar but more significant results (Fig. 5 B). 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. 5 C). 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. 5 D). 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. 5 E, F). Lentivirus-mediated gene silencing was used to knock down CCL2 in RBE and HuCCT1 cells (Fig. 5 G). 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. 5 H). 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. 5 I). 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. 6 A). 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. 6 B). 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. 6 C, 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. 6 E, 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. 7 A). 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. 7 B, 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. 7 D-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. Discussion 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. Materials and methods 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. Declarations 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. References Rizvi S, Gores GJ. 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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. Additional Declarations No competing interests reported. Supplementary Files Fig.S1.tif Fig.S2.tif Fig.S3.tif Fig.S4.tif TableS1.docx TableS2.docx supplement.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4099955","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280329324,"identity":"6ec38cb1-2322-4dc8-a4ec-a41e475ffaa6","order_by":0,"name":"Teng Cai","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Teng","middleName":"","lastName":"Cai","suffix":""},{"id":280329325,"identity":"d33b4779-a64f-4721-b4f1-1921f115b4a9","order_by":1,"name":"Jianye Dai","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jianye","middleName":"","lastName":"Dai","suffix":""},{"id":280329326,"identity":"f0c309f0-2332-46d1-8bed-c7814e018012","order_by":2,"name":"Yanyan Lin","email":"","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yanyan","middleName":"","lastName":"Lin","suffix":""},{"id":280329327,"identity":"05830e06-fb7f-4505-9723-36e54df47fe3","order_by":3,"name":"Zhongtian Bai","email":"","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhongtian","middleName":"","lastName":"Bai","suffix":""},{"id":280329328,"identity":"3f3a7ec5-4179-492c-aaa2-3fc9dd937b7d","order_by":4,"name":"Wenbo Meng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYLACxgYbGTCDhwQtaTwkazlMghbd9t7Dr3l3nOeRn5HA+OBtG4O8OSEtZmfOpVnznrnNwzgjgdlwbhuD4c4GQlpu5JgZ57bd5mGWSGCT5m1jSDA4QEjL/TcgLed42CQS2H8Tp+UGj/Hj3LYDPDxAW5iJ03Imx4z575lkHgmeh82Sc85JGG4gqOX4GeOPM3fYycm3Jx/88KbMRp6gLUDAJgGhGRuAhARh9UDA/IEoZaNgFIyCUTByAQBojjv/kKg5TAAAAABJRU5ErkJggg==","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":true,"prefix":"","firstName":"Wenbo","middleName":"","lastName":"Meng","suffix":""}],"badges":[],"createdAt":"2024-03-14 10:49:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4099955/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4099955/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53086247,"identity":"636d41d9-3ced-40bf-ac47-b1d43ed5bf28","added_by":"auto","created_at":"2024-03-20 12:00:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":400347,"visible":true,"origin":"","legend":"\u003cp\u003eNAT10 is significantly upregulated in human CCA. (A) NAT10 is upregulated in CCA as compared to adjacent normal tissue (TCGA data). (B) NAT10 is upregulated in STAD, COAD, and READ as compared to adjacent normal tissue (TCGA data). (C) There was no significant correlation between NAT10 expression and OS or DFS in CCA patients. (D) Representative images of NAT10 IHC staining on TMA. (E) NAT10 IHC staining scores of CCA tissue and matched normal tissues; the expression of NAT10 was indicated by the proportion of positive area. (F) Expression of NAT10 in different CCA cell lines and normal bile duct cells.\u003c/p\u003e\n\u003cp\u003eNAT10: N-acetyltransferase 10; CCA: cholangiocarcinoma; TCGA: The Cancer Genome Atlas; STAD: stomach adenocarcinoma; COAD: colon adenocarcinoma; READ: rectal adenocarcinoma; OS: overall survival; DFS: disease-free survival; IHC: immunohistochemistry; TMA: tissue microarray\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/aa7e03e594d373bf22bd900d.png"},{"id":53086250,"identity":"74197f18-55f4-4506-a19a-4c8d0926f8c8","added_by":"auto","created_at":"2024-03-20 12:00:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":995772,"visible":true,"origin":"","legend":"\u003cp\u003eKnocking down NAT10 inhibits ICC cell proliferation and tumor growth in mice. (A, B) NAT10-knockdown cell lines were constructed and verified at the mRNA and protein levels by qRT-PCR and WB, respectively. (C) CCK8 assays showed that knocking down NAT10 significantly inhibited the proliferation of ICC cells. (D, E) Live-cell imaging showed that knocking down NAT10 significantly inhibited the proliferation of ICC cells. (F) Knocking down NAT10 significantly reduced the colony-formation ability of ICC cells. (G-I) Knocking down NAT10 effectively inhibited the growth of ICC subcutaneous tumors. Tumor size was measured every 3 days. (J) Expression of NAT10 protein in subcutaneous tumors.\u003c/p\u003e\n\u003cp\u003eNAT10: N-acetyltransferase 10; ICC: intrahepatic cholangiocarcinoma; qRT-PCR: quantitative real-time polymerase chain reaction; WB: Western Blotting;\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/7ce0ec5a3d5c9c5881cf5c71.png"},{"id":53086251,"identity":"c9243813-c585-4fa9-97ab-f38e62b9a880","added_by":"auto","created_at":"2024-03-20 12:00:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":585984,"visible":true,"origin":"","legend":"\u003cp\u003eCCL2 is a downstream target of NAT10. (A) RNA sequencing identified differentially expressed genes in NAT10-knockdown cells compared with control cells. (B) GO analysis of the NAT10-knockdown downregulated genes revealed the potential functions of NAT10 in regulating apoptosis, cell surface receptor signaling pathways, immune response, and secretion. (C, D) Knocking down NAT10 reduced CCL2 expression at the mRNA and protein levels in ICC cells. (E) IHC showed that NAT10 knockdown decreased CCL2 in subcutaneous tumors. (F, G) NAT10 interacts with CCL2 mRNA (F) but not protein (G).\u003c/p\u003e\n\u003cp\u003eCCL2: C-C motif chemokine ligand 2; NAT10: N-acetyltransferase 10; GO: Gene Ontology; ICC: intrahepatic cholangiocarcinoma; IHC: immunohistochemistry\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/acaafa927f098ff0ee0026e3.png"},{"id":53086257,"identity":"b9b100c3-cb18-4107-b687-bac380f55ad8","added_by":"auto","created_at":"2024-03-20 12:00:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1130524,"visible":true,"origin":"","legend":"\u003cp\u003eKnocking down CCL2 inhibits ICC cell proliferation and tumor growth in mice. (A) CCL2-knockdown cell lines were constructed and verified at the protein level. (B) CCK8 assays showed that knockdown of CCL2 significantly inhibited the proliferation of ICC cells. (C, D) Live-cell imaging showed that knockdown of CCL2 significantly inhibited the proliferation of ICC cells. (E) Knockdown of CCL2 significantly reduced the colony-formation ability of ICC cells. (F-H) Knocking down CCL2 effectively inhibited the growth of ICC subcutaneous tumor in nude mice. Tumor size was measured every 3 days. (I, J) H\u0026amp;E staining (I) and CCL2 IHC (J) of subcutaneous tumors after CCL2 knockdown.\u003c/p\u003e\n\u003cp\u003eCCL2: C-C motif chemokine ligand 2; ICC: intrahepatic cholangiocarcinoma; H\u0026amp;E: hematoxylin and eosin; IHC: immunohistochemistry\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/322047b5e9c535186060a32a.png"},{"id":53086252,"identity":"6f03dab5-a586-4631-abce-cfec9ac553c6","added_by":"auto","created_at":"2024-03-20 12:00:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":793616,"visible":true,"origin":"","legend":"\u003cp\u003eNAT10 polarizes macrophages toward the M2 type through CCL2. (A) The supernatant of ICC cells polarized macrophages. (B) Co-culture of ICC cells and macrophages polarized macrophages. (C) Co-culture of ICC cells and macrophages led to the polarization of macrophages toward M2. (D) Immunofluorescence showed that CD86 expression increased and CD163 expression decreased in NAT10-knockdown tumors. (E, F) WB (E) and ELISA (F) showed that NAT10 knockdown decreased CCL2 expression levels in ICC cells and cell supernatant. (G) CCL2-knockdown cell lines were constructed and verified at the protein level. (H) Flow cytometry confirmed that CCL2 knockdown reduced the polarization of macrophages toward M2. (I) Immunofluorescence showed that CD86 expression increased and CD163 expression decreased in CCL2-knockdown tumors.\u003c/p\u003e\n\u003cp\u003eNAT10: N-acetyltransferase 10; CCL2: C-C motif chemokine ligand 2; ICC: intrahepatic cholangiocarcinoma; WB: Western Blotting; ELISA: enzyme-linked immunosorbent assay; M: medium; C: cell.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/5ee2d44a573a6acba05d4be5.png"},{"id":53087249,"identity":"108fd961-524f-4e6c-bc8e-2a9bd4142df8","added_by":"auto","created_at":"2024-03-20 12:08:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":377336,"visible":true,"origin":"","legend":"\u003cp\u003eBBR binds and inhibits CCL2. (A) Molecular docking simulation of BBR with NAT10 and CCL2. (B) Volcano plot of differentially expressed genes in HuCCT1 cells treated with BBR. (C, D) Expression of NAT10 and CCL2 at the mRNA and protein levels after BBR treatment. (E, F) BBR bound specifically to CCL2 protein with a specific concentration gradient trend; its affinity was 423.4 μM.\u003c/p\u003e\n\u003cp\u003eBBR: berberine; CCL2: C-C motif chemokine ligand 2; NAT10: N-acetaltransferase 10\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/e23494abd234e8523400be0e.png"},{"id":53087252,"identity":"15b5b459-3a2f-4fed-b1c9-e964d6cc4bf7","added_by":"auto","created_at":"2024-03-20 12:08:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":974931,"visible":true,"origin":"","legend":"\u003cp\u003eBBR inhibits ICC cell proliferation and tumor growth in nude mice. (A) IC50 value of BBR in ICC cells. (B, C) BBR significantly inhibited the proliferation of ICC cells. (D-F) BBR effectively inhibited the growth of ICC subcutaneous tumors in nude mice. Tumor size was measured every 3 days.\u003c/p\u003e\n\u003cp\u003eBBR: berberine; ICC: intrahepatic cholangiocarcinoma;\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/485b89b210685447a4e3c8fe.png"},{"id":53086254,"identity":"2712b6ad-3476-49bf-b2c5-78439b5c8ae9","added_by":"auto","created_at":"2024-03-20 12:00:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1536509,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of action of NAT10 and BBR in ICC and macrophage polarization.\u003c/p\u003e\n\u003cp\u003eNAT10: N-acetyltransferase 10; BBR: berberine; ICC: intrahepatic cholangiocarcinoma\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/b854958406e4a84b221fdadb.png"},{"id":53819465,"identity":"6f907062-d661-4245-8837-98b5eb0ad343","added_by":"auto","created_at":"2024-03-31 22:37:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6058770,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/2af322b4-4677-4d05-8254-ea97c9bd1cfb.pdf"},{"id":53086249,"identity":"011f2997-2ce7-47d3-9041-18eebf44d47a","added_by":"auto","created_at":"2024-03-20 12:00:46","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3531664,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/e53f4b4c388bb38fb7658166.tif"},{"id":53086256,"identity":"0e555d6a-97de-4e58-81b4-acca4ccbdeb9","added_by":"auto","created_at":"2024-03-20 12:00:46","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16997348,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/7dbadfc3679c6e6f10650b49.tif"},{"id":53086261,"identity":"46e44d7f-d8b3-419e-8f07-e803235e7caa","added_by":"auto","created_at":"2024-03-20 12:00:47","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14463244,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/7f4e670d6b30a4728f80aa4e.tif"},{"id":53086259,"identity":"0bb0325f-9751-4f5b-bb9d-df8fcd499861","added_by":"auto","created_at":"2024-03-20 12:00:47","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18333064,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/6fb47f266f625ea3d9773ce3.tif"},{"id":53086258,"identity":"fa22c39e-d5f2-4001-ae96-4eaafcb0e4c5","added_by":"auto","created_at":"2024-03-20 12:00:47","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":18040,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/beb08f6deb1acaf9c13512ac.docx"},{"id":53086262,"identity":"36c8e182-6d06-404a-894b-35ea4c7b4b09","added_by":"auto","created_at":"2024-03-20 12:00:47","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16087,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/81c123e519ec712a91b9be48.docx"},{"id":53086255,"identity":"7f4decb2-a2f1-4a1e-81d1-e7d9547e3f1b","added_by":"auto","created_at":"2024-03-20 12:00:46","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":22459,"visible":true,"origin":"","legend":"","description":"","filename":"supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-4099955/v1/64af7235749a41391a79765e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"NAT10 affects the progression of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating CCL2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCholangiocarcinoma (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 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. ICC accounts for 10% of all primary liver cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and its incidence is increasing [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Surgical resection is the only possible curative treatment, and the 5-year overall survival rate is 15\u0026ndash;40% [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, up to two-thirds of patients will relapse after surgical removal [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eIn 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 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], multiple studies have examined how NAT10 regulates tumor progression by modifying mRNA with ac4C [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. 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) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], which can be transformed into each other [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].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.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNAT10 is upregulated in ICC\u003c/h2\u003e \u003cp\u003eSurfing in The Cancer Genome Atlas (TCGA) database, we found NAT10 was significantly upregulated in CCA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E), and NAT10 expression was significantly correlated with tumor location, histological grade, and primary tumor stage (Supporting Table\u0026nbsp;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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNAT10 promotes the growth of ICC in vivo and in vitro\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). In addition, we constructed NAT10-overexpressing cell lines and validated them at the mRNA and protein levels (Supplementary Fig.\u0026nbsp;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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E, Supplementary Fig.\u0026nbsp;2D, E). Colony formation assay was performed to determine the long-term effects of NAT10 on ICC cell proliferation. After 8\u0026ndash;12 days, NAT10 knockdown significantly reduced colony formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), whereas NAT10 overexpression significantly increased colony formation (Supplementary Fig.\u0026nbsp;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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-I). NAT10 also promotes ICC growth in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubcutaneous tumor tissues from xenograft animal models were stained with hematoxylin and eosin (H\u0026amp;E) (Supplementary Fig.\u0026nbsp;2G), and the knockdown of NAT10 was confirmed by WB and IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, Supplementary Fig.\u0026nbsp;2H). Data shown that NAT10 promotes ICC proliferation and may be an oncogene of ICC.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCCL2 is a downstream target of NAT10\u003c/h3\u003e\n\u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Additionally, CCL2 expression was significantly decreased in NAT10-knockdown tumors in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and G). In summary, CCL2 is a downstream target directly regulated by NAT10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCCL2 promotes ICC growth in vitro and in vivo\u003c/h3\u003e\n\u003cp\u003eTo verify the tumor-promoting function of CCL2, we established stable CCL2-knockdown cell lines, which were validated by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Using CCK8 and live-cell imaging assays, we found that CCL2 knockdown significantly inhibited the proliferation of ICC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). Additionally, CCL2 knockdown significantly inhibited colony formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H). Therefore, CCL2 promotes ICC growth in vivo. We stained the subcutaneous tumor tissues of xenograft animal models with H\u0026amp;E (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), and the knockout of CCL2 was confirmed by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next infected NAT10-knockdown ICC cells with a \u003cem\u003elentivirus\u003c/em\u003e carrying CCL2 (Supplementary Fig.\u0026nbsp;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.\u0026nbsp;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.\u003c/p\u003e\n\u003ch3\u003eNAT10 polarizes macrophages toward the M2 type through its regulation of CCL2\u003c/h3\u003e\n\u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We also used a transwell chamber to co-culture ICC and RAW264.7 cells and obtained similar but more significant results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results indicate that ICC cells could polarize RAW264.7 cells toward the M2 type.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCCL2 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003eLentivirus-mediated gene silencing was used to knock down CCL2 in RBE and HuCCT1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). 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.\u003c/p\u003e\n\u003ch3\u003eBerberine can target binding and inhibit CCL2\u003c/h3\u003e\n\u003cp\u003eBased 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 \u0026minus;\u0026thinsp;8.3 kcal/mol and \u0026minus;\u0026thinsp;6.3 kcal/mol, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, 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 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAntitumor effects of BBR on ICC in vitro and in vivo\u003c/h3\u003e\n\u003cp\u003eTo 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 \u0026micro;M) and performed CCK8 assays. BBR significantly reduced the viability of both cell lines. This inhibition was time- and concentration-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;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.\u0026nbsp;4C, D). Taken together, the data suggest that BBR exerts antitumor effects by blocking ICC cells in the G0/G1 phase and inducing apoptosis.\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F). The tumor tissues were stained with H\u0026amp;E (Supplementary Fig.\u0026nbsp;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.\u0026nbsp;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.\u0026nbsp;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.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePost-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 [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], 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 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eThe 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 [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e],and on the other hand can act on tumor cells to promote tumor progression [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. 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\u0026ndash;mesenchymal transition (EMT), angiogenesis and extracellular matrix (ECM) remodeling [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Tumor cells and macrophages promote each other's progression through CCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). 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 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] but also mRNA [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eICC has no specific targeted drugs. Berberine is a small isoquinoline alkaloid, which can be extracted from coptidis and hydrastis canadensis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Berberine has been found to have therapeutic effects on many diseases [\u003cspan additionalcitationids=\"CR41 CR42 CR43 CR44\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. More and more studies have reported that berberine also has anti-tumor effects [\u003cspan additionalcitationids=\"CR47 CR48 CR49 CR50\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR53 CR54 CR55\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCholangiocarcinoma (CCA) specimens and cell lines\u003c/h2\u003e \u003cp\u003eThe 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\u0026deg;C. Macrophage RAW264.7 cells were purchased from Wuhan Procell Life Science \u0026amp; 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of stable knockdown and overexpressing cell lines\u003c/h2\u003e \u003cp\u003eNAT10 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 \u0026lsquo;an, China). Transfection was performed according to the manufacturer instructions. Stable clones were screened with 2 \u0026micro;g/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\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal 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\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were lysed in RIPA lysis buffer and centrifuged at 12 000 \u0026times;g at 4\u0026deg;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\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation, live-cell imaging, and colony formation assays\u003c/h2\u003e \u003cp\u003eFor the cell proliferation assays, 2 \u0026times; 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 \u0026times; 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\u0026ndash;12 days, the cells were fixed with 4% paraformaldehyde, subjected to crystal violet staining, and photographed, and the colonies were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003e 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\u0026ndash;5 weeks to analyze the effects of NAT10 and CCL2 on ICC. HuCCT1-shNC, HuCCT1-shNAT10#1, and HuCCT1-shCCL2 cells (1 \u0026times; 107/100 \u0026micro;L per mouse, n\u0026thinsp;=\u0026thinsp;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\u0026thinsp;=\u0026thinsp;0.5 \u0026times; L \u0026times; 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\u0026amp;E) staining, immunohistochemistry (IHC), immunofluorescence, and WB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E staining, IHC, and immunofluorescence\u003c/h2\u003e \u003cp\u003eTissue samples were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for H\u0026amp;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\u0026deg;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\u0026thinsp;=\u0026thinsp;π(i\u0026thinsp;+\u0026thinsp;1), where π is the percentage of positive cells and i is the staining intensity (0\u0026ndash;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.\u003c/p\u003e \u003cp\u003eFor immunofluorescence, xenograft tumor sections underwent dewaxing, hydration, antigen repair, and incubation in 3% H2O2 at 37\u0026deg;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNanopore Technologies (ONT) full-length transcriptome sequencing\u003c/h2\u003e \u003cp\u003eONT 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\u0026thinsp;\u0026ge;\u0026thinsp;1.5 and P\u0026thinsp;\u0026lt;\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation quantitative polymerase chain reaction (RIP-qPCR)\u003c/h2\u003e \u003cp\u003eHuCCT1 cells and the Imprint\u0026reg; 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 \u0026micro;g 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCoimmunoprecipitation (COIP) assay\u003c/h2\u003e \u003cp\u003eHuCCT1 cells were lysed on ice with immunoprecipitation lysis buffer for 30 minutes and then centrifuged at 4\u0026deg;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\u0026deg;C overnight. Then, 30 \u0026micro;L of beads were added, and the sample was rotated overnight at 4\u0026deg;C. The beads were washed three times with cracking buffer, and the supernatant was collected for western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe 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\u0026rsquo;s instructions. After adding the substrate solution, the plate was incubated at 37\u0026deg;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.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eThe 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\u0026thinsp;=\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing after BBR treatment\u003c/h2\u003e \u003cp\u003eCells 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\u0026thinsp;\u0026ge;\u0026thinsp;2 or \u0026le;\u0026thinsp;1/2 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were the screening criteria. Differentially expressed genes were analyzed using GO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://geneontology.org/\u003c/span\u003e\u003cspan address=\"http://geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.kegg.jp/\u003c/span\u003e\u003cspan address=\"http://www.kegg.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eSurface plasmon resonance (SPR)\u003c/h2\u003e \u003cp\u003eSPR 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eToxicity testing\u003c/h2\u003e \u003cp\u003eBBR (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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eICC 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\u0026rsquo;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\u0026deg;C for 30 minutes. Macrophage polarization was analyzed using an Agilent flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAntitumor effect of BBR in vivo\u003c/h2\u003e \u003cp\u003eHuCCT1 cells (1 \u0026times; 107) were suspended in 100 \u0026micro;L serum-free RPMI-1640 medium and injected subcutaneously into the right forelimb of male BALB/c nude mice aged 4\u0026ndash;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prime 8.0 and SPSS Statistics 20.0. All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and were analyzed by Student\u0026rsquo;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\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTC 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (82060551; 82060666).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRizvi S, Gores GJ. Pathogenesis, Diagnosis, and Management of Cholangiocarcinoma. Gastroenterology. 2013;145:1215\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRizvi S, Khan SA, Hallemeier CL, Kelley RK, Gores GJ. 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Mil Med Res. 2022;9:22.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NAT10, CCL2, intrahepatic cholangiocarcinoma, M2 polarization, berberine","lastPublishedDoi":"10.21203/rs.3.rs-4099955/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4099955/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntrahepatic 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.\u003c/p\u003e","manuscriptTitle":"NAT10 affects the progression of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating CCL2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-20 12:00:39","doi":"10.21203/rs.3.rs-4099955/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"20a7a032-8e45-49a3-8ca7-f1472b26e8ce","owner":[],"postedDate":"March 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-31T22:29:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-20 12:00:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4099955","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4099955","identity":"rs-4099955","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}