Introduction
Intrahepatic cholangiocarcinoma (ICC) emanates from the epithelial lining of the intrahepatic biliary tree, and is a malignant disease characterized by onset occult, rapid progression, high relapse rate, and high mortality [
1,
2]. As the second most common primary liver cancer, ICC accounts for about 3% of all gastrointestinal malignancies, and recent years have seen a rapid increase in ICC-related morbidity and mortality [
3,
4]. Thus, there is an urgent need to explore the molecular mechanisms mediating the development of ICC, which will lay the basis for identifying novel diagnostic biomarkers and validating therapeutic targets.
Tumor metastasis is one of the most important indicators of tumor progression and a leading cause of mortality in cancer patients. Besides, the tumor microenvironment (TME) has been shown to play a key role in driving tumor development and metastasis [
5]. ICC is more prone to intrahepatic spreading and metastasis as compared with extrahepatic cholangiocarcinoma. The ICC cells break through the wall of the intrahepatic bile duct and invade the liver parenchyma. The ICC liver microenvironment is mainly composed of liver parenchymal cells, which account for 85%, and non-parenchymal cells [
6]. Previous data have demonstrated that liver cells can modulate the TME via the secretion of cytokines, chemokines, and other tumor-regulating factors which are vital for tumor metastasis [
7‐
9]. Other studies have shown that various cytokines and chemokines such as CCL2 and CXCL12 in the liver microenvironment mediate many physiological and pathological processes in cholangiocarcinoma [
10,
11].
Chemokines, a family of small proteins (8–10 kDa) that interact with specific G-protein–coupled chemokine receptors, are key components of the TME [
12]. The chemokines play an important role in processes controlled at epigenetic, transcriptional, and post-transcriptional regulation levels, which affect the occurrence and development of tumor cells [
13]. In addition, there has been an association between extracellular signals and N6-methyladenosine (m
6A) modification [
14,
15]. For example, TGF-β can directly modulate the m
6A dynamics which trigger cancer proliferation and metastasis [
16,
17].
The m
6A methylation occurs on the sixth N of adenylate (A) in RNA, which is the most universal and abundant epigenetic modification in eukaryotic mRNA [
18,
19]. The m
6A RNA methylation is a dynamic and reversible process mainly mediated by the methyltransferase complex. The process is mediated by m
6A demethylases and is related to diverse cellular functions by recognizing and binding the m
6A reader proteins [
20]. The m
6A methyltransferase complex consists of METTL3, METTL14, VIRMA (vir-like m
6A methyltransferase associated), and WTAP, while the m
6A demethylases comprise FTO and AlkBH5. m
6A methyltransferase includes YTH domain-containing family proteins (YTHDFs, comprising YTHDF1/2/3) and insulin-like growth factor 2 binding proteins (IGF2BPs, comprising IGF2BP1/2/3), which can specifically recognize m
6A modification sites and recruit related proteins to regulate the stability of mRNA, nuclear output or translation efficiency. Another study suggested that the m
6A modification and m
6A binding proteins participate in a range of physiological and pathological processes such as DNA damage [
21], embryonic development [
22], immune response [
23], tumorigenesis, or tumor progression [
24,
25]. Besides, m
6A methylation plays an essential role in the promotion and metastasis of many types of malignancies such as gastric cancer [
26], hepatocellular cancer [
27], ovarian cancer [
28], breast cancer [
29], and colorectal cancer [
30,
31], thus is a potential target for cancer therapy. For instance, VIRMA promotes tumor development in breast cancer [
32], while METTL3-mediated m
6A modification of HDGF mRNA modulates the progression of gastric cancer [
26]. To date, however, data on the biological roles and mechanisms of m
6A modification in ICC remain unclear.
In this study, we analyzed the interplay between ICC and liver microenvironment and investigated the role of m6A modification in ICC. Our study demonstrated that CCL3, which is secreted by hepatocytes, promotes tumor metastasis by fueling m6A modification in ICC. Moreover, we showed that VIRMA was a major modulator in the m6A modification, which correlated with poor outcomes in ICC patients and promoted ICC metastasis. In addition, SIRT1 was shown to be a critical downstream target of VIRMA, which fuels tumor metastasis. Taken together, our data enhanced our understanding of the interaction between hepatocytes and ICC cells, and uncovered the molecular mechanism of VIRMA-mediated m6A modification in ICC metastasis.
Materials and methods
Tissue specimens and clinical data
Ethical approval for this study was provided by the Institutional Review Board of Sun Yat-sen Memorial Hospital. A total of 110 paraffin-embedded ICC specimens and corresponding adjacent normal tissues were obtained from Sun Yat-sen Memorial Hospital, Sun Yat-sen University between January 2011 and December 2020.
Cell culture
Immortalized normal liver epithelial cells (THLE3) and human normal bile duct epithelial cell lines (HIBEpiC) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured as specified by respective manufacturers. Two intrahepatic cholangiocarcinoma cell lines (HuCCT1 and RBE) were acquired from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. The cell lines were grown at 37 °C under 5% CO2 in a 1640 medium supplemented with 10% fetal bovine serum (FBS, Hyclone, USA). The cells were seeded at a density of 5 × 105 cells in a T25 culture flask and passaged every 4–5 days.
Transwell assays
Here, we employed both the transwell migration and invasion assays. The ICC cells were inoculated in the upper transwell chamber (Corning, USA) with an indicated density of 3–5 × 104 cells per well in the 24-well plates, with or without Matrigel (BD Biosciences, USA), and incubated for 24 h. Thereafter, unmigrated and uninvaded ICC cells were removed with cotton swabs. The ICC cells on the bottom surface were then fixed in methanol for 10 min, at room temperature, followed by staining with 0.5% crystal violet. The experiments were carried out three times and in triplicate.
Quantitative real-time PCR
Total RNA was extracted from tissues and cells using TRIzol Reagent (Invitrogen, USA), following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using PrimeScript RT reagent Kit and SYBR Premix Ex Taq (Takara, Japan), and employed primer sequences as shown in Additional file
1: Table S1. Results were normalized to GAPDH expression and calculated using the 2
–ΔΔCt method. The experiments were repeated at least three times.
Plasmid construction
Full-length VIRMA (Ensembl: ENSG00000164944) cDNA was designed and synthesized by GenePharma (Shanghai, China), and then the cloned fragments were ligated into pcDNA3.1. Lipofectamine 3000 (Invitrogen, USA) was served as the role of transfecting the plasmids into the ICC cells, following the manufacturer’s protocol. Small hairpin RNA (shRNA) of VIRMA (shVIRMA) and negative control (shNC) were designed as shown in Additional file
2: Table S2 and synthesized by GenePharma (Shanghai, China). In order to acquire stable VIRMA-knockdown ICC cell lines, the cells were infected with lentiviral particles and then selected under 8 μg/ml puromycin pressure.
Animal experiments
All animal experiments were approved by the Animal Research Committee of Sun Yat-sen University Cancer Center. Female Balb/c nude mice (Balb/c-nu, weighing ~ 15–20 g), were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). To generate a subcutaneous xenograft tumor model, we injected 5 × 106 transfected HuCCT1 cells, which were suspended in 0.2 ml of PBS, into the flank of nude mice (five mice per group). On the other hand, an orthotopic xenograft tumor model was established by inoculating 3 × 106 transfected HuCCT1 cells in 50 μl of PBS into the liver of nude mice (five mice per group). For the treated groups, HuCCT1 cells were treated with CCL3 (100 ng/ml) for 6 h before inoculation. For subcutaneous injection, the intratumoral multiple-point injection of CCL3 (20 mg/kg) diluted in 50 μl PBS was performed every 5 days. The control groups were treated with PBS. Subcutaneous tumor size was measured twice a week. After in vivo fluorescence imaging, all the study mice were sacrificed with the in vivo imaging system (IVIS) spectrum after four weeks. The mice tumors and organs were dissected, photographed, weighed, and stained.
Additional methods and experimental details are described in Additional file
4.
Discussion
Metastasis is the main clinical challenge in ICC. Tumor metastasis is affected by the TME, a unique environment that arises because of tumor progression. Most studies on the mechanism of ICC metastasis have focused on the analysis of the ICC cells, but only a few have assessed the role of other cells in TME. The liver cancer microenvironment is composed of cells such as immune cells, fibroblasts, and liver parenchymal cells as well as extracellular matrix components [
33]. Previous studies have demonstrated that ICC development is affected by the liver microenvironment, which promotes tumor proliferation, infiltration, and metastasis by releasing cytokines and regulating the transcription and translation of tumor-related genes [
34,
35].
In this study, we showed that the co-culture of the ICC cells with hepatocytes significantly enhanced the migration and invasion ability of the cells
. Moreover, to clarify the molecular interaction between hepatocytes and ICC cells, we performed a cytokine microarray analysis and showed that CCL3 is the main chemokine that is involved in the interaction. CCL3, also referred to as macrophage inflammatory protein-1 (MIP-1α), belongs to the C–C chemokine family. Studies have shown that CCL3 contributes to the development and progression of various malignancies, such as esophageal cancer, breast cancer, osteosarcoma, leukemia, and multiple myeloma [
36]. As expected, our data demonstrated that recombinant human CCL3 obviously promoted migration and invasion of the ICC cells.
Thereafter, we analyzed the mechanisms of CCL3 activity in the communication between hepatocytes and ICC. Functional enrichment analysis showed that the genes that interacted with CCL3 were enriched for functions related to RNA metabolism. We further demonstrated that chemical modification occurs at the RNA level, which affects gene-regulatory networks at the epigenetic level. Previous studies demonstrated an association between extracellular signals such as cytokines or chemokines and m
6A modification [
14,
15]. The m
6A RNA modification has been identified as a source of epigenetic regulation in DNA modification. The m
6A modification accounts for more than 80% of RNA modification and is a reversible and dynamic process regulated by m
6A methyltransferase, demethylases, and readers [
37]. m
6A methylation of mRNA has been reported to play significant roles in many RNA functions, such as RNA splicing, RNA processing, translation regulation, and RNA decay [
18‐
20,
38,
39]. However, data on the role of m
6A modification in ICC remains limited. In this study, there was an increased m
6A modification in ICC cells co-cultured with hepatocytes, which was potentially affecting cancer metastasis. We showed that CCL3 was the key factor mediating the interaction between the liver microenvironment and ICC cells. Subsequently, we characterized methyltransferase VIRMA and showed that it is the aberrant m
6A modification in ICC cells co-cultured with hepatocytes.
VIRMA is an important member of the m
6A methyltransferase complex and is mainly located in the nucleosome and cytoplasm. As a key regulator of m
6A methylation, VIRMA was shown to form a complex with WTAP/METTL3/METTL14 heterodimer to catalyze m
6A formation [
25,
40]. Recent studies have shown that VIRMA is upregulated in several malignancies such as breast cancer [
32], lung cancer [
41], germ cell tumor [
42], and liver cancer [
43], and is involved in tumor proliferation and metastasis. So far, however, there are limited studies that have evaluated the biological function and mechanism of VIRMA-mediated m
6A modification in ICC. In this study, we showed that VIRMA was significantly upregulated in ICC tissues, which correlated with poor prognosis and thus regarded as a prognostic factor for tumor recurrence in ICC. Moreover, our in vitro and in vivo assays demonstrated that VIRMA promotes malignant development and progression of ICC, and its expression showcased a significant correlation with various metastatic, angiogenic and proliferation gene markers. These findings suggested that VIRMA may function as a tumor-promoting gene and might be a potential clinical prognostic biomarker and therapeutic target for ICC.
Analysis of the RNA-Seq and MeRIP-Seq data identified SIRT1 (Sirtuin type 1), as a crucial downstream target of VIRMA. Our RNA pull-down and RIP assays demonstrated that SIRT1 was positively regulated by VIRMA and modified by VIRMA-mediated m
6A methylation. Thereafter, we conducted GO and KEGG enrichment analyses to explore the major biological functions and downstream signaling pathways of the genes from the RNA-Seq and MeRIP-Seq analyses, respectively. SIRTI, a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, plays important roles in oncogenesis via transcription, translation, and post-translational modifications [
44]. SIRT1 was shown to be a potential tumor promoter based on its role in negatively regulating many tumors suppressors [
45,
46]. In sync with previous studies, our findings showed that SIRT1 facilitates the proliferation and migration of tumor cells in ICC. As previously demonstrated, SIRT1 modulates various pathways such as the p53 signaling pathway [
47,
48] and FoxO signaling pathway [
44] to control cell cycle, metabolism, proliferation, differentiation, and epigenetics. These data agree with that obtained by GO and KEGG enrichment analysis of MeRIP-seq. Therefore, CCL3/VIRMA/SIRT1 axis may be one of the important mechanisms underlying tumor evolution and metastasis and is a potential new therapeutic target for ICC.
In our study, we only employed the m6A dot blot assay to quantify the m6A levels of ICC, and showed that SIRT1 is a downstream target of VIRMA. Thus, there is a need for further studies which would take the following variables into account: (1) examine the m6A levels of ICC using a colorimetric strategy or liquid chromatography-mass spectrometry (LC–MS), (2) detect whether VIRMA functions independently of its m6A catalytic activity in cancer progression, and (3) develop a peptide inhibitor to target VIRMA domain and explore whether it may be beneficial in the treatment of ICC.
In conclusion, our study highlights the critical role of VIRMA-mediated m6A modification in ICC progression and metastasis. Mechanistically, we demonstrated that CCL3 is secreted by hepatocytes and may promote metastasis of ICC cells by regulating m6A methylation. The regulation of m6A methylation is mediated by VIRMA, which epigenetically promotes SIRT1 expression through an m6A methylation-dependent mechanism. Our results suggested that the interaction between hepatocytes and ICC cells might offer a possible interventional target for ICC. Besides, the m6A modification on tumor metastasis will contribute to further studies that would explore molecular mechanisms and identify efficient treatment strategies against ICC.
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