Revised: April 4, 2026
Accepted: May 11, 2026
Published online: June 27, 2026
Processing time: 120 Days and 7.6 Hours
Aberrant methionine (Met) metabolism, ferroptosis, and epithelial-mesenchymal transition (EMT) have been recognized as critical drivers of tumor progression. However, their crosstalk and underlying regulatory mechanisms in hepatocellular carcinoma (HCC) remain unclear.
To explore the effect of Met metabolic dysregulation on ferroptosis and EMT within HCC cells and its underlying mechanisms.
Met levels in HCC cells were measured, and alterations in ferroptosis- and EMT-related markers were evaluated following Met depletion or supplementation. ARNT2 levels were assessed under altered Met conditions. ARNT2’s expression pattern in HCC tissues and its correlation with patient prognosis was analyzed by bioinformatics tools. The expression of ferroptosis/EMT markers and UBE2Z were examined after ARNT2 knockdown. UBE2Z levels in HCC tissues were mea
The results demonstrated that, with prolonged culture duration, Met levels in HCC cells decreased more markedly compared to normal hepatocytes. Met de
Met deficiency inhibits HCC progression by inducing ferroptosis and suppressing EMT via the ARNT2/UBE2Z signaling, while Met supplementation exerts the opposite effect.
Core Tip: Aberrant methionine (Met) metabolism critically promotes the progression of hepatocellular carcinoma (HCC). Met deficiency may induce ferroptosis and inhibit epithelial-mesenchymal transition through the ARNT2/UBE2Z axis, thereby suppressing HCC progression, whereas Met supplementation reverses these effects. Furthermore, high expression levels of ARNT2 and UBE2Z predict unfavorable prognosis in HCC patients. Collectively, these findings indicate that the Met-ARNT2/UBE2Z pathway may serve as a novel anti-HCC therapeutic target.
- Citation: Yin DD, Jiang WY, Huang YT, Zhou SP, Zhang YC. Methionine depletion inhibits hepatocellular carcinoma by inducing ferroptosis and suppressing epithelial-mesenchymal transition through the ARNT2/UBE2Z pathway. World J Hepatol 2026; 18(6): 120427
- URL: https://www.wjgnet.com/1948-5182/full/v18/i6/120427.htm
- DOI: https://dx.doi.org/10.4254/wjh.120427
Hepatocellular carcinoma (HCC) occupies 75%-85% of all primary liver cancers and ranks fifth in malignancies with regard to global morbidity and third among factors inducing cancer-related death[1]. This may mainly be due to the low early diagnosis rate of HCC and the limited treatment effects in advanced stages[1]. Despite significant progress in HCC treatment over the past few years, especially in systemic and local therapies, challenges such as difficulty in selecting treatment options, drug resistance and recurrence, individual variability, and high treatment costs persist[2]. Conse
The occurrence and development of HCC involve multiple complex biological processes, among which the regulation of ferroptosis and epithelial-mesenchymal transition (EMT) exerts crucial effects on the invasion, metastasis, and drug resistance of HCC[3,4]. EMT endows HCC cells with enhanced migratory and invasive capabilities, whereas ferroptosis, the cell death pattern dependent on iron-catalyzed lipid peroxidation, has significant implications for tumor therapy[5]. Ferroptosis is closely associated with EMT, sharing key genes that influence the prognosis of HCC[6]. In recent years, increasing evidence has indicated that abnormal amino acid metabolism is closely linked to tumor initiation and progression[7,8]. Methionine (Met), an essential amino acid, has a critical effect on cell growth and metabolism[9]. Abnormal Met metabolism affects DNA and RNA methylation modifications, which are key mechanisms regulating cellular ferroptosis and EMT[10-12]. However, how Met depletion affects ferroptosis and EMT of HCC cells, particularly the pathways through which Met depletion regulates these processes and modulates the biological behavior of HCC, remain unclear.
ARNT2 is a key helix-loop-helix transcription factor that is highly responsive to a wide range of physiological and external stimuli and directly regulates the transcription of related genes[13]. ARNT2 has recently been suggested to exert a crucial effect on cell migration and invasion. These processes are key features of EMT and are important for biological events such as embryonic development, tissue repair, and cancer progression[14,15]. In addition, ARNT2 has been identified as a crucial regulator of cardiac fibrosis[16]. The tumor microenvironment often features hypoxic or low-oxygen conditions. ARNT is a component of the hypoxia-inducible factor pathway, mediating adaptive responses that support tumor cell survival under hypoxic conditions[17]. Notably, previous reports have indicated that met-choline-deficient can increase the levels of hypoxia-inducible factor-alpha in cells[18]. This finding suggests a complex link between intracellular Met metabolism and ARNT2 function of ARNT2.
In this study, we explore into the effects of Met deficiency vs supplementation on ferroptosis and EMT within HCC cells. Furthermore, we also examined the regulatory function of ARNT2 in this intricate process, along with its down
Human HCC cells, including Huh7 (RRID: CVCL_0336) and Hep3B (RRID: CVCL_0326), were provided by Shanghai Cyagen Biosciences Inc. (China) and cultivated within 1640 medium that contained 10 g/L penicillin/streptomycin antibiotics and 100 g/L certified fetal bovine serum (C04001-500, Vivacell), or in 1640 medium without Met (C0893-500 mL, Beyotime). Human hepatocytes HHL-5 (RRID: CVCL_S956) were obtained from Shanghai Qingqi Biotechnology Development Co. (China). All cells were authenticated by STR profiling within the last three years and were confirmed to be mycoplasma-free. The cells were cultured in high-glucose DMEM complete medium supplemented with 10 g/L penicillin/streptomycin antibiotics and 100 g/L certified fetal bovine serum (C04001-500, Vivacell). All cells received culture under 37 °C and 50 mL/L CO2 conditions.
Erastin (a ferroptosis inducer, HY-15763), ferrostatin-1 (Fer-1, a strong, selective ferroptosis inhibitor, HY-100579), necro
After removing the culture medium, cells were rinsed using phosphate-buffered saline (PBS), followed by addition of RIPA lysis buffer including protease inhibitors for 30 minutes of cell lysis on ice. Subsequently, cells were centrifuged (12000 rpm, 30 minutes, 4 °C) to collect supernatants as protein samples. Pierce™ BCA Protein Assay Kits (23225, Thermo Scientific™, United States) were adopted for determining total protein content. Following separation through SDS-PAGE, proteins were subjected to transfer on PVDF membranes. Thereafter, these membranes received 1 hour of blocking using 50 mg/mL defatted milk under ambient temperature, prior to overnight primary antibody incubation under 4 °C, involving ACSL4 (1:2500, HUABIO, ET7111-43, China), GPX4 (1:5000, HUABIO, ET1706-45, China), E-cadherin (1:1000, CST, 9782, United States), N-cadherin (1:1000, CST, 9782, United States), ARNT2 (1:1000, UpingBio, YP-Ab-01539, China), and UBE2Z (1:1000, Abmart, PS08611S, China). After incubation, TBST was introduced for membrane washing before additional 1 hour of secondary antibody incubation under ambient temperature. After further washing with TBST, chemiluminescence imaging was performed. Target protein expression was quantified using β-actin as an internal con
After removing medium, cells underwent rapid washing thrice with pre-cooled PBS. A small volume of PBS was then added, and a cell scraper was used to collect cells before transfer into centrifuge tubes. The cells were centrifuged for 5 minutes (4 °C, 500 × g). After removal of the supernatant, the cells were washed with PBS and centrifuged again for 5 minutes (4 °C, 1000 × g). The PBS-containing cell suspension was then counted. A suspension containing 1 × 107 cells was added in the 2 mL sterile centrifuge tube before centrifugation (4 °C, 10 minutes, 1000 × g). After discarding supernatants, total protein level was analyzed by BCA kit. Following relevant reagent kit protocols, Met (YPJ1223, UpingBio, China) and S-adenosylmethionine (SAM) (YPJ1224, UpingBio, China) concentrations were determined and subsequently calculated based on standard curves. Results were expressed as the content of Met and SAM per unit protein weight (mg/ng) in the original samples.
Cells (1 × 103/well) were inoculated in 96-well plates before 24 hours, 48 hours, and 72 hours of culture in a 50 mL/L CO2 incubator at 37 °C. Medium was removed at respective time points and substituted by fresh complete medium. Subse
The DCFH-DA stock solution (S0033S, Beyotime, China) received dilution using serum-free medium at 1:1000 to 10 μM. After collection, cell suspension was obtained within this DCFH-DA solution at 1-20 million cells/mL before 20 minutes of incubation at 37 ºC. To guarantee sufficient probe-cell contact, this suspension was slightly inverted before mixing at 3-5 minutes intervals. Serum-free medium was added to rinse cells thrice for removing unincorporated DCFH-DA. At last, the fluorometer was employed for measuring fluorescence intensities.
After removal of the culture medium, cells received PBS washing before counting. RIPA lysis buffer was added at a ratio of 0.1 mL per 1 million cells. Following lysis on ice, the mixed samples were centrifuged (12000 × g, 10 minutes) to obtain the supernatant, and total protein content was determined using the BCA protein assay kit. A sample (0.1 mL) was added to a 1 mL centrifuge tube, followed by addition of malondialdehyde (MDA) detection working solution (0.2 mL, S0131S, Beyotime, China). After mixing, the samples were heated at 100 ºC for 15 minutes. The mixtures were then cooled to room temperature within the water bath before 10 min of centrifugation (1000 × g) under ambient temperature. Subsequently, the supernatants (200 μL) were added into the 96-well plate, while absorbance values were determined at 532 nm with the microplate reader. Results were expressed as MDA content per unit protein weight (μmoL/mg) in the original samples.
Cells (1 × 106) were collected and homogenized in PBS (500 μL). After homogenization, the samples received centrifugation (10000 × g, 4 °C, 10 minutes) to collect supernatants, which were kept on ice for subsequent analysis. A portion of the supernatant was reserved for measuring total protein level by BCA kit. Reduced glutathione (GSH) colorimetric assay was then performed using the assay kit (E-BC-K030-M, Elabscience, China). OD measurements were completed at 405 nm, with GSH level being determined according to the standard curve.
When the medium was removed, cells received PBS washing. A cell suspension was then prepared by trypsin digestion, followed by centrifugation (600 × g, 5 minutes) under ambient temperature to remove supernatants. An appropriate volume of BODIPY 581/591 staining working solution (S0043S, Beyotime, China) was introduced till reaching 1-10 million cells/mL. These cells then underwent 25 minutes of incubation under 37 ºC in a cell culture incubator. Subse
Following medium removal, cells were rinsed by PBS. Following trypsin digestion, a cell suspension was prepared and counted. Cells (5 × 107) were collected and rinsed once with cold PBS (1 mL), followed by centrifugation (14000 rpm, 4 minutes) to discard the supernatant. The Iron assay kit (I291, Dojindo, Japan) was prepared according to the manufacturer’s instructions. Subsequently, assay buffer (1.3 mL) was added, and the samples were sonicated for 5 minutes. The mixed samples were centrifuged again (16000 × g, 10 minutes), and the supernatant (1.3 mL) was aliquoted into separate tubes for preparation of ferrous iron, total iron samples, and sample blanks. Assay buffer (20 μL) was introduced into sample blank and ferrous iron tubes, while reducer solution (20 μL) into total iron sample tube; addi
After removal of the medium, PBS was added for cell washing. Subsequently, medium (1 mL/well) was introduced to the 6-well plate. JC-1 staining working solution (1 mL, BL711A, Biosharp, China) was then poured and mixed thoroughly. After 20 minutes of incubation under 37 °C, the supernatant was removed. Following washing by JC-1 staining buffer twice, the medium (2 mL) was poured, and the fluorescence microscope was employed for cell observation. Alteration of mitochondrial membrane potential was represented by the green-to-red fluorescence ratio.
After removing the medium, cells were rinsed by PBS. Following trypsin digestion and resuspension, the cell suspension was obtained and counted. Cells (1000/well) were later inoculated in the 6-well plate before culture under 37 °C and 50 mL/L CO2 conditions. After colonies formed and the number of cells within each colony exceeded 50, supernatants were removed before cell washing by PBS. Following 30 minutes of 40 g/L paraformaldehyde fixation, cells were rinsed by PBS prior to 15 minutes of 1 g/L crystal violet staining. At last, image capturing and colony counting were completed.
Three equally spaced horizontal lines were drawn on the bottom of each well of a 6-well plate using a black marker. Cells (8 × 105/well) were seeded into the 6-well plate and cultured for 24 hours. Using a 10 μL pipette tip, three perpendicular scratches were made across the pre-drawn lines to create cell wounds. Subsequently, serum-free medium was introduced to culture cells for 24 hours. Finally, images were obtained using an inverted microscope to evaluate wound closure.
After 24 hours of starvation through substituting original medium by serum-free medium, a serum-free cell suspension (density, 1 × 106 cells/mL) was prepared. Afterwards, cell suspension (100 μL) was introduced into the upper Transwell chamber (14342-D, LABSELECT, China) pre-coated with Matrigel (3D200-005, BDBIO, China). Complete medium containing 100 g/L serum (600 μL) was introduced into the bottom chamber. The medium was eliminated following 24 hours of 50 mL/L CO2 incubation under 37 °C, while cells were gently rinsed by PBS thrice. These cells then received 10 minutes of 40 g/L paraformaldehyde fixation and 10 minutes of 1 g/L crystal violet staining. At last, migrated cells were counted and photographs were taken with the inverted microscope for analysis.
In the ULCAN online database (https://ualcan.path.uab.edu/analysis.html), the ARNT2 and UBE2Z gene expression levels were analyzed using the “Expression” module based on different clinical parameters. The association between ARNT2 and UBE2Z gene levels was explored using the “Correlation” module. Furthermore, the relation of UBE2Z gene level with patient survival was evaluated with “Survival” module. ARNT2 gene expression was also analyzed using UCSC Xena (https://xena.ucsc.edu/) and GEPIA 2 (http://gepia2.cancer-pku.cn/#analysis) online databases. Additio
A total of 14 liver cancer tissue samples and 14 matched non-carcinoma samples were obtained as paraffin-embedded blocks from the Department of Pathology, the First Affiliated Hospital of Anhui University of Science and Technology, from January 1, 2021, to November 31, 2024. These specimens were processed into tissue microarrays (TMAs). However, due to the extremely small volume of cancer tissue in two cases, the corresponding cancer tissue spots on the TMAs were missing. All 14 liver cancer patients had confirmed pathological diagnoses, and none had received preoperative radio
For statistical analysis framework, all experimental protocols were subjected to three independent replications to ensure data reproducibility. Each replication was conducted under an identical experimental condition with separate sample preparations to minimize systematic errors. We adopted the two-tailed unpaired Student’s t-test in independent group analyses. P < 0.05 stood for significant differences, and each test was conducted using GraphPad Prism 10 to ensure computational accuracy. Before conducting the t-test, Shapiro-Wilk test was utilized for assessing data normality, while appropriate non-parametric alternatives (such as the Mann-Whitney U test) were pre-specified for non-normally-distributed variables to maintain analytical rigor.
Firstly, intracellular Met and SAM levels were precisely measured. The results showed that both Met (Figure 1A) and SAM (Figure 1B) levels in HCC cells significantly decreased after prolonged culture compared with the control group. This phenomenon indicates that HCC cells excessively consume Met during their growth compared to normal hepato
Given that the physiological concentration of Met in human serum is about 30 μm[19], and considering that tumor cells consume substantial amounts of Met while high concentrations (100 μm) have minimal effects on tumor cell death[7], the impact of Met supplementation (100 μm) for 72 hours on ferroptosis of HCC cells was further investigated. Western blot was first performed to examine ACSL4 and GPX4 levels. As discovered, the Met+ group exhibited significantly reduced ACSL4 levels, whereas GPX4 expression was notably increased relative to control group (Figure 2A). Next, the in
Overall, these results indicate that Met supplementation inhibits ferroptosis in HCC cells, as reflected by changes in ferroptosis-related proteins, decreased lipid peroxidation, reduced ROS levels, lower Fe2+ content, and increased mitochondrial membrane potential.
To further analyze how Met supplementation and deficiency affected EMT of HCC cells, this study designed two experimental groups: One group cultured HCC cells were cultured for 72 hours in Met-free medium to mimic Met deficiency, and in medium supplemented with Met (100 μm) for 72 hours to simulate a Met-sufficient environment.
The occurrence of EMT is associated with multiple biological changes, including alterations in EMT marker expression and enhanced cell proliferation, invasion, and migration. E-cadherin and N-cadherin, two key EMT proteins. Our results (Figure 3A) indicated that, the Met+ group showed the markedly lower E-cadherin level whereas notably higher N-cadherin level than control group, indicating activation of EMT. In contrast, the Met- group showed E-cadherin up-regulation but N-cadherin down-regulation, suggesting suppression of EMT. Subsequently, cell proliferation activity was evaluated. As shown in Figure 3B, the Met+ group formed more and larger colonies relative to control group, suggesting enhanced proliferation, whereas the Met- group exhibited the opposite trend. Scratch healing assay results (Figure 3C) showed that the Met+ group had a significantly faster wound closure rate than the control group, indicating that Met supplementation enhances HCC cell migration. In contrast, the Met- group showed some degree of inhibition in cell migration. The Transwell assay results (Figure 3D) further confirmed that the Met+ group had significantly more cells migrating from the upper to lower Transwell chambers compared with the control group, confirming enhanced invasion capacity, whereas the Met- group exhibited reduced invasion ability. In summary, our findings indicate that Met supplementation promotes EMT in HCC cells, whereas Met deficiency effectively suppresses this process.
To validate this hypothesis, we investigated how Met deficiency and supplementation affect ARNT2 expression in HCC cells. As discovered, ARNT2 level markedly increased in Met+ group, but declined in Met- group relative to control group (Figure 4A). Additionally, through GEPIA 2 and UCSC Xena databases analysis, ARNT2 levels within HCC tissues generally increased relative to non-carcinoma samples (Figure 4B and Supplementary Figure 1A). By ULCAN and GEPIA 2 databases, the ARNT2 level was positively related to the tumor grade 3 in HCC (Figure 4C and Supplementary Figure 1B), suggesting its potential as a marker of disease progression. Further analysis showed that, ARNT2 expression differed significantly between HCC and normal tissues in TP53 non-mutant status (Figure 4D). We also explored the relationship between ARNT2 expression and its promoter methylation. The results indicated that, compared with the normal group, ARNT2 promoter methylation levels were reduced in HCC tissues in both the primary tumor group (Figure 4E) and the grade 3 group (Figure 4F), but increased in the TP53 NonMutant group (Figure 4G). In survival analysis, high ARNT2 expression was negatively associated with recurrence-free survival, disease-free survival, and progression-free survival, whereas statistically significant association was not detected with overall survival (Figure 4H-K). ARNT2 expression was higher in liver cancer TMA (A1, A4, A6, B1, B3, B5, B7, C1, C3, C5, C7, and D1) compared with adjacent non-tumor tissues (A2, A3, A5, A7, A8, B2, B4, B6, B8, C2, C4, C6, C8 and D2) (P < 0.01, Figure 4L). Furthermore, predictive analysis using the JASPAR database revealed strong binding sites for ARNT2 in the promoter regions of key molecules involved in EMT signaling pathways[20,21], such as Smad2/3, Wnt3A, YAP, and Notch1 (Tables 1, 2, 3, 4 and 5), strongly suggesting that ARNT2 may participate in regulating EMT. Consequently, Met probably regulates HCC cell EMT and progression through modulating ARNT2.
| Matrix ID | Name | Relative score | Start | End | Strand | Predicted sequence |
| MA1464.2 | MA1464.2.ARNT2 | 0.9659331479380997 | 1374 | 1381 | + | CCCACGTG |
| MA1464.2 | MA1464.2.ARNT2 | 0.9447176944922032 | 1372 | 1379 | - | CCCACGTG |
| Matrix ID | Name | Relative score | Start | End | Strand | Predicted sequence |
| MA1464.2 | MA1464.2.ARNT2 | 0.9053040287573585 | 1826 | 1833 | + | CCCACGTG |
| MA1464.2 | MA1464.2.ARNT2 | 0.9053040287573585 | 1828 | 1835 | - | CCCACGTG |
| Matrix ID | Name | Relative score | Start | End | Strand | Predicted sequence |
| MA1464.2 | MA1464.2.ARNT2 | 0.9041148759925752 | 59 | 66 | + | GTCACGAG |
| Matrix ID | Name | Relative score | Start | End | Strand | Predicted sequence |
| MA1464.2 | MA1464.2.ARNT2 | 1.0000000012185972 | 1796 | 1803 | + | GTCACGTG |
| MA1464.2 | MA1464.2.ARNT2 | 0.9208872643353855 | 1798 | 1805 | - | CGCACGTG |
| Matrix ID | Name | Relative score | Start | End | Strand | Predicted sequence |
| MA1464.2 | MA1464.2.ARNT2 | 0.9447176944922032 | 1167 | 1174 | + | AGCACGTG |
| MA1464.2 | MA1464.2.ARNT2 | 0.9053040287573585 | 1169 | 1176 | - | CCCACGTG |
To further clarify how ARNT2 affects ferroptosis and EMT in HCC cells, ARNT2 was silenced and the corresponding ferroptosis and EMT markers were analyzed. As shown in Figure 5A, ARNT2 silencing markedly upregulated ACSL4 expression while downregulating GPX4 relative to control group. Additionally, from Figure 5B, MDA levels were signifi
Regarding EMT-related markers, ARNT2 silencing up-regulated E-cadherin but down-regulated N-cadherin in HCC cells (Figure 5E). Furthermore, as shown in Figure 5F and G, cell proliferation and invasion abilities were significantly reduced relative to control group after ARNT2 silencing. From the above findings, ARNT2 silencing inhibits EMT process in HCC cells. In summary, these findings demonstrate that ARNT2 silencing promotes ferroptosis while suppressing EMT in HCC cells.
To explore those potential downstream mechanisms for ARNT2 to regulate ferroptosis and EMT in HCC cells, we first conducted a comprehensive analysis using the ULCAN database. The results indicated that UBE2Z ranked among the top three genes in HCC (Figure 6A) and was significantly positively correlated with ARNT2 expression (Figure 6B). As revealed by further data mining, HCC patients showing UBE2Z up-regulation showed markedly shortened survival times, suggesting that UBE2Z may serve as an important prognostic marker (Figure 6C). Compared with non-carcinoma liver samples, UBE2Z expression markedly increased within HCC tissues (Figure 6D). This increase also showed a positive association with TP53 mutation status (Figure 6E), cancer stage (Figure 6F), histological subtype (Figure 6G), and tumor grade (Figure 6H), indicating its important role in HCC progression. Subsequently, ARNT2 was experimentally silenced, and UBE2Z expression was examined. The results showed that ARNT2 silencing significantly reduced UBE2Z expression, supporting the hypothesis that ARNT2 may act as an upstream regulator of UBE2Z (Figure 6I). Consistent with the ARNT2 expression pattern, UBE2Z was highly expressed in liver cancer TMA (A1, A4, A6, B1, B3, B5, B7, C1, C3, C5, C7, and D1) compared with adjacent non-tumor tissues (A2, A3, A5, A7, A8, B2, B4, B6, B8, C2, C4, C6, C8 and D2) (P < 0.01, Figure 6J).
In summary, our study preliminarily reveals that ARNT2 may promote EMT in HCC by positively regulating UBE2Z expression and may also influence iron-related processes, thereby influencing the biological behavior of HCC cells and patient prognosis. Such results help understand the molecular mechanisms underlying HCC and offer potential targets for future targeted therapies.
The current study delved into the intricate regulatory mechanisms of Met in HCC cells, particularly in relation to ferroptosis and EMT, and further clarified the potential roles of the ARNT2 protein and its downstream effector UBE2Z within this regulatory network. Specifically, the following key findings were identified: (1) Under conditions of Met deficiency, the ferroptosis process in HCC cells was promoted, whereas EMT was significantly suppressed. Conversely, the replenishment of Met inhibited ferroptosis while enhancing EMT; (2) The supplementation of Met upregulated ARNT2 expression, which indirectly inhibited ferroptosis in HCC cells and promoted EMT, while Met deficiency down
Disturbances in amino acid metabolism have been widely recognized as key factors driving the malignant progression of various cancers, including lung[22], colorectal[23], liver[24], breast[25], and gastric cancers[26]. In-depth explorations in the field of liver cancer have unveiled specific impacts of individual amino acid metabolic pathways in HCC. For example, Wang et al[27] demonstrated the pivotal role of branched-chain amino acid aminotransferase 2 in regulating ferroptosis in HCC cells. Xu et al[28] discovered the importance of HNF4α in modulating sulfur amino acid metabolism and cellular sensitivity to Met restriction in liver cancer. Nakagawa et al[29] further indicated that alterations in intrace
This study demonstrated that adequate Met levels significantly inhibit ferroptosis in HCC cells, manifested by alte
Notably, the physiological concentration of Met in human serum is about 30 μm[19]. Although tumor cells exhibit high Met consumption, previous studies have shown that elevated Met concentrations (100 μm) have minimal effects on tumor cell death[7]. Accordingly, we further investigated the effects of 100 μm Met supplementation for 72 hours on ferroptosis in HCC cells. This concentration was selected based on commonly used in vitro protocols in liver cancer and tumor metabolism studies, ensuring sufficient Met availability for cell growth and reliable phenotypic assessment[7]. In future studies, we will further validate our findings using physiological and supraphysiological concentrations to evaluate their clinical relevance. Met supplementation significantly upregulated ARNT2 expression in HCC cells, whereas Met defi
On the other hand, UBE2Z is a well-characterized member of the E2 ubiquitin-conjugating enzyme family that regulates protein ubiquitination and stability, thereby influencing multiple biological processes involved in tumorigenesis[33,34]. As a core component of the ubiquitin-proteasome system, UBE2Z catalyzes the covalent attachment of ubiquitin moieties to target proteins, marking them for proteasomal degradation or altering their functional modification[35,36]. Previous work by Shi et al[37] showed that silencing UBE2Z suppresses tumor cell proliferation, migration, and invasion through downregulating MMP2 and MMP9. Given its enzymatic function, we propose two mutually nonexclusive mechanisms by which UBE2Z may act in HCC: First, UBE2Z may promote the K48-linked ubiquitination and protea
While this work analyzed the effects of Met on ferroptosis and EMT of HCC cells and preliminarily revealed the potential involvement of the ARNT2 transcription factor and its downstream molecule UBE2Z in this complex process, several limitations remain. Specifically, regarding the precise role of UBE2Z in HCC, our understanding is primarily based on predictions and analyses from online databases, lacking direct experimental validation to confirm its actual functions and impacts. Moreover, although we observed significant regulatory effects of Met and ARNT2 on ferroptosis and EMT in HCC cells in vitro, these findings have not yet been validated in vivo, such as in animal models. Furthermore, the expression mechanism of ARNT2 in HCC, which is only predicted and analyzed based on online databases, may be related to its promoter methylation level; however, whether Met regulates ARNT2 expression through DNA or RNA methylation has not been investigated. Additionally, the specific regulatory mechanisms between ARNT2 and UBE2Z, including how they interact, under what conditions this interaction occurs, and how this interplay affects the biological behavior of HCC cells, remain insufficiently explored. Of note, emerging evidence suggests that metabolic status regulates the stemness properties of HCC cells[38]. As a central regulator of cellular adaptation to hypoxia, HIF-1α plays a critical role in cancer metabolic reprogramming[39,40]. Dysregulation of the HIF-1α pathway is tightly related to enhanced proliferation, invasiveness, metastatic potential, and therapeutic resistance in HCC[39,40]. In addition, the HIF-1α/ARNT complex has been reported to promote immune metabolic imbalance and enhance inflammatory responses[41]. Collectively, these findings underscore the critical interplay between HIF-1α-mediated metabolic reprogramming and the malignant phenotype of HCC. Further exploration of the regulatory network linking HIF-1α, metabolism, and cancer stemness offers a novel strategy for diagnosing and treating HCC.
There are several limitations in the present work. First, functional experiments were carried out at the cellular level in vitro. Although the results provide preliminary evidence for the biological functions and potential mechanisms of the target gene, conclusions derived solely from in vitro assays can not completely indicate the complicated physio-patho
This study indicates that Met deficiency inhibits HCC progression by inducing ferroptosis and suppressing EMT via the ARNT2/UBE2Z signaling pathway, whereas Met supplementation exerts opposite effect. This discovery not only offers novel potential targets for HCC treatment but also broadens the horizons for therapeutic strategy considerations. To deepen our understanding of this mechanism, future studies should focus on experimental validation of the ARNT2-UBE2Z signaling pathway in HCC to achieve a more comprehensive understanding of its functions. Additionally, exploring and evaluating the effectiveness and safety of targeted therapeutic strategies against this pathway will be important for advancing HCC treatment. Furthermore, integrating multi-omics data, including other key molecular markers and detailed clinical information, to establish a more precise and comprehensive HCC prognosis assessment system holds great significance for guiding clinical practice and facilitating the development of individualized treatment plans. This will aid in better managing HCC patients, enhancing treatment outcomes, and ultimately enhance patient outcomes and quality of life.
We sincerely appreciate the invaluable support provided by the Central Laboratory of the First Hospital of Anhui University of Science and Technology for the research platform, which was crucial for the completion of this study.
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