Emodin Induces Oxidative Stress and Ferroptosis in Hepatocellular Carcinoma Cells via Inactivating miR- 4465/NFE2L3/HMGCR/GPX4 Signaling Axis

Emodin Induces Oxidative Stress and Ferroptosis in Hepatocellular Carcinoma Cells via Inactivating miR- 4465/NFE2L3/HMGCR/GPX4 Signaling Axis | 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 Article Emodin Induces Oxidative Stress and Ferroptosis in Hepatocellular Carcinoma Cells via Inactivating miR- 4465/NFE2L3/HMGCR/GPX4 Signaling Axis Zhiran Ding, Menghua Zheng, Yu Li, Bingbing Mou, Boxin Qin, Lu Qiu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6007918/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Ferroptosis is a form of programmed cell death characterized by iron-dependent lipid peroxidation. Targeting ferroptosis is considered a novel strategy for cancer treatment. The benefits of using natural products to treat tumors have drawn more attention. Emodin, a natural anthraquinone derivative, has been shown to exert anti-tumor effects by promoting the generation and accumulation of reactive oxygen species (ROS), inducing apoptosis, autophagy, and cell cycle arrest. The molecular processes behind Emodin-mediated ferroptosis in hepatocellular carcinoma (HCC) cells were examined in our work. Emodin caused ferroptosis and suppressed growth in HCC cells in vitro. Emodin could increase ROS and lipid peroxidation, meanwhile decreasing glutathione (GSH), mitochondrial membrane potential, glutathione peroxidase 4 (GPX4), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression, these effects could be reversed by Ferostatin-1 (Fer-1, an inhibitor of ferroptosis) and NFE2-like bZIP transcription factor 3 (NFE2L3). Mechanistically, Emodin enhances the expression of miR-4465 , thereby suppressing NFE2L3 expression. The interaction between NFE2L3 and the HMGCR promoter is diminished, which subsequently downregulates GPX4 expression via the mevalonate pathway, leading to ferroptosis. Overexpression of NFE2L3 could alleviate Emodin-induced ferroptosis in HCC cells. Moreover, NFE2L3 knockout markedly reduced the expression of HMGCR and GPX4 in the Nfe2l3 −/− mouse model. Emodin also caused ferroptosis and inhibited tumor development in a xenograft mice model. In conclusion, these results suggested that Emodin induces ferroptosis by inactivating the NFE2L3/HMGCR/GPX4 pathway in HCC cells. Emodin may be a promising candidate for the development of anticancer drugs and offers new strategies for cancer therapy. Biological sciences/Molecular biology Health sciences/Medical research Health sciences/Molecular medicine Emodin Ferroptosis Oxidative stress NFE2L3 HMGCR Hepatocellular carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Hepatocellular carcinoma (HCC) is one of the major malignant tumors threatening human health worldwide, with a high incidence and mortality rate. 1 Due to the complexity of its biological processes, which involve various factors such as the activation of oncogenes, disruption of redox balance, abnormal cell differentiation, and angiogenesis, 2 , 3 the molecular mechanism underlying the occurrence and development of HCC is not yet fully understood. 4 Ferroptosis is an iron and reactive oxygen species (ROS)-dependent mode of programmed cell death caused by excessive lipid peroxidation and subsequent rupture of the plasma membrane, 5 which plays a key role in the onset, progression, and phenotypic transformation of HCC. 6 It is widely accepted that ferroptosis is implicated in multiple types of cancers, and the induction of ferroptosis can suppress cancer cell growth and overcome resistance to chemotherapy. Recent studies have focused on inducing ferroptosis to inhibit HCC development, which can support new anticancer strategies for cancer therapy. 7 In eukaryotes, the CNC-bZIP family functions as a transcription factor that regulates cellular redox homeostasis. Its members include NFE2L1, NFE2L2, NFE2L3, NFE2, Bach 1, and Bach 2. 8 The CNC-bZIP family members can form a heterodimeric complex with small MAF (sMAF) proteins and then bind to antioxidant response elements (AREs) on the promoters of target genes to promote transcription and regulate a variety of biological processes, including cell death, cell proliferation ,and metabolism. 9 NFE2L2 is a key regulator of oxidative stress. 10 Numerous studies have shown that target genes of NFE2L2 are involved in the regulation of ferroptosis, such as glutathione peroxidase 4 (GPX4) and heme oxygenase-1 (HO-1). 11 , 12 Recent studies have indicated that NFE2L1 promotes the expression of proteasome genes to maintain proteasome activity and inhibits ferroptosis by regulating the function of proteasomes and GPX4. 13 NFE2L3 has been shown to have high homology with the structures of NFE2L2 and NFE2L1 and is highly expressed in various malignant tumors. 14 However, it remains unclear whether NFE2L3 is involved in regulating ferroptosis in tumor cells, especially in HCC. Recently, traditional Chinese medicine and natural products have attracted increased attention for cancer treatment due to their various advantages, including enduring efficacy, multi-targeting capabilities, and reduced toxicity. Emodin, a natural anthraquinone derivative found in many widely used Chinese medicinal herbs, such as aloe and rhubarb, 15 has strong anti-inflammatory, anti-cancer, and antimicrobial activities. 16 Previous studies indicated that Emodin has an anti-tumor effect on multiple cancers, especially in liver, 17 lung, 18 colon, 19 breast, 20 ovarian and pancreatic cancer. 21 , 22 Recent studies have shown that Emodin activates NCOA4-mediated ferritinophagy and induces ferroptosis in colorectal cancer. 23 However, it is unclear whether Emodin induces ferroptosis in the anti-cancer effect on HCC and its specific mechanism. The present study demonstrated that Emodin inhibited HCC cell growth both in vivo and in vitro through inducing ferroptosis, and the regulatory axis miR4465/NFE2L3/HMGCR was involved in this process. Our results revealed a new insight into the mechanism of Emodin-induced ferroptosis and provided a valuable candidate for the development of anticancer drugs. Materials and methods Cell culture All cells (MHCC97H, HepG2, and HEK-293T) were cultured in complete medium (BasalMedia, #L110KJ) at 37°C with 5% CO 2 . Transient transfection and lentivirus transfection siRNA, plasmid, and miRNA were transfected into cells using Lipofectamine® 3000 in Opti-MEM (BasalMedia, #L530JV). Cells were infected with NEF2L3 knockdown or overexpression lentivirus (Cyagen) using puromycin resistance (2 µg/ml) and fluorescence as selection markers. Cell counting kit-8 (CCK-8) assay The CCK-8 kit (SparkJade, #CT0001-B) assayed cell viability after treatment with six concentrations of Emodin at 0, 5, 10, 20, 40, and 80 µM for 0, 24, 48, and 72 h. The half-maximal inhibitory concentration (IC 50 ) was then calculated from the absorbance. Intracellular ROS, lipid peroxidation, and mitochondrial membrane potential detection The fluorescence intensity detected by the DCFH-DA (Beyotime, #S0033), BODIPY C11 581/591 (Thermo Fisher, #D3861), or Mito-tracker red CMXRos (Beyotime, #C1049B) kits was used to assess cellular ROS, lipid peroxidation, and mitochondrial membrane potential levels. Glutathione detection The absorbance of the samples was measured at 412 nm with an enzyme marker (Beyotime, #S0053) according to the kit instructions, and then the intracellular GSH and GSSG content was calculated. Cellular iron content assay The intracellular iron colorimetric assay kit (APPLYGEN, #E1042) was used to determine the cellular iron ion concentration. Transmission electron microscopy (TEM) The ultrastructure of the cells was examined with a transmission electron microscope (JEM-1400-FLASH, Jeol, Japan) at 2 µm and 500 nm scales. Western blot (WB) Western blot (WB) Total protein was extracted in RIPA lysis buffer containing a protease inhibitor cocktail, after which the protein concentration was quantified using a BCA protein quantification kit (Vazyme, #E112-01/02). 40 µg of protein was electrophoresed and transferred to a PVDF membrane, incubated overnight at 4°C with the indicated primary antibody, and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Finally, images were captured with chemiluminescent reagents and analyzed using ImageJ 1.8.0 software ( https://imagej.net/ij/download.html)‌ . Due to the large number of groups and antibodies, long membranes were cut and then incubated with different antibodies and the original complete blot is shown in Supplementary Fig. 4. The antibodies used in this study are shown in Supplementary Table 1. RNA isolation and RT-qPCR Experimental cells were subjected to isolate total RNA using TRIzol® Reagent (Thermo Fisher, #15596026). For mRNA RT-qPCR, 1 µg total RNA was added in a reverse-transcriptase reaction to generate the first strand of cDNA by using the HiScript® Ⅲ RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme, #R323-01). For miRNA RT-qPCR, the miRNA 1st Strand cDNA Synthesis kit (by tailing A) (Vazyme, #MR201-01) and the miRNA 1st Strand cDNA Synthesis kit (by stem-loop) (Vazyme, #MR101-02) were used to reversely transcribe the total RNA. The synthesized cDNA served as a template for qPCR using ChamQ Universal SYBR qPCR Master Mix (Vazyme, #Q711-02). β-actin or U6 was selected as the internal standard control, the relative expression level of the target gene was normalized to controls using the comparative CT method (2 −ΔΔCt ). All primers and oligos used for RT-qPCR were synthesized by Tsingke Biotech (Chengdu, China). Dual-luciferase reporter assay Equal numbers of HEK-293T cells were seeded, and growth was observed in each well of 12-well plates. After reaching 70% confluence, the cells were co-transfected for 8 h with an indicated luciferase plasmid (pGL3) together with one of the expression constructs (pCMV3-3Flag or pCMV3-NFE2L3-3Flag), the pRL-TK plasmid served as an internal control for transfection efficiency. In addition, pmirGLO plasmid with wild-type NFE2L3 3′-UTR or mutant NFE2L3 3′-UTR was co-transfected with miR-4465 mimic or mimic control into HEK-293T cells, respectively. After being cultured in a fresh complete medium for 24 h, the cells were harvested, and luciferase activities were measured using a dual-luciferase reporter assay (Vazyme, #DL101-01) according to the protocol. ChIP-qPCR analysis HTK-293T cells were seeded in 10 cm dishes. After reaching 70% confluence, the cells were transfected for 8 h with pCMV3-3Flag and pCMV3-N3-3Flag plasmids, respectively. After being cultured in a fresh complete medium with 10 µM MG132 for 4 h, the experiment was carried out according to the protocol of the kit (CST, #91820). To quantify the binding of NFE2L3 to the target regions of the HMGCR promoter, RT-qPCR was performed using the primers described in Supplementary Table 2. Nfe2l3 −/− mice and subcutaneous tumor xenografts in nude mice Nfe2l3 −/− mice were constructed, identified, and raised by our laboratory, as previously described. 24 Four-week-old male BALB/c nude mice were obtained from the Laboratory Animal Center of North Sichuan Medical College (Nanchong, China). Tumors were established in the backs of mice by subcutaneously injecting 5 × 10 6 HepG2 cells resuspended in 200 µl of PBS (n = 5 mice each). The mice were monitored daily for palpable tumor formation, and tumor volume was calculated using the formula V = 0.523 × (short axis) 2 × (long axis). When the tumor volume was about 100 mm 3 , the group was divided into control (DMSO), Emodin low dose group (25 mg/kg BW), and Emodin high dose group (50 mg/kg BW), and the intraperitoneal injections were performed every other day. After 14 days, the mice were killed by cervical dislocation; the tumor tissue was excised, weighed, and photographed. All mice were housed and treated in accordance with the standard protocols at the Laboratory Animal Center of North Sichuan Medical College (Nanchong, China). Hematoxylin-eosin staining and immunohistochemical analysis Tissue samples from mice were fixed in 4% paraformaldehyde and dehydrated in a gradient of ethanol and xylene, the samples were then embedded in paraffin and cut into 5 µm sections. Next, tissue sections were incubated with anti-NFE2L3 antibody (1:100), anti-HMGCR antibody (1:400, Proteintech, 13533-1-AP), and anti-GPX4 antibody (1:200) at 4°C overnight. Images were acquired at 200× and 400× under the microscope. Statistical analysis All data are expressed as mean ± SD of at least three independent experiments. GraphPad Prism 8 software was used for plotting and statistical analysis. Unpaired t-tests were used to compare two groups, while one-way ANOVA was used to compare two or more groups. Statistical significance was defined as P < 0.05. In the figures, * P < 0.05, ** P < 0.01, and *** P < 0.001. Results Emodin promotes ROS elevation and induces ferroptosis in HCC cells To investigate the potential of Emodin in inducing cellular ROS accumulation and ferroptosis in HCC, we utilized HepG2 and MHCC97H cell lines. Initially, we assessed the in vitro inhibition of HCC cell growth following exposure to various concentrations and durations of Emodin. As depicted in Fig. 1 A and Supplementary Fig. 1A, Emodin suppressed cell viabilities and proliferation, with IC 50 values of 56.9 µM and 51.46 µM, respectively. Considering the IC 50 and previous literature reports, we employed the 24 h treatment with 40 µM Emodin for further studies. We then examined the endogenous ROS levels in cells treated with Emodin and RSL3. DCFH-DA staining showed a significant increase in ROS production subsequent to Emodin treatment (Fig. 1 B-C), and flow cytometry results showed that Emodin significantly increased lipid peroxidation (Fig. 1 D). Additionally, we observed a reduction in the concentration of GSH (Fig. 1 E) and a substantial increase in iron levels after Emodin treatment (Fig. 1 F). Cells undergoing ferroptosis typically exhibit diminished mitochondrial membrane potential and aberrant mitochondrial structure. We detected and observed the mitochondrial membrane potential and mitochondrial microstructure after treatment with Emodin. As shown in Fig. 1 G, exposure to Emodin led to a reduction in mitochondrial membrane potential. Moreover, TEM observations revealed that the mitochondria appeared shrunken with increased membrane density, characteristic of the mitochondrial morphology associated with ferroptosis (Fig. 1 H). Beyond morphological changes, we also evaluated the expression of molecular markers of ferroptosis, including System Xc − , GPX4, and TFRC. The WB and RT-qPCR analyses demonstrated that TFRC expression was upregulated, whereas the expression of System Xc − and GPX4 was downregulated after Emodin treatment (Fig. 2 A-B). These findings suggest that Emodin enhances ROS accumulation and disrupts redox balance, thereby inducing ferroptosis in HCC cells. NFE2L3 partially inhibits ferroptosis induced by Emodin in HCC cells To test whether the redox imbalance caused by Emodin was related to the CNC-bZIP family, we examined the expression of NFE2L1, NFE2L2, and NFE2L3 in HCC cells. As shown in Fig. 2 C-D, the mRNA expression of NFE2L1 and NFE2L2 increased, whereas the expression of NFE2L2 protein decreased, with minimal change in NFE2L1 protein expression. However, the expression of NFE2L3 mRNA has decreased, and the protein expression exhibited a gradient reduction (Fig. 2 C-D). Interestingly, GPX4 expression was increased in NFE2L3 overexpressing cells and decreased in NFE2L3 knockout cells, yet there was no notable alteration in System Xc − expression (Fig. 2 E). These findings suggest that Emodin suppresses the expression of NFE2L3, which may play a role in the regulation of ferroptosis. To confirm this conclusion, HCC cells overexpressing NFE2L3 were treated with Emodin and Fer-1. As illustrated, overexpression of NFE2L3 and Fer-1 groups resulted in reduced levels of ROS (Fig. 3 A) and significantly higher levels of GSH compared to the control group (Fig. 3 B). Additionally, fluorescence and TEM results indicated that the overexpression of NFE2L3 partially restored the membrane potential (Fig. 3 C), and mitochondria exhibit a normal elliptical structure with relatively intact intima and cristae (Fig. 3 D). Furthermore, confocal microscopy results indicated that Emodin treatment efficiently induced lipid peroxidation in HCC cells, and the overexpression of NFE2L3 reduces lipid peroxidation of cell membranes (Fig. 4 A-B). Furthermore, in the experimental group treated with both Fer-1 and Emodin, we observed that Fer-1 could counteract the morphological changes of ferroptosis induced by Emodin and restore the expression of System Xc − and GPX4 (Fig. 4 C). Furthermore, to ascertain whether NFE2L3 mediates Emodin-induced ferroptosis, we assessed the expression of NFE2L3, GPX4, and System Xc − in NFE2L3 overexpressing cells post-treatment with Emodin. The WB results showed that Emodin treatment decreased the expression of NFE2L3, GPX4, and System Xc − , while the overexpression of NFE2L3 reversed the GPX4 expression, with no significant change in System Xc − expression (Fig. 4 D). Together, these results imply that NFE2L3 partially inhibits ferroptosis induced by Emodin in HCC cells. NFE2L3 regulates GPX4 expression by binding to the HMGCR promoter Since GPX4 plays a key role in regulating ferroptosis, we speculated that NFE2L3 might regulate GPX4 transcription by binding to the promoter, thereby inhibiting cellular ferroptosis. However, analysis of the GPX4 promoter did not reveal any ARE elements. Interestingly, our previous transcriptome sequencing data analysis found that the steroid biosynthesis signaling pathway was significantly enriched in stable overexpressing NFE2L3 cells (Fig. 5 A-B). 24 The synthesis precursors of steroid biomolecules involve the mevalonate pathway, which directly affects the expression and synthesis of GPX4 (Fig. 5 C). The key enzyme HMGCR in the mevalonate pathway has been shown to play an important role in ferroptosis and is one of the molecular markers of ferroptosis. We propose the hypothesis that NFE2L3 regulates the expression of HMGCR and mediates the synthesis of GPX4, and thus involved in ferroptosis. To verify this hypothesis, we detected the expression of HMGCR in overexpressing and knockdown NFE2L3 cells, respectively. RT-qPCR and WB results showed that knockdown of NFE2L3 downregulated the expression of HMGCR, while overexpressing NFE2L3 promoted the expression of HMGCR (Fig. 5 D-F). This is consistent with the results reported in existing literature, where overexpressing NFE2L3 upregulates the expression of HMGCR and activates the enzyme activity of HMGCR. 25 Next, we interfered with HMGCR in NFE2L3 overexpressing cells, and the results showed that interfering with HMGCR could down-regulate the expression of GPX4 (Fig. 5 G-H). We further detected the expression of HMGCR in cells treated with Emodin and RSL3. WB results showed that the expression of HMGCR in cells treated with Emodin and RSL3 decreased in a concentration-dependent manner (Fig. 6 A). Meanwhile, in Emodin-treated cells, overexpression of NFE2L3 could restore the expression of HMGCR and GPX4 (Fig. 6 B). This was consistent with the results of the Fer-1 treatment group. These results suggest that NFE2L3 regulates the expression of GPX4 mediated by HMGCR and participates in the process of ferroptosis in HCC cells. Furthermore, we analyzed and found three ARE binding sites within the 2000 bp upstream promoter sequence of HMGCR. In order to verify whether NFE2L3 interacts with the HMGCR promoter through these sites, we constructed mutants for different sites, named Mut-1, Mut-2, and Mut-3, respectively (Fig. 6 C). The results of the luciferase experiment showed that the Mut-1 in the HMGCR promoter significantly reduced the fluorescence intensity, while the Mut-2 and Mut-3 did not remarkably change the fluorescence (Fig. 6 D). Furthermore, a ChIP-qPCR assay was used to detect the interaction between NFE2L3 and HMGCR promoter, and the results showed that all three ARE sites interacted with NFE2L3 (Fig. 6 E). Overall, these results confirmed that NFE2L3 regulated HMGCR transcriptional expression by interacting with its promoter. Emodin reduces NFE2L3 expression through miR-4465 Emodin induces ferroptosis in HCC cells by downregulating the NFE2L3/HMGCR/GPX4 signaling pathway. NFE2L3 binds to the HMGCR promoter and regulates its expression. However, the molecular mechanisms by which Emodin downregulates NFE2L3 are unclear. Initially, we speculated that Emodin regulated the stability of the NFE2L3 protein. Therefore, CHX experiments were used to observe the stability of NFE2L3 after Emodin treatment. The results indicated that the expression of NFE2L3 decreased following exposure to different concentrations of Emodin (10, 40 µM), but the stability did not change significantly (Supplementary Fig. 2A). In addition, we detected the expression of proteasome genes. RT-qPCR results showed that the expression of proteasome genes was largely reduced after Emodin treatment (Supplementary Fig. 2B-C), which was consistent with the conclusion that NFE2L3 regulated the expression of proteasome genes. 24 , 26 It has been reported that a decrease in proteasome activity is one of the molecular characteristics of ferroptosis. 27 What mechanism does Emodin employ to downregulate NFE2L3? Through database analysis, we discovered a binding site in the 3'-UTR of NFE2L3 mRNA for the miR-26 family (Fig. 7 A). Additionally, we assessed the expression of the miR-26 family in HCC cells treated with Emodin using RT-qPCR. The outcomes indicated that following Emodin treatment, miR-4465 expression significantly increased in both HepG2 and MHCC97H cells; miR-26b expression significantly increased only in HepG2 cells, whereas the expression of other miRNAs remained stable (Fig. 7 B-C). Does Emodin inhibit NFE2L3 expression by upregulating miR-4465 ? To verify whether miR-4465 regulates the expression of NFE2L3, we synthesized miR-4465 mimics and inhibitors. RT-qPCR and WB results showed that the mRNA expression of NFE2L3 was downregulated after treatment with miR-4465 mimics and upregulated after treatment with miR-4465 inhibitors (Fig. 7 D-F, 7 K). Similarly, we analyzed the effect of the miR-4465 inhibitor on NFE2L3 expression after Emodin treatment. RT-qPCR results showed that the miR-4465 inhibitor could partially restore the down-regulation of NFE2L3 mRNA expression induced by Emodin (Fig. 7 G-H). WB and RT-qPCR results also showed that NFE2L3 expression after miR-4465 inhibitor treatment was higher than that of the control group (Fig. 7 K). To further verify that miR-4465 regulates NFE2L3 expression by binding to mRNA 3'-UTR, we constructed a luciferase reporter vector of NFE2L3 mRNA 3'-UTR and its mutants. The results of the luciferase reporter experiment showed that the fluorescence intensity of the UTR WT group was significantly decreased after adding miR-4465 mimics. After mutating the binding site, there was no significant change in fluorescence intensity between the mimics and NC group (Fig. 7 I). At the same time, we also detected the luciferase reporter experiment after Emodin treatment, and the experimental results showed a slight increase in fluorescence intensity in the mutants (Fig. 7 J). However, we also found that compared with the control group, the fluorescence intensity of Emodin treatment was significantly reduced, suggesting that Emodin may also inhibit gene transcription at this concentration. In summary, we demonstrated that Emodin downregulated the NFE2L3/HMGCR/GPX4 signal pathway through miR-4465 , which mediated the ferroptosis of HCC cells in vitro. The expression of NFE2L3/HMGCR/GPX4 decreased in Nfe2l3 −/− mice and Emodin reduces the expression of NFE2L3 in subcutaneous transplanted tumor tissue of nude mice To further identify the regulation of Emodin on ferroptosis of HCC cells in vivo, we successfully constructed Nfe2l3 knockout mice using CRISPR/Cas9 technology in the laboratory. WB, IHC, and RT-qPCR experiments were conducted to assess the expression of HMGCR and GPX4 in Nfe2l3 −/− mice. The results indicated that the expression of HMGCR and GPX4 in the liver tissue of Nfe2l3 −/− mice was downregulated (Fig. 8 A-B; Supplementary Fig. 3A-B), confirming that NFE2L3 regulates the expression of HMGCR/GPX4 signaling molecules. Additionally, in the HCC cell xenograft model in nude mice, the body weight of the mice remained stable throughout the treatment period (Supplementary Fig. 3D), and the final tumor volume was smaller than that of the control group (Fig. 8 C; Supplementary Fig. 3C). Compared with the control group, both the tumor volume and weight in the high-dose Emodin group and the low-dose Emodin group were significantly suppressed (Fig. 8 D-E), and the tumor tissues showed extensive necrosis (Supplementary Fig. 3E). Further, IHC results demonstrated that the expressions of NEF2L3, HMGCR, and GPX4 were decreased following Emodin treatment (Fig. 8 F). These results suggest that Emodin suppresses tumor growth and induces ferroptosis of HCC cells in vivo. Discussion Despite significant advances in the prevention and treatment of HCC, existing treatment methods and available drugs still have remarkable limitations. 15 As a natural anthraquinone, Emodin has been extensively studied for its anti-cancer properties across various cancers. In this study, we elucidated that Emodin induces oxidative stress and ferroptosis in HCC cells. Phenotypically, Emodin reduced the level of GSH and mitochondrial membrane potential, induced the presence of Fe 2+ , lipid peroxidation, and ROS accumulation, and caused mitochondrial wrinkling in HCC cells. Emodin downregulated the expression of SLC7A11 and GPX4, and upregulated the expression of TFRC, which are key regulators of ferroptosis. Mechanically, Emodin inactivated the NFE2L3/HMGCR/GPX4 signaling axis. In addition, Emodin promoted the expression of miR-4465 , which inhibits the transcription of NFE2L3, thereby inducing ferroptosis in HCC cells and achieving the therapeutic effect of tumor treatment (Fig. 9 ). In summary, our study provides new insights into the molecular mechanisms associated with Emodin-induced ferroptosis and its role in the anti-tumor treatment, and the combination with chemotherapeutic agents may provide new avenues for enhancing the sensitivity of chemotherapy patients to these drugs. Ferroptosis is mainly regulated by the Fenton reaction of excess intracellular free iron, which produces large amounts of ROS. These ROS catalyze the peroxidation of unsaturated fatty acids, ultimately resulting in cell membrane rupture and cell death. 5 Research has indicated that oxidative damage to lipids plays a role in cancer development and contributes to mortality from acute coronary syndrome (ACS). 28 Tumor cells, characterized by high glycolysis, high oxidative phosphorylation, and high ROS production, 29 , 30 are more susceptible to ferroptosis. 31 Consequently, these insights pave the way for new cancer treatment strategies and the exploration of novel ferroptosis inducers. In our experiments, we showed that Emodin induced ROS accumulation, but some previous studies have shown that Emodin promotes ROS generation in a concentration-dependent manner, with high concentrations promoting ROS generation and low concentrations reducing ROS levels. Therefore, we tested the effect of lower concentrations of Emodin on ROS. Within the concentration range of our experimental drugs, Emodin promoted ROS accumulation, and the expression of NFE2L3, System Xc − , and GPX4 decreased with increasing Emodin concentration (Supplementary Fig. 1B-C). Therefore, further exploration is needed to determine whether low concentrations of Emodin reduce ROS levels in HCC cells. In this study, we elucidated the mechanism of Emodin-mediated ferroptosis and demonstrated that it acts as an inducer of ferroptosis in HCC. Ferroptosis is significantly regulated by System Xc − and GPX4. The antiporter function of System Xc − aids in maintaining redox homeostasis by controlling the production of GSH. Ferroptosis is caused by decreased GPX4 expression and malfunction. GPX4 is an antioxidant enzyme that aids in the repair of oxidized lipids 12 . We observed that the levels of System Xc − and GSH in Emodin-treated cells were reduced, suggesting that the SLC7A11 pathway was inhibited during Emodin-mediated ferroptosis. Concurrently, Emodin downregulated the expression of NFE2L3 and HMGCR, inactivating the mevalonate pathway, which resulted in the reduced GPX4 levels and a weakened antioxidant function. In addition, we noted that Emodin promoted the accumulation of Fe 2+ and the expression of TFRC, although we did not explore the mechanisms by which Emodin facilitates the accumulation of iron ions in our study. Recent research has indicated that Emodin can activate NCOA4-mediated iron autophagy, thereby contributing to the elevation of Fe 2+ levels. 23 Our research indicates that Emodin therapy reduces the expression of NFE2L3, GPX4, and SLC7A11. Since NFE2L3 forms a transcriptional complex with SREBP2 to induce HMGCR expression, 25 and HMGCR has been shown to play an important role in the mevalonate pathway and ferroptosis. 32 , 33 Our further investigation reveals that the HMGCR promoter region has a binding site for NFE2L3 and that interfering with HMGCR results in a decrease in GPX4. Consequently, it is evident that NFE2L3 binds to the HMGCR promoter and activates transcription, thereby promoting the synthesis of GPX4 through the mevalonate pathway and enhancing the antioxidant activity. MicroRNAs are a class of evolutionarily conserved endogenous 20–23 nucleotide small non-coding RNAs, which can cause mRNA inhibition of translation or degradation by complementary binding to the 3' untranslated regions of target mRNA. 34 Emodin suppresses HCC development, proliferation, and autophagy via controlling microRNA expression, as earlier research has indicated. 35 , 36 The miR-26 family is a group of broadly conserved miRNAs, including miR-26a , miR-26b , miR-1297 , and miR-4465 , which share an identical seed region sequence "UCAAGUA". 37 MiR-4465 directly targets the oncogene enhancer of zeste homolog 2 (EZH2) to inhibit tumor growth and spread in non-small cell lung cancer. 38 Additionally, miR-4465 acts as a tumor suppressor in nasopharyngeal carcinoma (NPC), and histone deacetylase 7 (HDAC7) promotes the carcinogenicity of NPC cells by inhibiting the expression of miR-4465 . 39 However, the role of miR-4465 in ferroptosis has been scarcely reported. Our results showed that Emodin upregulates miR-4465 expression, and miR-4465 inhibitor remarkably increased NFE2L3 expression in HCC cells, supporting that NFE2L3 is a direct target of miR-4465 in HCC cells after treatment with Emodin. Thus, this presents a novel method for enhancing anti-tumor therapy. However, it remains essential to assess the differential expression of NFE2L3 and miR-4465 across various patients to implement suitable therapeutic strategies. Conclusion Our findings indicate that Emodin enhances the expression of miR-4465 , thereby suppressing NFE2L3 expression. The interaction between NFE2L3 and the HMGCR promoter is diminished, which subsequently downregulates GPX4 expression via the mevalonate pathway, leading to Emodin-induced ferroptosis in HCC cells. Declarations Funding This work was supported by the Natural Science Foundation of Sichuan (No. 2022NSFSC0731); the Cooperation Project of Nanchong Science and Technology (No. 20SXQT0023, No. 22SXQT0221); the Research Development Fund of North Sichuan Medical College (CBY22-JQ01, CBY24-QNA19); and the Research Development Fund of Affiliated Hospital of North Sichuan Medical College (2023PTZK010). Author contributions All authors contributed to the study conception and design. RYG and DZR: developed the original hypothesis, designed the experiments, and wrote the manuscript. DZR, ZMH and LY: performed in vivo and in vitro experiments. DZR, MBB and QBX: analyzed the data. RYG, QL and YXS: supervised, planned and designed the research. Ethics declarations All animal experiments were performed according to the National Guidelines for Animal Usage in Research (China) and were approved by the Animal Ethics Committee of North Sichuan Medical College (NSMC-2021049). The study is reported in accordance with ARRIVE guidelines. Availability of data and materials Data is provided within the manuscript or supplementary information files. Declaration of competing interest The authors declare no potential competing interests. Acknowledgements We would like to thank Dr. Jian Yang, School of Basic Medical Sciences and Forensic Medicine, North Sichuan Medical College, for providing material supports. Dr. Xinrong He and Jia Tao, Department of Pathology, North Sichuan Medical College, for their assistance in pathological analysis. We also gratefully acknowledge Dr. Qingyong Hu, School of Basic Medical Sciences and Forensic Medicine, North Sichuan Medical College, for their many kind suggestions. References Bray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74 , 229-263, (2024). Zhu, J. et al. 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NRF3 upregulates gene expression in SREBP2-dependent mevalonate pathway with cholesterol uptake and lipogenesis inhibition. iScience 24 , 103180, (2021). Waku, T. et al. NRF3-POMP-20S Proteasome Assembly Axis Promotes Cancer Development via Ubiquitin-Independent Proteolysis of p53 and Retinoblastoma Protein. Mol Cell Biol 40 , e00597-00519, (2020). Kotschi, S. et al. NFE2L1-mediated proteasome function protects from ferroptosis. Mol Metab 57 , 101436, (2022). Karakayali, M. et al. Serum malondialdehyde levels at admission as a predictor of inhospital mortality in patients with acute coronary syndrome. Coron Artery Dis, (2024). Yang, H.-Y. et al. Emodin suppresses oxaliplatin-induced neuropathic pain by inhibiting COX2/NF-κB mediated spinal inflammation. J Biochem Mol Toxicol 37 , e23229, (2023). Xing, M. et al. Emodin disrupts the Notch1/Nrf2/GPX4 antioxidant system and promotes renal cell ferroptosis. J Appl Toxicol 43 , 1702-1718, (2023). Liu, Y. e. et al. The diversified role of mitochondria in ferroptosis in cancer. Cell Death Dis 14 , 519, (2023). Wang, H. et al. BRCC36 Deubiquitinates HMGCR to Regulate the Interplay Between Ferroptosis and Pyroptosis. Adv Sci (Weinh) 11 , e2304263, (2024). Qiu, L., Zhou, R., Zhou, L., Yang, S. & Wu, J. CAPRIN2 upregulation by LINC00941 promotes nasopharyngeal carcinoma ferroptosis resistance and metastatic colonization through HMGCR. Front Oncol 12 , 931749, (2022). Farazi, T. A., Spitzer, J. I., Morozov, P. & Tuschl, T. miRNAs in human cancer. J Pathol 223 , 102-115, (2011). Wu, W. et al. Emodin regulates the autophagy via the miR-371a-5p/PTEN axis to inhibit hepatic malignancy. Biochem Biophys Res Commun 619 , 1-8, (2022). Yin, J., Zhao, X., Chen, X. & Shen, G. Emodin suppresses hepatocellular carcinoma growth by regulating macrophage polarization via microRNA-26a/transforming growth factor beta 1/protein kinase B. Bioengineered 13 , 9548-9563, (2022). Li, C. et al. miR-26 family and its target genes in tumorigenesis and development. Crit Rev Oncol Hematol 157 , 103124, (2021). Sun, J., Tian, X., Lu, S. Q. & Hu, H. B. MicroRNA-4465 suppresses tumor proliferation and metastasis in non-small cell lung cancer by directly targeting the oncogene EZH2. Biomed Pharmacother 96 , 1358-1362, (2017). Li, Q. G. et al. HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis. Cell Death Dis 11 , 322, (2020). Additional Declarations No competing interests reported. Supplementary Files RevisedsupplementaryMaterial.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 10 Apr, 2025 Reviews received at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers invited by journal 31 Mar, 2025 Submission checks completed at journal 31 Mar, 2025 First submitted to journal 19 Mar, 2025 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-6007918","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":436557119,"identity":"32062e46-b5e3-4485-8ea3-dbfb373ac3f5","order_by":0,"name":"Zhiran Ding","email":"","orcid":"","institution":"Institute of Basic Medicine, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhiran","middleName":"","lastName":"Ding","suffix":""},{"id":436557122,"identity":"c00b731a-5186-4eeb-8f1e-fcdfa72c5a9c","order_by":1,"name":"Menghua Zheng","email":"","orcid":"","institution":"Institute of Basic Medicine, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Menghua","middleName":"","lastName":"Zheng","suffix":""},{"id":436557125,"identity":"3cfbd126-0047-4b7f-b42b-649f3f34b430","order_by":2,"name":"Yu Li","email":"","orcid":"","institution":"Institute of Basic Medicine, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Li","suffix":""},{"id":436557129,"identity":"647ab4d4-6b8d-44a6-98e9-261119828a5b","order_by":3,"name":"Bingbing Mou","email":"","orcid":"","institution":"School of Public Health, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Bingbing","middleName":"","lastName":"Mou","suffix":""},{"id":436557131,"identity":"83966342-4c3a-4d48-8265-2f7fdd2af909","order_by":4,"name":"Boxin Qin","email":"","orcid":"","institution":"Institute of Basic Medicine, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Boxin","middleName":"","lastName":"Qin","suffix":""},{"id":436557133,"identity":"55828285-e1e7-41ae-9b91-e0564e29730d","order_by":5,"name":"Lu Qiu","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Qiu","suffix":""},{"id":436557135,"identity":"6b52f5f6-3a5c-4005-9e32-96443abd9a98","order_by":6,"name":"Xuesong Yang","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xuesong","middleName":"","lastName":"Yang","suffix":""},{"id":436557136,"identity":"8c2d58fe-1cd5-4c7b-a3bf-a0ea4f1ab2bb","order_by":7,"name":"Yonggang Ren","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYLCCCgYGOSiTmUgtZxgYjEnXkthAtBbzGekXbxxsO5y+nf904geGCuvEBvazB/BqkbmRU2wB1JK7c0buZgmGM+mJDTx5CXi1SEjkpEl/BGrZcIN3GwNj2+HEBgkeA4JaJEAOMzh/FqjlH1Fa0o+BtCQYHMgFamkgRgvPG2aLA+fSDTfcAPol4Vi6cRtPDgEt7OkPbxwos5YHOmzjhw811rL97Gfwa2EQyDGQYGRrhnASgJgNv3og4D/+QILhTx1BdaNgFIyCUTCCAQDqMUtJGKRLBQAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Basic Medicine, North Sichuan Medical College","correspondingAuthor":true,"prefix":"","firstName":"Yonggang","middleName":"","lastName":"Ren","suffix":""}],"badges":[],"createdAt":"2025-02-11 13:38:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6007918/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6007918/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79736752,"identity":"c9cdffc1-d3ac-46fe-81eb-0756975204c3","added_by":"auto","created_at":"2025-04-02 07:16:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":382093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmodin promotes ROS elevation and lipid peroxidation, reduces mitochondrial function, and triggers ferroptosis in HCC cells. (A)\u003c/strong\u003e Cell viability and IC50 were assessed using the CCK8 assay after treatment with different concentrations of Emodin for 24 h. (\u003cstrong\u003eB-C)\u003c/strong\u003e ROS levels were detected by fluorescence microscopy and flow cytometry using the DCFH-DA probe after treatment with Emodin for 24 h. Scale bars, 200 μm. (\u003cstrong\u003eD)\u003c/strong\u003e Lipid peroxidation levels were assessed via flow cytometry using BODIPY C11 581/591 after treatment with Emodin for 24 h. (\u003cstrong\u003eE-F)\u003c/strong\u003e GSH and Fe\u003csup\u003e2+\u003c/sup\u003e levels were detected by GSH detection and cellular iron content assay. (\u003cstrong\u003eG)\u003c/strong\u003e Mitochondrial membrane potential was determined by Mito-Tracker Red CMXRos staining after treatment with Emodin for 24 h. (\u003cstrong\u003eH)\u003c/strong\u003e Mitochondrial morphology was observed via TEM after Emodin treatment. Emodin: 40 μM, RSL3: 8 μM, FAC: 150 μM, Error bars represent means ± SD of at least three independent experimental replicates with * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/bfe2b615d78ddc6de68b23aa.jpg"},{"id":79736743,"identity":"650688d4-2dab-4fef-a5e9-0381329aaf50","added_by":"auto","created_at":"2025-04-02 07:16:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":269563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmodin inhibits NFE2L3 expression and promotes ferroptosis in HCC cells. (A)\u003c/strong\u003eTFRC, SLC7A11, and GPX4 protein levels were detected by cropped western blot after 24 h treatment with different concentrations of Emodin and RSL3.\u003cstrong\u003e (B) \u003c/strong\u003eThe mRNA levels of \u003cem\u003eTFRC, SLC7A11\u003c/em\u003e, and \u003cem\u003eGPX4\u003c/em\u003e in MHCC97H and HepG2 cells treated with 40 μM Emodin for 24 h were detected by RT-qPCR.\u003cstrong\u003e (C) \u003c/strong\u003eNFE2L3 protein was detected by western blot after 24 h treatment with different concentrations of Emodin and RSL3. (\u003cstrong\u003eD) \u003c/strong\u003eThe mRNA levels of N\u003cem\u003eFE2L1, NFE2L2\u003c/em\u003e, and \u003cem\u003eNFE2L3\u003c/em\u003e in MHCC97H and HepG2 cells treated with 40 μM Emodin for 24 h were detected by RT-qPCR.\u003cstrong\u003e (E)\u003c/strong\u003e Western blot was performed to detect SLC7A11 and GPX4 protein in HCC cells transfected with NFE2L3 lentivirus and plasmid. lvNC: negative control overexpression lentivirus, lvN3: NFE2L3 overexpression lentivirus, shNC: negative control short-hairpin lentivirus; shN3-1: NFE2L3 short-hairpin lentivirus-1; shN3-2: NFE2L3 short-hairpin lentivirus-2, EV: pCMV3-3Flag, N3: pCMV3-NFE2L3-3Flag, ns: no significant difference, Error bars represent means ± SD of at least three independent experimental replicates with * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/94821ab041ab8734659f1106.jpg"},{"id":79737944,"identity":"ad180568-9ed3-4344-8bf7-b76add8a7581","added_by":"auto","created_at":"2025-04-02 07:24:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":288610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNFE2L3 partially inhibited the Emodin-induced ferroptosis in HCC cells.\u003c/strong\u003e (\u003cstrong\u003eA)\u003c/strong\u003e HCC cells were divided into DMSO, Emodin, EV+Emodin, N3+Emodin, and Emodin+Fer-1, and then subjected to ROS assay using the DCFH-DA probe. Scale bars, 200 μm. (\u003cstrong\u003eB)\u003c/strong\u003e GSH levels were measured via GSH detection assay in HepG2. (\u003cstrong\u003eC)\u003c/strong\u003e HCC cells were treated with EV+Emodin, N3+Emodin, and Emodin+Fer-1, the mitochondrial membrane potential was detected by laser confocal microscopy. (\u003cstrong\u003eD) \u003c/strong\u003eMitochondrial morphology of MHCC97H cells after treatments with EV+Emodin, N3+Emodin, and Emodin+Fer-1 was observed via TEM. EV: pCMV3-3Flag, N3: pCMV3-NFE2L3-3Flag. Error bars represent means and ± SD of at least three independent experimental replicates with * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. ns: not significant difference.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/9461cb7ff078a64b3cc448b4.jpg"},{"id":79736742,"identity":"12e5e6ed-a29d-4c9d-abcf-aa21ce9b5244","added_by":"auto","created_at":"2025-04-02 07:16:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":235957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNFE2L3 partially inhibited the Emodin-induced ferroptosis in HCC cells. (A-B) \u003c/strong\u003eMHCC97H and HepG2 cells were divided into DMSO, Emodin, EV+Emodin, N3+Emodin and Emodin+Fer-1, and the levels of lipid peroxidation were detected by laser confocal microscopy.\u003cstrong\u003e (C-D)\u003c/strong\u003e The expression of NFE2L3, SLC7A11, and GPX4 in HCC cells with different treatments was detected via western blot. Fer-1: 20 μM. Error bars represent means ± SD of at least three independent experimental replicates with * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. ns: not significant difference.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/55a1e80c1c255a666476c25c.jpg"},{"id":79736745,"identity":"dbb573ec-90a2-42ed-a11b-de742eda44db","added_by":"auto","created_at":"2025-04-02 07:16:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":310378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNFE2L3 regulates GPX4 expression through HMGCR. (A)\u003c/strong\u003e PCA analysis results of transcriptome sequencing data from lvNC and lvN3 cells. Each point represents a sample. (\u003cstrong\u003eB)\u003c/strong\u003e Association between NFE2L3 expression and steroid biosynthesis pathway, based on GSEA analysis. (\u003cstrong\u003eC)\u003c/strong\u003e Schematic representation of the mevalonate pathway. (\u003cstrong\u003eD-F)\u003c/strong\u003e RT-qPCR and western blot were used to detect HMGCR expression in HCC cells transfected with NFE2L3 lentivirus. (\u003cstrong\u003eG-H)\u003c/strong\u003e Overexpressing NFE2L3 HCC cells was transfected with siHMGCR, and the expression of HMGCR and GPX4 was detected by RT-qPCR and western blot. Error bars represent means ± SD of at least three independent experimental replicates with * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. ns: not significant difference.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/092b4c4782edeb278680cd0c.jpg"},{"id":79736761,"identity":"1845803a-4a42-4a46-a74b-c3b7334991f8","added_by":"auto","created_at":"2025-04-02 07:16:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":238151,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNFE2L3 binds to the ARE site of the HMGCR promoter region. (A-B)\u003c/strong\u003e Western blot was performed to detect HMGCR protein expression. ns: no significant difference. (\u003cstrong\u003eC)\u003c/strong\u003eSchematic diagram of the location of the ARE sites in the HMGCR promoter region. (\u003cstrong\u003eD)\u003c/strong\u003e Detecting the interaction between the HMGCR promoter and NFE2L3 using a dual luciferase activity assay in HEK-293T cells. (\u003cstrong\u003eE)\u003c/strong\u003eChIP­qPCR was used to analyze the combination of NFE2L3 and HMGCR promoter. Fer-1: 20 μM, EV: pCMV3-3Flag, N3: pCMV3-NFE2L3-3Flag. Error bars represent means ± SD of at least three independent experimental replicates with * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05. ns: not significant difference.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/7e9b17244bfa2fa111735d82.jpg"},{"id":79736748,"identity":"4dc3ee4f-70c5-4bab-b581-ef191301955f","added_by":"auto","created_at":"2025-04-02 07:16:10","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":302539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmodin reduces NFE2L3 expression through \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emiR-4465\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (\u003cstrong\u003eA)\u003c/strong\u003e Schematic representation of the binding site of \u003cem\u003eNFE2L3\u003c/em\u003e3’-UTR to \u003cem\u003emiR-26\u003c/em\u003e family members. (\u003cstrong\u003eB-C)\u003c/strong\u003e RT-qPCR was used to detect expression of \u003cem\u003emiR-26\u003c/em\u003e family members after Emodin treatment. (\u003cstrong\u003eD-E) \u003c/strong\u003eRT-qPCR was performed to detect \u003cem\u003emiR-4465\u003c/em\u003e expression level after transfection with 50 nM \u003cem\u003emiR-4465\u003c/em\u003e mimic, 100 nM \u003cem\u003emiR-4465\u003c/em\u003e inhibitor, or their respective controls.\u003cstrong\u003e (F) \u003c/strong\u003eRT-qPCR was performed to detect \u003cem\u003eNFE2L3\u003c/em\u003eexpression level after transfection with 50 nM \u003cem\u003emiR-4465\u003c/em\u003e mimic, 100 nM \u003cem\u003emiR-4465\u003c/em\u003einhibitor, or their respective controls. (\u003cstrong\u003eG) \u003c/strong\u003e\u003cem\u003emiR-4465\u003c/em\u003e expression was detected via RT-qPCR in HCC cells transfected with 100 nM \u003cem\u003emiR-4465\u003c/em\u003einhibitor or control after Emodin treatment. (\u003cstrong\u003eH)\u003c/strong\u003e \u003cem\u003eNFE2L3\u003c/em\u003eexpression was detected via RT-qPCR in HCC cells transfected with 100 nM \u003cem\u003emiR-4465\u003c/em\u003einhibitor or control after Emodin treatment. (\u003cstrong\u003eI) \u003c/strong\u003eLuciferase activity of WT or mutant \u003cem\u003eNFE2L3\u003c/em\u003e 3'-UTR with \u003cem\u003emiR-4465\u003c/em\u003e mimic or control was measured via dual luciferase activity assay in HEK-293T cells. (\u003cstrong\u003eJ) \u003c/strong\u003eLuciferase activity in HEK-293T cells transfected with WT and mutant \u003cem\u003eNFE2L3\u003c/em\u003e 3'-UTR plasmids and then treated with Emodin.\u003cstrong\u003e (K)\u003c/strong\u003e NFE2L3 expression in HCC cells transfectied with 50 nM \u003cem\u003emiR-4465\u003c/em\u003e mimic, 100 nM \u003cem\u003emiR-4465\u003c/em\u003e inhibitor or controls was examined via western blot. Emodin: 40 μM. Error bars represent means ± SD of at least three independent experimental replicates with * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. ns: not significant difference.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/c5e84ee77040c9fac730ab68.jpg"},{"id":79736782,"identity":"a434c15a-2355-4600-8afe-aafcb0d8c647","added_by":"auto","created_at":"2025-04-02 07:16:12","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":504677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of NFE2L3/HMGCR/GPX4 decreased in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNfe2l3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice and Emodin reduces the expression of NFE2L3 in subcutaneous transplanted tumor tissue of nude mice. (A-B)\u003c/strong\u003e The expression of NFE2L3, HMGCR and GPX4 in liver tissue from \u003cem\u003eNfe2l3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice was detected via western blot and IHC. (\u003cstrong\u003eC)\u003c/strong\u003e Images of subcutaneously transplanted HCC tumor tissue in nude mice treated with DMSO, low and high concentrations of Emodin. (\u003cstrong\u003eD-E)\u003c/strong\u003e The growth curve of tumor volume and tumor weight was measured after treatment with DMSO, low and high concentrations of Emodin in the xenograft mouse model. of subcutaneously transplanted HCC tumor tissue from nude mice. (\u003cstrong\u003eF)\u003c/strong\u003e The expression of NFE2L3, HMGCR and GPX4 from subcutaneous transplanted tumor tissue was assessed by IHC staining. Error bars represent means ± SD of at least three independent experimental replicates with * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/dd773032e4cebbf72143091b.jpg"},{"id":79736754,"identity":"7599dae5-b779-41f3-97f8-e85ab2f8e48e","added_by":"auto","created_at":"2025-04-02 07:16:10","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":91087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of Emodin induces ferroptosis in HCC cells through the miR-4465/NFE2L3/HMGCR pathway.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/923585b5bafcf3bf25e696f8.jpg"},{"id":79737952,"identity":"00e5619b-5ace-4327-87db-d69e98a978bd","added_by":"auto","created_at":"2025-04-02 07:24:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3971717,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/3e9a9826-1a82-46c5-849a-8280553fe7eb.pdf"},{"id":79736756,"identity":"39735f48-c151-49c0-8bce-a8c3251debcd","added_by":"auto","created_at":"2025-04-02 07:16:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1728680,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedsupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6007918/v1/81d7169a9e63587eb7061a32.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emodin Induces Oxidative Stress and Ferroptosis in Hepatocellular Carcinoma Cells via Inactivating miR- 4465/NFE2L3/HMGCR/GPX4 Signaling Axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the major malignant tumors threatening human health worldwide, with a high incidence and mortality rate.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Due to the complexity of its biological processes, which involve various factors such as the activation of oncogenes, disruption of redox balance, abnormal cell differentiation, and angiogenesis,\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e the molecular mechanism underlying the occurrence and development of HCC is not yet fully understood.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Ferroptosis is an iron and reactive oxygen species (ROS)-dependent mode of programmed cell death caused by excessive lipid peroxidation and subsequent rupture of the plasma membrane,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e which plays a key role in the onset, progression, and phenotypic transformation of HCC.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e It is widely accepted that ferroptosis is implicated in multiple types of cancers, and the induction of ferroptosis can suppress cancer cell growth and overcome resistance to chemotherapy. Recent studies have focused on inducing ferroptosis to inhibit HCC development, which can support new anticancer strategies for cancer therapy.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn eukaryotes, the CNC-bZIP family functions as a transcription factor that regulates cellular redox homeostasis. Its members include NFE2L1, NFE2L2, NFE2L3, NFE2, Bach 1, and Bach 2.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The CNC-bZIP family members can form a heterodimeric complex with small MAF (sMAF) proteins and then bind to antioxidant response elements (AREs) on the promoters of target genes to promote transcription and regulate a variety of biological processes, including cell death, cell proliferation ,and metabolism.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e NFE2L2 is a key regulator of oxidative stress.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Numerous studies have shown that target genes of NFE2L2 are involved in the regulation of ferroptosis, such as glutathione peroxidase 4 (GPX4) and heme oxygenase-1 (HO-1).\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Recent studies have indicated that NFE2L1 promotes the expression of proteasome genes to maintain proteasome activity and inhibits ferroptosis by regulating the function of proteasomes and GPX4.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e NFE2L3 has been shown to have high homology with the structures of NFE2L2 and NFE2L1 and is highly expressed in various malignant tumors.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e However, it remains unclear whether NFE2L3 is involved in regulating ferroptosis in tumor cells, especially in HCC.\u003c/p\u003e \u003cp\u003eRecently, traditional Chinese medicine and natural products have attracted increased attention for cancer treatment due to their various advantages, including enduring efficacy, multi-targeting capabilities, and reduced toxicity. Emodin, a natural anthraquinone derivative found in many widely used Chinese medicinal herbs, such as aloe and rhubarb,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e has strong anti-inflammatory, anti-cancer, and antimicrobial activities.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Previous studies indicated that Emodin has an anti-tumor effect on multiple cancers, especially in liver,\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e lung,\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e colon,\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e breast,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e ovarian and pancreatic cancer.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Recent studies have shown that Emodin activates NCOA4-mediated ferritinophagy and induces ferroptosis in colorectal cancer.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e However, it is unclear whether Emodin induces ferroptosis in the anti-cancer effect on HCC and its specific mechanism.\u003c/p\u003e \u003cp\u003eThe present study demonstrated that Emodin inhibited HCC cell growth both in vivo and in vitro through inducing ferroptosis, and the regulatory axis miR4465/NFE2L3/HMGCR was involved in this process. Our results revealed a new insight into the mechanism of Emodin-induced ferroptosis and provided a valuable candidate for the development of anticancer drugs.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eAll cells (MHCC97H, HepG2, and HEK-293T) were cultured in complete medium (BasalMedia, #L110KJ) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransient transfection and lentivirus transfection\u003c/h3\u003e\n\u003cp\u003esiRNA, plasmid, and miRNA were transfected into cells using Lipofectamine\u0026reg; 3000 in Opti-MEM (BasalMedia, #L530JV). Cells were infected with NEF2L3 knockdown or overexpression lentivirus (Cyagen) using puromycin resistance (2 \u0026micro;g/ml) and fluorescence as selection markers.\u003c/p\u003e\n\u003ch3\u003eCell counting kit-8 (CCK-8) assay\u003c/h3\u003e\n\u003cp\u003eThe CCK-8 kit (SparkJade, #CT0001-B) assayed cell viability after treatment with six concentrations of Emodin at 0, 5, 10, 20, 40, and 80 \u0026micro;M for 0, 24, 48, and 72 h. The half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) was then calculated from the absorbance.\u003c/p\u003e\n\u003ch3\u003eIntracellular ROS, lipid peroxidation, and mitochondrial membrane potential detection\u003c/h3\u003e\n\u003cp\u003eThe fluorescence intensity detected by the DCFH-DA (Beyotime, #S0033), BODIPY C11 581/591 (Thermo Fisher, #D3861), or Mito-tracker red CMXRos (Beyotime, #C1049B) kits was used to assess cellular ROS, lipid peroxidation, and mitochondrial membrane potential levels.\u003c/p\u003e\n\u003ch3\u003eGlutathione detection\u003c/h3\u003e\n\u003cp\u003eThe absorbance of the samples was measured at 412 nm with an enzyme marker (Beyotime, #S0053) according to the kit instructions, and then the intracellular GSH and GSSG content was calculated.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCellular iron content assay\u003c/h2\u003e \u003cp\u003eThe intracellular iron colorimetric assay kit (APPLYGEN, #E1042) was used to determine the cellular iron ion concentration.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eThe ultrastructure of the cells was examined with a transmission electron microscope (JEM-1400-FLASH, Jeol, Japan) at 2 \u0026micro;m and 500 nm scales.\u003c/p\u003e\n\u003ch3\u003eWestern blot (WB)\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot (WB)\u003c/div\u003e \u003cp\u003eTotal protein was extracted in RIPA lysis buffer containing a protease inhibitor cocktail, after which the protein concentration was quantified using a BCA protein quantification kit (Vazyme, #E112-01/02). 40 \u0026micro;g of protein was electrophoresed and transferred to a PVDF membrane, incubated overnight at 4\u0026deg;C with the indicated primary antibody, and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Finally, images were captured with chemiluminescent reagents and analyzed using ImageJ 1.8.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/download.html)\u0026zwnj;\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/download.html)\u0026zwnj;\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Due to the large number of groups and antibodies, long membranes were cut and then incubated with different antibodies and the original complete blot is shown in Supplementary Fig.\u0026nbsp;4. The antibodies used in this study are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and RT-qPCR\u003c/h2\u003e \u003cp\u003eExperimental cells were subjected to isolate total RNA using TRIzol\u0026reg; Reagent (Thermo Fisher, #15596026). For mRNA RT-qPCR, 1 \u0026micro;g total RNA was added in a reverse-transcriptase reaction to generate the first strand of cDNA by using the HiScript\u0026reg; Ⅲ RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) kit (Vazyme, #R323-01). For miRNA RT-qPCR, the miRNA 1st Strand cDNA Synthesis kit (by tailing A) (Vazyme, #MR201-01) and the miRNA 1st Strand cDNA Synthesis kit (by stem-loop) (Vazyme, #MR101-02) were used to reversely transcribe the total RNA. The synthesized cDNA served as a template for qPCR using ChamQ Universal SYBR qPCR Master Mix (Vazyme, #Q711-02). β-actin or U6 was selected as the internal standard control, the relative expression level of the target gene was normalized to controls using the comparative CT method (2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e). All primers and oligos used for RT-qPCR were synthesized by Tsingke Biotech (Chengdu, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eEqual numbers of HEK-293T cells were seeded, and growth was observed in each well of 12-well plates. After reaching 70% confluence, the cells were co-transfected for 8 h with an indicated luciferase plasmid (pGL3) together with one of the expression constructs (pCMV3-3Flag or pCMV3-NFE2L3-3Flag), the pRL-TK plasmid served as an internal control for transfection efficiency. In addition, pmirGLO plasmid with wild-type \u003cem\u003eNFE2L3\u003c/em\u003e 3\u0026prime;-UTR or mutant \u003cem\u003eNFE2L3\u003c/em\u003e 3\u0026prime;-UTR was co-transfected with \u003cem\u003emiR-4465\u003c/em\u003e mimic or mimic control into HEK-293T cells, respectively. After being cultured in a fresh complete medium for 24 h, the cells were harvested, and luciferase activities were measured using a dual-luciferase reporter assay (Vazyme, #DL101-01) according to the protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eChIP-qPCR analysis\u003c/h2\u003e \u003cp\u003eHTK-293T cells were seeded in 10 cm dishes. After reaching 70% confluence, the cells were transfected for 8 h with pCMV3-3Flag and pCMV3-N3-3Flag plasmids, respectively. After being cultured in a fresh complete medium with 10 \u0026micro;M MG132 for 4 h, the experiment was carried out according to the protocol of the kit (CST, #91820). To quantify the binding of NFE2L3 to the target regions of the HMGCR promoter, RT-qPCR was performed using the primers described in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNfe2l3\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice and subcutaneous tumor xenografts in nude mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eNfe2l3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were constructed, identified, and raised by our laboratory, as previously described.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Four-week-old male BALB/c nude mice were obtained from the Laboratory Animal Center of North Sichuan Medical College (Nanchong, China). Tumors were established in the backs of mice by subcutaneously injecting 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e HepG2 cells resuspended in 200 \u0026micro;l of PBS (n\u0026thinsp;=\u0026thinsp;5 mice each). The mice were monitored daily for palpable tumor formation, and tumor volume was calculated using the formula V\u0026thinsp;=\u0026thinsp;0.523 \u0026times; (short axis)\u003csup\u003e2\u003c/sup\u003e \u0026times; (long axis). When the tumor volume was about 100 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, the group was divided into control (DMSO), Emodin low dose group (25 mg/kg BW), and Emodin high dose group (50 mg/kg BW), and the intraperitoneal injections were performed every other day. After 14 days, the mice were killed by cervical dislocation; the tumor tissue was excised, weighed, and photographed. All mice were housed and treated in accordance with the standard protocols at the Laboratory Animal Center of North Sichuan Medical College (Nanchong, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-eosin staining and immunohistochemical analysis\u003c/h2\u003e \u003cp\u003eTissue samples from mice were fixed in 4% paraformaldehyde and dehydrated in a gradient of ethanol and xylene, the samples were then embedded in paraffin and cut into 5 \u0026micro;m sections. Next, tissue sections were incubated with anti-NFE2L3 antibody (1:100), anti-HMGCR antibody (1:400, Proteintech, 13533-1-AP), and anti-GPX4 antibody (1:200) at 4\u0026deg;C overnight. Images were acquired at 200\u0026times; and 400\u0026times; under the microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of at least three independent experiments. GraphPad Prism 8 software was used for plotting and statistical analysis. Unpaired t-tests were used to compare two groups, while one-way ANOVA was used to compare two or more groups. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. In the figures, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEmodin promotes ROS elevation and induces ferroptosis in HCC cells\u003c/h2\u003e \u003cp\u003eTo investigate the potential of Emodin in inducing cellular ROS accumulation and ferroptosis in HCC, we utilized HepG2 and MHCC97H cell lines. Initially, we assessed the in vitro inhibition of HCC cell growth following exposure to various concentrations and durations of Emodin. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;1A, Emodin suppressed cell viabilities and proliferation, with IC\u003csub\u003e50\u003c/sub\u003e values of 56.9 \u0026micro;M and 51.46 \u0026micro;M, respectively. Considering the IC\u003csub\u003e50\u003c/sub\u003e and previous literature reports, we employed the 24 h treatment with 40 \u0026micro;M Emodin for further studies. We then examined the endogenous ROS levels in cells treated with Emodin and RSL3. DCFH-DA staining showed a significant increase in ROS production subsequent to Emodin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C), and flow cytometry results showed that Emodin significantly increased lipid peroxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Additionally, we observed a reduction in the concentration of GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and a substantial increase in iron levels after Emodin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCells undergoing ferroptosis typically exhibit diminished mitochondrial membrane potential and aberrant mitochondrial structure. We detected and observed the mitochondrial membrane potential and mitochondrial microstructure after treatment with Emodin. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, exposure to Emodin led to a reduction in mitochondrial membrane potential. Moreover, TEM observations revealed that the mitochondria appeared shrunken with increased membrane density, characteristic of the mitochondrial morphology associated with ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Beyond morphological changes, we also evaluated the expression of molecular markers of ferroptosis, including System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e, GPX4, and TFRC. The WB and RT-qPCR analyses demonstrated that TFRC expression was upregulated, whereas the expression of System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e and GPX4 was downregulated after Emodin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). These findings suggest that Emodin enhances ROS accumulation and disrupts redox balance, thereby inducing ferroptosis in HCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNFE2L3 partially inhibits ferroptosis induced by Emodin in HCC cells\u003c/h2\u003e \u003cp\u003eTo test whether the redox imbalance caused by Emodin was related to the CNC-bZIP family, we examined the expression of NFE2L1, NFE2L2, and NFE2L3 in HCC cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D, the mRNA expression of \u003cem\u003eNFE2L1\u003c/em\u003e and \u003cem\u003eNFE2L2\u003c/em\u003e increased, whereas the expression of NFE2L2 protein decreased, with minimal change in NFE2L1 protein expression. However, the expression of \u003cem\u003eNFE2L3\u003c/em\u003e mRNA has decreased, and the protein expression exhibited a gradient reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). Interestingly, GPX4 expression was increased in NFE2L3 overexpressing cells and decreased in NFE2L3 knockout cells, yet there was no notable alteration in System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These findings suggest that Emodin suppresses the expression of NFE2L3, which may play a role in the regulation of ferroptosis. To confirm this conclusion, HCC cells overexpressing NFE2L3 were treated with Emodin and Fer-1. As illustrated, overexpression of NFE2L3 and Fer-1 groups resulted in reduced levels of ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and significantly higher levels of GSH compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Additionally, fluorescence and TEM results indicated that the overexpression of NFE2L3 partially restored the membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and mitochondria exhibit a normal elliptical structure with relatively intact intima and cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, confocal microscopy results indicated that Emodin treatment efficiently induced lipid peroxidation in HCC cells, and the overexpression of NFE2L3 reduces lipid peroxidation of cell membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, in the experimental group treated with both Fer-1 and Emodin, we observed that Fer-1 could counteract the morphological changes of ferroptosis induced by Emodin and restore the expression of System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e and GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, to ascertain whether NFE2L3 mediates Emodin-induced ferroptosis, we assessed the expression of NFE2L3, GPX4, and System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e in NFE2L3 overexpressing cells post-treatment with Emodin. The WB results showed that Emodin treatment decreased the expression of NFE2L3, GPX4, and System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e, while the overexpression of NFE2L3 reversed the GPX4 expression, with no significant change in System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Together, these results imply that NFE2L3 partially inhibits ferroptosis induced by Emodin in HCC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNFE2L3 regulates GPX4 expression by binding to the HMGCR promoter\u003c/h2\u003e \u003cp\u003eSince GPX4 plays a key role in regulating ferroptosis, we speculated that NFE2L3 might regulate GPX4 transcription by binding to the promoter, thereby inhibiting cellular ferroptosis. However, analysis of the GPX4 promoter did not reveal any ARE elements. Interestingly, our previous transcriptome sequencing data analysis found that the steroid biosynthesis signaling pathway was significantly enriched in stable overexpressing NFE2L3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B).\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The synthesis precursors of steroid biomolecules involve the mevalonate pathway, which directly affects the expression and synthesis of GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The key enzyme HMGCR in the mevalonate pathway has been shown to play an important role in ferroptosis and is one of the molecular markers of ferroptosis. We propose the hypothesis that NFE2L3 regulates the expression of HMGCR and mediates the synthesis of GPX4, and thus involved in ferroptosis. To verify this hypothesis, we detected the expression of HMGCR in overexpressing and knockdown NFE2L3 cells, respectively. RT-qPCR and WB results showed that knockdown of NFE2L3 downregulated the expression of HMGCR, while overexpressing NFE2L3 promoted the expression of HMGCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). This is consistent with the results reported in existing literature, where overexpressing NFE2L3 upregulates the expression of HMGCR and activates the enzyme activity of HMGCR.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we interfered with HMGCR in NFE2L3 overexpressing cells, and the results showed that interfering with HMGCR could down-regulate the expression of GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-H). We further detected the expression of HMGCR in cells treated with Emodin and RSL3. WB results showed that the expression of HMGCR in cells treated with Emodin and RSL3 decreased in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Meanwhile, in Emodin-treated cells, overexpression of NFE2L3 could restore the expression of HMGCR and GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This was consistent with the results of the Fer-1 treatment group. These results suggest that NFE2L3 regulates the expression of GPX4 mediated by HMGCR and participates in the process of ferroptosis in HCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we analyzed and found three ARE binding sites within the 2000 bp upstream promoter sequence of HMGCR. In order to verify whether NFE2L3 interacts with the HMGCR promoter through these sites, we constructed mutants for different sites, named Mut-1, Mut-2, and Mut-3, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The results of the luciferase experiment showed that the Mut-1 in the HMGCR promoter significantly reduced the fluorescence intensity, while the Mut-2 and Mut-3 did not remarkably change the fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Furthermore, a ChIP-qPCR assay was used to detect the interaction between NFE2L3 and HMGCR promoter, and the results showed that all three ARE sites interacted with NFE2L3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Overall, these results confirmed that NFE2L3 regulated HMGCR transcriptional expression by interacting with its promoter.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEmodin reduces NFE2L3 expression through\u003c/b\u003e \u003cb\u003emiR-4465\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEmodin induces ferroptosis in HCC cells by downregulating the NFE2L3/HMGCR/GPX4 signaling pathway. NFE2L3 binds to the HMGCR promoter and regulates its expression. However, the molecular mechanisms by which Emodin downregulates NFE2L3 are unclear. Initially, we speculated that Emodin regulated the stability of the NFE2L3 protein. Therefore, CHX experiments were used to observe the stability of NFE2L3 after Emodin treatment. The results indicated that the expression of NFE2L3 decreased following exposure to different concentrations of Emodin (10, 40 \u0026micro;M), but the stability did not change significantly (Supplementary Fig.\u0026nbsp;2A). In addition, we detected the expression of proteasome genes. RT-qPCR results showed that the expression of proteasome genes was largely reduced after Emodin treatment (Supplementary Fig.\u0026nbsp;2B-C), which was consistent with the conclusion that NFE2L3 regulated the expression of proteasome genes.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e It has been reported that a decrease in proteasome activity is one of the molecular characteristics of ferroptosis.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWhat mechanism does Emodin employ to downregulate NFE2L3? Through database analysis, we discovered a binding site in the 3'-UTR of \u003cem\u003eNFE2L3\u003c/em\u003e mRNA for the \u003cem\u003emiR-26\u003c/em\u003e family (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Additionally, we assessed the expression of the \u003cem\u003emiR-26\u003c/em\u003e family in HCC cells treated with Emodin using RT-qPCR. The outcomes indicated that following Emodin treatment, \u003cem\u003emiR-4465\u003c/em\u003e expression significantly increased in both HepG2 and MHCC97H cells; \u003cem\u003emiR-26b\u003c/em\u003e expression significantly increased only in HepG2 cells, whereas the expression of other miRNAs remained stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). Does Emodin inhibit NFE2L3 expression by upregulating \u003cem\u003emiR-4465\u003c/em\u003e? To verify whether \u003cem\u003emiR-4465\u003c/em\u003e regulates the expression of NFE2L3, we synthesized \u003cem\u003emiR-4465\u003c/em\u003e mimics and inhibitors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRT-qPCR and WB results showed that the mRNA expression of \u003cem\u003eNFE2L3\u003c/em\u003e was downregulated after treatment with \u003cem\u003emiR-4465\u003c/em\u003e mimics and upregulated after treatment with \u003cem\u003emiR-4465\u003c/em\u003e inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK). Similarly, we analyzed the effect of the \u003cem\u003emiR-4465\u003c/em\u003e inhibitor on NFE2L3 expression after Emodin treatment. RT-qPCR results showed that the \u003cem\u003emiR-4465\u003c/em\u003e inhibitor could partially restore the down-regulation of \u003cem\u003eNFE2L3\u003c/em\u003e mRNA expression induced by Emodin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-H). WB and RT-qPCR results also showed that NFE2L3 expression after \u003cem\u003emiR-4465\u003c/em\u003e inhibitor treatment was higher than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eTo further verify that \u003cem\u003emiR-4465\u003c/em\u003e regulates NFE2L3 expression by binding to mRNA 3'-UTR, we constructed a luciferase reporter vector of \u003cem\u003eNFE2L3\u003c/em\u003e mRNA 3'-UTR and its mutants. The results of the luciferase reporter experiment showed that the fluorescence intensity of the UTR WT group was significantly decreased after adding \u003cem\u003emiR-4465\u003c/em\u003e mimics. After mutating the binding site, there was no significant change in fluorescence intensity between the mimics and NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). At the same time, we also detected the luciferase reporter experiment after Emodin treatment, and the experimental results showed a slight increase in fluorescence intensity in the mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). However, we also found that compared with the control group, the fluorescence intensity of Emodin treatment was significantly reduced, suggesting that Emodin may also inhibit gene transcription at this concentration. In summary, we demonstrated that Emodin downregulated the NFE2L3/HMGCR/GPX4 signal pathway through \u003cem\u003emiR-4465\u003c/em\u003e, which mediated the ferroptosis of HCC cells in vitro.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression of NFE2L3/HMGCR/GPX4 decreased in\u003c/b\u003e \u003cb\u003eNfe2l3\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice and Emodin reduces the expression of NFE2L3 in subcutaneous transplanted tumor tissue of nude mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further identify the regulation of Emodin on ferroptosis of HCC cells in vivo, we successfully constructed \u003cem\u003eNfe2l3\u003c/em\u003e knockout mice using CRISPR/Cas9 technology in the laboratory. WB, IHC, and RT-qPCR experiments were conducted to assess the expression of HMGCR and GPX4 in \u003cem\u003eNfe2l3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. The results indicated that the expression of HMGCR and GPX4 in the liver tissue of \u003cem\u003eNfe2l3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice was downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B; Supplementary Fig.\u0026nbsp;3A-B), confirming that NFE2L3 regulates the expression of HMGCR/GPX4 signaling molecules.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, in the HCC cell xenograft model in nude mice, the body weight of the mice remained stable throughout the treatment period (Supplementary Fig.\u0026nbsp;3D), and the final tumor volume was smaller than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC; Supplementary Fig.\u0026nbsp;3C). Compared with the control group, both the tumor volume and weight in the high-dose Emodin group and the low-dose Emodin group were significantly suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-E), and the tumor tissues showed extensive necrosis (Supplementary Fig.\u0026nbsp;3E). Further, IHC results demonstrated that the expressions of NEF2L3, HMGCR, and GPX4 were decreased following Emodin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). These results suggest that Emodin suppresses tumor growth and induces ferroptosis of HCC cells in vivo.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite significant advances in the prevention and treatment of HCC, existing treatment methods and available drugs still have remarkable limitations.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e As a natural anthraquinone, Emodin has been extensively studied for its anti-cancer properties across various cancers. In this study, we elucidated that Emodin induces oxidative stress and ferroptosis in HCC cells. Phenotypically, Emodin reduced the level of GSH and mitochondrial membrane potential, induced the presence of Fe\u003csup\u003e2+\u003c/sup\u003e, lipid peroxidation, and ROS accumulation, and caused mitochondrial wrinkling in HCC cells. Emodin downregulated the expression of SLC7A11 and GPX4, and upregulated the expression of TFRC, which are key regulators of ferroptosis. Mechanically, Emodin inactivated the NFE2L3/HMGCR/GPX4 signaling axis. In addition, Emodin promoted the expression of \u003cem\u003emiR-4465\u003c/em\u003e, which inhibits the transcription of NFE2L3, thereby inducing ferroptosis in HCC cells and achieving the therapeutic effect of tumor treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In summary, our study provides new insights into the molecular mechanisms associated with Emodin-induced ferroptosis and its role in the anti-tumor treatment, and the combination with chemotherapeutic agents may provide new avenues for enhancing the sensitivity of chemotherapy patients to these drugs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFerroptosis is mainly regulated by the Fenton reaction of excess intracellular free iron, which produces large amounts of ROS. These ROS catalyze the peroxidation of unsaturated fatty acids, ultimately resulting in cell membrane rupture and cell death.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Research has indicated that oxidative damage to lipids plays a role in cancer development and contributes to mortality from acute coronary syndrome (ACS).\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Tumor cells, characterized by high glycolysis, high oxidative phosphorylation, and high ROS production,\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e are more susceptible to ferroptosis.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Consequently, these insights pave the way for new cancer treatment strategies and the exploration of novel ferroptosis inducers. In our experiments, we showed that Emodin induced ROS accumulation, but some previous studies have shown that Emodin promotes ROS generation in a concentration-dependent manner, with high concentrations promoting ROS generation and low concentrations reducing ROS levels. Therefore, we tested the effect of lower concentrations of Emodin on ROS. Within the concentration range of our experimental drugs, Emodin promoted ROS accumulation, and the expression of NFE2L3, System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e, and GPX4 decreased with increasing Emodin concentration (Supplementary Fig.\u0026nbsp;1B-C). Therefore, further exploration is needed to determine whether low concentrations of Emodin reduce ROS levels in HCC cells.\u003c/p\u003e \u003cp\u003eIn this study, we elucidated the mechanism of Emodin-mediated ferroptosis and demonstrated that it acts as an inducer of ferroptosis in HCC. Ferroptosis is significantly regulated by System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e and GPX4. The antiporter function of System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e aids in maintaining redox homeostasis by controlling the production of GSH. Ferroptosis is caused by decreased GPX4 expression and malfunction. GPX4 is an antioxidant enzyme that aids in the repair of oxidized lipids \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We observed that the levels of System Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e and GSH in Emodin-treated cells were reduced, suggesting that the SLC7A11 pathway was inhibited during Emodin-mediated ferroptosis. Concurrently, Emodin downregulated the expression of NFE2L3 and HMGCR, inactivating the mevalonate pathway, which resulted in the reduced GPX4 levels and a weakened antioxidant function. In addition, we noted that Emodin promoted the accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e and the expression of TFRC, although we did not explore the mechanisms by which Emodin facilitates the accumulation of iron ions in our study. Recent research has indicated that Emodin can activate NCOA4-mediated iron autophagy, thereby contributing to the elevation of Fe\u003csup\u003e2+\u003c/sup\u003e levels.\u003csup\u003e23\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur research indicates that Emodin therapy reduces the expression of NFE2L3, GPX4, and SLC7A11. Since NFE2L3 forms a transcriptional complex with SREBP2 to induce HMGCR expression,\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and HMGCR has been shown to play an important role in the mevalonate pathway and ferroptosis.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Our further investigation reveals that the HMGCR promoter region has a binding site for NFE2L3 and that interfering with HMGCR results in a decrease in GPX4. Consequently, it is evident that NFE2L3 binds to the HMGCR promoter and activates transcription, thereby promoting the synthesis of GPX4 through the mevalonate pathway and enhancing the antioxidant activity.\u003c/p\u003e \u003cp\u003eMicroRNAs are a class of evolutionarily conserved endogenous 20\u0026ndash;23 nucleotide small non-coding RNAs, which can cause mRNA inhibition of translation or degradation by complementary binding to the 3' untranslated regions of target mRNA.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Emodin suppresses HCC development, proliferation, and autophagy via controlling microRNA expression, as earlier research has indicated.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e The \u003cem\u003emiR-26\u003c/em\u003e family is a group of broadly conserved miRNAs, including \u003cem\u003emiR-26a\u003c/em\u003e, \u003cem\u003emiR-26b\u003c/em\u003e, \u003cem\u003emiR-1297\u003c/em\u003e, and \u003cem\u003emiR-4465\u003c/em\u003e, which share an identical seed region sequence \"UCAAGUA\".\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eMiR-4465\u003c/em\u003e directly targets the oncogene enhancer of zeste homolog 2 (EZH2) to inhibit tumor growth and spread in non-small cell lung cancer.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Additionally, \u003cem\u003emiR-4465\u003c/em\u003e acts as a tumor suppressor in nasopharyngeal carcinoma (NPC), and histone deacetylase 7 (HDAC7) promotes the carcinogenicity of NPC cells by inhibiting the expression of \u003cem\u003emiR-4465\u003c/em\u003e. \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e However, the role of \u003cem\u003emiR-4465\u003c/em\u003e in ferroptosis has been scarcely reported. Our results showed that Emodin upregulates \u003cem\u003emiR-4465\u003c/em\u003e expression, and \u003cem\u003emiR-4465\u003c/em\u003e inhibitor remarkably increased NFE2L3 expression in HCC cells, supporting that NFE2L3 is a direct target of \u003cem\u003emiR-4465\u003c/em\u003e in HCC cells after treatment with Emodin. Thus, this presents a novel method for enhancing anti-tumor therapy. However, it remains essential to assess the differential expression of NFE2L3 and \u003cem\u003emiR-4465\u003c/em\u003e across various patients to implement suitable therapeutic strategies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings indicate that Emodin enhances the expression of \u003cem\u003emiR-4465\u003c/em\u003e, thereby suppressing NFE2L3 expression. The interaction between NFE2L3 and the HMGCR promoter is diminished, which subsequently downregulates GPX4 expression via the mevalonate pathway, leading to Emodin-induced ferroptosis in HCC cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Sichuan (No. 2022NSFSC0731); the Cooperation Project of Nanchong Science and Technology (No. 20SXQT0023, No. 22SXQT0221); the Research Development Fund of North Sichuan Medical College (CBY22-JQ01, CBY24-QNA19); and the Research Development Fund of Affiliated Hospital of North Sichuan Medical College (2023PTZK010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. RYG and DZR: developed the original hypothesis, designed the experiments, and wrote the manuscript. DZR, ZMH and LY:\u0026nbsp;performed in vivo and in vitro experiments. DZR, MBB and QBX: analyzed the data. RYG, QL and YXS: supervised, planned and designed the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed according to the National Guidelines for Animal Usage in Research (China) and were approved by the Animal Ethics Committee of North Sichuan Medical College (NSMC-2021049).\u0026nbsp;The study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Jian Yang, School of Basic Medical Sciences and Forensic Medicine, North Sichuan Medical College, for providing material supports. Dr. Xinrong He and Jia Tao, Department of Pathology, North Sichuan Medical College, for their assistance in pathological analysis. We also gratefully acknowledge Dr. Qingyong Hu, School of Basic Medical Sciences and Forensic Medicine, North Sichuan Medical College, for their many kind suggestions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin \u003cstrong\u003e74\u003c/strong\u003e, 229-263, (2024).\u003c/li\u003e\n\u003cli\u003eZhu, J. et al. MYBL1 induces transcriptional activation of ANGPT2 to promote tumor angiogenesis and confer sorafenib resistance in human hepatocellular carcinoma. Cell Death Dis \u003cstrong\u003e13\u003c/strong\u003e, 727, (2022).\u003c/li\u003e\n\u003cli\u003eFebbraio, M. A. et al. Preclinical Models for Studying NASH-Driven HCC: How Useful Are They? 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HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis. Cell Death Dis \u003cstrong\u003e11\u003c/strong\u003e, 322, (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Emodin, Ferroptosis, Oxidative stress, NFE2L3, HMGCR, Hepatocellular carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-6007918/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6007918/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFerroptosis is a form of programmed cell death characterized by iron-dependent lipid peroxidation. Targeting ferroptosis is considered a novel strategy for cancer treatment. The benefits of using natural products to treat tumors have drawn more attention. Emodin, a natural anthraquinone derivative, has been shown to exert anti-tumor effects by promoting the generation and accumulation of reactive oxygen species (ROS), inducing apoptosis, autophagy, and cell cycle arrest. The molecular processes behind Emodin-mediated ferroptosis in hepatocellular carcinoma (HCC) cells were examined in our work. Emodin caused ferroptosis and suppressed growth in HCC cells in vitro. Emodin could increase ROS and lipid peroxidation, meanwhile decreasing glutathione (GSH), mitochondrial membrane potential, glutathione peroxidase 4 (GPX4), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression, these effects could be reversed by Ferostatin-1 (Fer-1, an inhibitor of ferroptosis) and NFE2-like bZIP transcription factor 3 (NFE2L3). Mechanistically, Emodin enhances the expression of \u003cem\u003emiR-4465\u003c/em\u003e, thereby suppressing NFE2L3 expression. The interaction between NFE2L3 and the HMGCR promoter is diminished, which subsequently downregulates GPX4 expression via the mevalonate pathway, leading to ferroptosis. Overexpression of NFE2L3 could alleviate Emodin-induced ferroptosis in HCC cells. Moreover, NFE2L3 knockout markedly reduced the expression of HMGCR and GPX4 in the \u003cem\u003eNfe2l3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003emouse model. Emodin also caused ferroptosis and inhibited tumor development in a xenograft mice model. In conclusion, these results suggested that Emodin induces ferroptosis by inactivating the NFE2L3/HMGCR/GPX4 pathway in HCC cells. Emodin may be a promising candidate for the development of anticancer drugs and offers new strategies for cancer therapy.\u003c/p\u003e","manuscriptTitle":"Emodin Induces Oxidative Stress and Ferroptosis in Hepatocellular Carcinoma Cells via Inactivating miR- 4465/NFE2L3/HMGCR/GPX4 Signaling Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 07:15:59","doi":"10.21203/rs.3.rs-6007918/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-04-10T23:19:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-31T22:47:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331166699290818574925450309638919318702","date":"2025-03-31T20:48:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275614071189200398351606584082691773221","date":"2025-03-31T20:24:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T20:23:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-31T12:27:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-19T12:49:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"130e7e53-19d4-4a6c-a409-ed4504f83e36","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":46483134,"name":"Biological sciences/Molecular biology"},{"id":46483135,"name":"Health sciences/Medical research"},{"id":46483136,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2025-04-11T10:38:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-02 07:15:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6007918","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6007918","identity":"rs-6007918","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}