Abstract
Background
Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with limited clinical treatment options. Although the natural compound curcumin has demonstrated potential anti-tumor effects, its specific regulatory mechanisms in HCC remain incompletely understood.
Purpose
This study aimed to investigate the molecular mechanism by which curcumin suppresses the malignant biological characteristics of HCC through microRNA (miR)-134 targeting nitric oxide synthase 3 (Nos3), thereby regulating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway.
Materials and Methods
Cells were divided into a blank control group (Group A), an HCC group (Group B), and a curcumin intervention group (Group C). Group A consisted of normal human hepatocyte L-02 cells without treatment; Group B comprised human HCC BEL-7402 cells without treatment; Group C involved BEL-7402 cells treated with 50 µmol/L curcumin. Cells were cultured for 48 h. Cell proliferation was assessed using the cell counting kit-8 (CCK-8) assay, protein expression by Western blot, messenger ribonucleic acid (mRNA) levels by quantitative polymerase chain reaction (qPCR), and migration and invasion abilities by the Transwell assay.
Results
Compared with Group B, Group C exhibited reduced cell proliferation, decreased migration and invasion, elevated expression of apoptotic proteins (Fas, BCL2-associated X protein [Bax], Fas ligand [FasL]), and lower B-cell lymphoma 2 (Bcl-2) expression. Group C showed higher relative expression of miR-134 and Nos3 protein, while AKT and PI3K mRNA and protein levels were lower than in Group B.
Conclusion
Curcumin may target Nos3 via miR-134, reducing HCC cell viability, suppressing proliferation, migration, and invasion, and inducing apoptosis. Its mechanism may involve modulation of the PI3K/AKT signaling pathway.
Introduction
Hepatocellular carcinoma (HCC) ranks as the sixth most common cancer and the third leading cause of cancer-related mortality, often arising from chronic liver diseases such as cirrhosis. Multiple risk factors, including hepatitis B and C viral load, diabetes, albumin expression, age at sustained virological response, alcohol consumption, and smoking, influence HCC development and progression (Liu et al., 2023; Wang, Tian, et al., 2023). Currently, no highly effective treatment exists for HCC patients, with radiotherapy and surgical resection being the most employed interventions (Huang et al., 2023). In recent years, the advancement of targeted therapies has expanded treatment options for various malignancies. Therefore, in-depth research into the pathophysiology and etiology of HCC is crucial for identifying novel therapeutic targets to alleviate clinical symptoms, delay disease progression, and improve patients’ quality of life. Natural plants and resources have gained increasing attention due to their abundance and potential anti-tumor properties. Curcumin, a natural antioxidant derived from the traditional Chinese herb Curcuma longa, regulates the cell cycle, proliferation, and oxygen free radical scavenging while inhibiting tumor growth. It also exhibits potent anti-viral and hepatoprotective effects (Li et al., 2024). Previous studies have demonstrated that curcumin suppresses HCC progression through multiple pathways, including JAK2/STAT3 and MAPK signaling (Liu et al., 2024). However, the precise mechanism involving microRNA (miR)-134 and nitric oxide synthase 3 (Nos3) in HCC regulation remains unclear.
MicroRNAs (miRNAs) are a class of highly conserved short non-coding RNAs involved in the regulation of cellular biological activities. Studies confirmed that miR-134 is aberrantly expressed in HCC patients and may play a role in HCC development and progression (Huang et al., 2022). For instance, miR-134 can suppress HCC cell proliferation by targeting ITGB1 (Bi et al., 2023), highlighting its potential as a regulatory factor. Additionally, the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway is a critical intracellular signaling pathway with broad functional roles, governing processes such as cell division, proliferation, migration, and apoptosis (Yang et al., 2024). Overactivation of this pathway is closely associated with the growth, survival, and invasiveness of cancer cells in endometrial cancer, breast cancer, and gastric cancer (Lu et al., 2023). In HCC, it has also been implicated in tumor invasion and metastasis, making it a key regulatory node for anti-tumor therapy.
This study investigated the mechanism by which curcumin suppresses the malignant biological characteristics of HCC by targeting Nos3 via miR-134 and modulating the PI3K/AKT pathway. The findings aimed to provide potential therapeutic targets for the clinical treatment of HCC.
Materials and Methods
Materials and Reagents
The human HCC cell line BEL-7402 was derived from a surgically resected moderately differentiated HCC specimen of a 52-year-old male patient without prior radiotherapy or chemotherapy. The normal human hepatocyte line L-02 originated from histologically normal liver tissue of a 35-year-old healthy male donor without a history of liver disease. Both cell lines were obtained from American Type Culture Collection (ATCC) (USA) and authenticated by STR profiling (purity >98%).
Curcumin (≥98% purity, Nanjing Duosifu Biotechnology) served as an experimental intervention. Preliminary studies in a diethylnitrosamine (DEN)-induced rat HCC model demonstrated that intraperitoneal injection of 50 mg/kg curcumin significantly reduced tumor nodules (p < .05) and downregulated PI3K/AKT pathway proteins, justifying the selected 50 µmol/L in vitro concentration.
Other experimental reagents included immunohistochemical goat anti-rabbit secondary antibody (Shanghai Beyotime Biotechnology Co., Ltd.), quantitative polymerase chain reaction (qPCR) test kit (Shanghai Beyotime), CCK-8 test kit (Shanghai Beyotime), Annexin V-FITC/PI apoptosis test kit (Shanghai Beyotime), Transwell cell (Corning Company of the USA), and artificial reconstruction basement membrane gel (Matrix, BD Company of the USA). AKT, PI3K, Nos3, Fas, BCL2-associated X protein (Bax), Fas ligand (FasL), and B-cell lymphoma 2 (Bcl-2) primary antibodies were all purchased from Abcam Biotechnology Ltd., in the UK, with a dilution ratio of 1:1,000.
Methods
Cell Culture and Treatment
Both normal human hepatocytes L-02 and HCC cells BEL-7402 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. When reaching 80% confluence, cells were digested with 0.25% trypsin and maintained in a 37 °C, 5% CO2 incubator. Three experimental groups were established with six replicates each: Group A (Control): Untreated L-02 normal hepatocytes; Group B (HCC): Untreated BEL-7402 HCC cells; Group C (Curcumin): BEL-7402 HCC cells treated with 50 µmol/L curcumin.
Protein Expression Detection Using Western Blot Technique
Cells were adjusted to a density of 1×10⁶ cells/well in 6-well plates and cultured at 37 °C with 5% CO2. BEL-7402 HCC cells were treated with 50 µmol/L curcumin for 48 h. Cells were homogenized in RIPA lysis buffer containing 1% PMSF on ice for 30 min, followed by centrifugation at 12,000 × g for 15 min at 4 °C (Eppendorf 5418R centrifuge). Supernatants were collected for analysis. Protein concentration was determined by the bicinchoninic acid (BCA) assay. Proteins were separated on 10% separating/5% stacking gels (80V for 30 min, then 120V for 60 min) and transferred to PVDF membranes (300 mA for 90 min; Bio-Rad Mini Trans-Blot system). Membranes were blocked with milk and probed with secondary antibodies. Protein bands were visualized using a chemiluminescence system (GE Healthcare ImageQuant LAS 4000). Experimental procedures followed Miyazaki et al. (2023).
Quantitative Polymerase Chain Reaction Determination of the Appropriate Amount of Messenger Ribonucleic Acid Expression
Cells were seeded in 6-well plates at a density of 1 × 106 cells/well and cultured at 37 °C with 5% CO2. BEL-7402 HCC cells were treated with 50 µmol/L curcumin for 48 h. Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA with PrimeScript RT Reagent Kit (Takara). qPCR was performed using SYBR Green PCR Master Mix (Takara) on a 7500 real-time PCR system (Applied Biosystems) under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. GAPDH served as the internal reference gene, and relative messenger RNA (mRNA) expression levels were calculated using the 2⁻∇∇Ct method.
Cell Counting Kit-8 Identifies the Capacity for Cell Division and Apoptosis
BEL-7402 HCC cells were treated with 50 µmol/L curcumin and incubated at 37 °C with 5% CO2 for 48 h. Cell proliferation was then measured using a microplate reader. The experimental procedure followed the method described by Shao et al. (2023).
Using the Transwell Technique to Detect the Capacity of Cells to Migrate and Invade
BEL-7402 HCC cells were treated with 50 µmol/L curcumin for 48 h. For the migration assay, cells (5 × 105/well) were seeded in the upper chamber of Transwell inserts (without Matrigel), with 600 µL medium containing 10% FBS in the lower chamber. For the invasion assay, upper chambers were pre-coated with 50 µL Matrigel (1:8 dilution) and incubated at 37 °C for 4 h to solidify before cell seeding. After 48 h, cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. Non-migrated/invaded cells were removed with cotton swabs. Migrated/invaded cells were counted in five random fields under an Olympus CX31 microscope and averaged.
Statistical Methods
Data were analyzed using SPSS 22.0 and presented as mean ± standard deviation (x̄ ± s). Independent variables were “group (A–C)” and “treatment (with/without curcumin),” while dependent variables included cell proliferation activity, migrated/invaded cell counts, and protein/mRNA expression levels. Multiple group comparisons were performed using one-way analysis of variance (ANOVA), with post hoc LSD-t tests for pairwise comparisons. Statistical significance was set at p < .05. a: p < .05 versus control group; b: p < .05 versus HCC group.
Results
Effects of Curcumin on Proliferation, Migration and Invasion of Hepatocellular Carcinoma Cells
In Table 1, compared with the blank control group (Group A), the HCC group (Group B) exhibited significantly increased cell proliferation activity, migrated cell count, and invaded cell count (a p < .05). The curcumin intervention group (Group C) demonstrated significantly decreased values in these three parameters compared with Group B (b p < .05). However, Group C still showed higher levels than Group A (c p < .05), indicating that curcumin partially reversed the malignant phenotype of HCC cells without complete restoration to normal hepatocyte levels.
Curcumin’s Effects on Hepatocellular Carcinoma (HCC) Cell Metastasis and Proliferation.
Curcumin’s Impact on the Expression of Proteins Associated with Apoptosis in Hepatocellular Carcinoma Cells
In Table 2, compared with Group A, Group B showed no significant differences in the expression of pro-apoptotic proteins (Fas, Bax, FasL; p > .05), but exhibited a slight decrease in anti-apoptotic protein Bcl-2 expression (p > .05). Compared with Group B, Group C demonstrated significantly increased expression of Fas, Bax, and FasL (b p < .05), along with significantly reduced Bcl-2 expression (b p < .05). Notably, Group C showed significantly higher Fas, Bax, and FasL expressions than Group A (c p < .05), while Bcl-2 expression was significantly lower than in Group A (c p < .05). These findings suggest that curcumin significantly enhances the apoptotic tendency of HCC cells.
Effects of Curcumin on the Expression of Apoptotic Proteins in Hepatocellular Carcinoma (HCC) Cells.
Effect of Curcumin on miR-134 and Nos3 Expression in Hepatocellular Carcinoma Cells
In Table 3, compared with Group A, Group B exhibited significantly decreased expression of Nos3 protein and miR-134 (a p < .05). Compared with Group B, Group C showed significantly increased expression of both markers (b p < .05). Notably, Nos3 protein expression in Group C was significantly higher than in Group A (c p < .05), while miR-134 expression showed no significant difference from Group A (p > .05). These results indicate that curcumin upregulates the expression of both Nos3 and miR-134, with a more pronounced effect on Nos3 upregulation.
Effect of Curcumin on the Expression of MicroRNA (miR)-134 and Nitric Oxide Synthase 3 (Nos3) in Hepatocellular Carcinoma (HCC) Cells.
Curcumin’s Impact on the Relative Messenger Ribonucleic Acid Expression of PI3K and AKT in Hepatocellular Carcinoma Cells
In Table 4, compared with Group A, Group B demonstrated significantly increased relative expression levels of AKT and PI3K mRNA (a p < .05). Compared with Group B, Group C showed significantly decreased expression of both markers (b p < .05). However, the AKT and PI3K mRNA expression levels in Group C remained significantly higher than those in Group A (c p < .05), indicating that curcumin downregulates the transcriptional activity of the PI3K/AKT pathway without complete restoration to normal levels.
Effect of Curcumin on Protein Kinase B (AKT) and Phosphatidylinositol 3-kinase (PI3K) Messenger RNA (mRNA) in Hepatocellular Carcinoma (HCC) Cells.
Effect of Curcumin on AKT and PI3K Levels in Hepatocellular Carcinoma Cells
In Table 5, compared with Group A, Group B showed significantly increased protein expression levels of AKT and PI3K (a p < .05). Compared with Group B, Group C exhibited significantly decreased expression of both proteins (b p < .05). However, the AKT and PI3K protein expression in Group C remained significantly higher than in Group A (c p < .05), demonstrating that curcumin inhibits PI3K/AKT pathway protein expression, consistent with the observed mRNA level changes.
Effect of Curcumin on Protein Kinase B (AKT) and Phosphatidylinositol 3-kinase (PI3K) Levels in Hepatocellular Carcinoma (HCC) Cells.
Discussion
HCC, accounting for 70%–90% of primary liver cancers and ranking as the sixth most common malignancy globally, demonstrates poor prognosis due to its highly invasive, metastatic, and recurrent nature (Yu et al., 2024). Our experimental data comparing Group A (normal hepatocytes L-02), Group B (HCC cells BEL-7402), and Group C (BEL-7402 + 50 µmol/L curcumin) revealed significantly higher proliferation, migration, and invasion in Group B versus Group A (p < .05), while Group C showed marked reduction in these parameters compared to Group B (p < .05), directly confirming curcumin’s inhibitory effects on HCC malignant phenotypes. Current HCC treatment primarily relies on radiotherapy and surgical resection, yet most patients present at inoperable stages with limited radiotherapy sensitivity (Gupta et al., 2024). The mechanisms identified in this study suggest curcumin’s potential as an adjuvant therapy—this natural compound exhibits multi-target characteristics (Li et al., 2023) capable of coordinately regulating multiple pathways to suppress tumor progression, aligning with herbal medicine’s advantages in cancer treatment by “reducing chemoresistance while enhancing efficacy” (Su et al., 2024).
As the primary active component of turmeric, curcumin has demonstrated well-established safety and polypharmacological properties (Srinivas et al., 2024), including immunomodulatory, anti-viral, anti-inflammatory, and antioxidant effects. Our study revealed significantly increased expression of pro-apoptotic proteins (Fas, Bax, FasL) and decreased anti-apoptotic Bcl-2 in Group C versus Group B (p < .05). These results align with Wang, Wu, et al. (2023)’s findings regarding curcumin-induced apoptosis through XRCC4 downregulation in HCC cells. Importantly, our work further elucidates curcumin’s dual apoptotic regulation: (a) activation of the extrinsic pathway via Fas/FasL signaling; (b) modulation of the intrinsic pathway through the Bax/Bcl-2 ratio. This dual-pathway activation demonstrates curcumin’s high efficacy in apoptosis induction. While numerous in vitro and in vivo studies have confirmed curcumin’s anti-proliferative and pro-apoptotic effects across various cancers (Onifade et al., 2023), our HCC model provides tumor-specific molecular evidence, highlighting both pathway conservation and tumor-type specificity in its mechanism of action.
miRNAs, endogenous non-coding RNAs of 19–25 nt in length, regulate gene expression by binding to the 3′-untranslated regions (3′-UTR) of target mRNAs, playing crucial roles in tumorigenesis (Hsueh et al., 2024; Huang et al., 2021). Our study demonstrated significantly lower miR-134 expression in Group B compared to Group A (p < .05), while Group C showed significantly higher miR-134 levels than Group B (p < .05), suggesting miR-134 may be a key mediator of curcumin’s effects. This aligns with (Hu et al., 2021), who confirmed miR-134’s low expression in HCC and its anti-proliferative effects when overexpressed. Additionally, Yin et al. (2022) reported that miR-134 regulates target genes through interactions with sponge RNAs (e.g., circRNA ZNF609), providing a mechanistic context for the observed miR-134/Nos3 interaction in our study. Notably, while Yu et al. (2024) and Wongworawat et al. (2024) reported that “FENDRR overexpression + miR-134 downregulation” inhibits HCC proliferation and promotes apoptosis—seemingly contradictory to our findings—this reflects the context-dependent nature of miR-134 regulation. The biological effects of miR-134 appear to depend on its downstream targets: When targeting tumor suppressors, miR-134 downregulation may be anti-tumorigenic (Yu et al.’s, 2024 model); when targeting oncogenes or pathway regulators (e.g., Nos3 in our study), miR-134 upregulation exerts anti-tumor effects.
NOS3, a member of the NOS family primarily regulating vascular permeability and vasodilation, demonstrates increased activity in tumor tissues that promotes vascular density and blood perfusion, thereby facilitating tumor invasion and metastasis (Zeng et al., 2024). Our study revealed significantly decreased Nos3 expression in Group B compared to Group A (p < .05), while Group C exhibited significantly higher Nos3 levels than Group B (p < .05). These results align with Nos3’s potential tumor-suppressive function. We hypothesize that curcumin-induced miR-134 upregulation may relieve suppression on Nos3 (possibly by targeting its negative regulators), thereby enhancing its vascular “normalization” effects: Reducing pathological angiogenesis and consequently decreasing HCC cell migration and invasion (consistent with reduced invasion counts in Group C). This vascular regulation mechanism corroborates findings by Omeroglu Ulu et al. (2023), who demonstrated that “curcumin combined with other compounds inhibits HCC through angiogenesis modulation,” confirming that vascular microenvironment regulation represents a crucial pathway for curcumin’s anti-metastatic effects.
The PI3K/AKT signaling pathway serves as a central regulatory axis for cell proliferation, migration, and apoptosis (Du et al., 2024), with its overactivation being closely associated with malignant progression in various cancers, including endometrial, breast, and gastric cancers (Sun et al., 2024). Our study demonstrated significantly elevated expression of both AKT and PI3K (mRNA and protein levels) in Group B compared to Group A (p < .05), while Group C showed a significant reduction in these markers versus Group B (p < .05). These results confirm curcumin’s inhibitory effect on PI3K/AKT pathway activation in HCC cells. As AKT represents the direct downstream target of PI3K, its phosphorylation level reflects PI3K activity (Aliyari et al., 2025). The concurrent downregulation of AKT and PI3K observed in our study suggests curcumin may act on upstream pathway components (e.g., inhibiting PI3K activation via Nos3). This finding contrasts with Zhu et al. (2024)’s report that “AURKB promotes HCC progression through PI3K/AKT/mTOR pathway,” instead revealing a negative regulatory mechanism whereby curcumin suppresses PI3K/AKT via the miR-134/Nos3 axis, providing an inhibitory intervention strategy for targeting this pathway.
The present study demonstrates that curcumin upregulates miR-134 to promote Nos3 expression, thereby suppressing PI3K/AKT pathway overactivation, ultimately reducing HCC cell viability, inhibiting proliferation/migration/invasion, and inducing apoptosis. This mechanism integrates miRNA regulation, vascular microenvironment modulation, and classical signaling pathways, providing a multidimensional perspective for understanding curcumin’s anti-HCC effects. These findings establish an experimental foundation for developing combination therapies based on miR-134 mimics or Nos3 activators.
Conclusion
This study confirms that curcumin can upregulate miR-134 expression to specifically enhance Nos3 protein production, thereby inhibiting activation of the PI3K/AKT signaling pathway. These effects ultimately reduce the viability of HCC cells (BEL-7402), suppress their proliferation, migration and invasive capabilities, while inducing apoptosis. The elucidated mechanism provides experimental evidence supporting curcumin’s potential as a therapeutic agent for HCC and establishes a foundation for developing treatment strategies targeting miR-134, Nos3, or the PI3K/AKT pathway.
Footnotes
Abbreviations
AKT: Protein kinase B; ANOVA: Analysis of variance; ATCC: American Type Culture Collection; Bax: BCL2-associated X protein; BCA: Bicinchoninic acid; Bcl-2: B-cell lymphoma 2; CCK-8: Cell counting kit-8; Fas: Cell surface death receptor; FasL: Fas ligand; HCC: Hepatocellular carcinoma; miR: MicroRNA; mRNA: Messenger ribonucleic acid; NO: Nitric oxide; Nos3: Nitric oxide synthase 3; NOS: Nitric oxide synthase; PI3K: Phosphatidylinositol 3-kinase; qPCR: Quantitative polymerase chain reaction.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical Approval and Informed Consent
Manuscript is approved by all authors for publication.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Hainan Provincial Natural Science Foundation (No. 824MS170).
