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. 2026 Jun 24;40(9):5566–5580. doi: 10.1002/ptr.70375

Curcumol Inhibits the Invasion and Migration of Breast Cancer via miR‐206/NCL/ERα36

Xiaojuan Li 1, Muzhao Tian 2, Luwei Zhou 2, Hongfang Ding 1, Riqiu Zhang 1, Xu Chen 2, Juan Wang 1,✉
PMCID: PMC13548921  PMID: 42338311

ABSTRACT

Breast cancer (BC) is a highly heterogeneous and aggressive disease with high mortality. Curcumol, a major bioactive component from Curcumae kwangsiensis, has shown antitumor activity by targeting nucleolin (NCL) and its interaction with ERα36. NCL is an RNA‐binding protein that interacts with miR‐206, a tumor‐suppressive miRNA that inhibits EMT and invasion by regulating target gene expression. However, whether curcumol's effect on BC invasion and migration, as well as the NCL/ERα36 interaction, is regulated by miR‐206 remains unclear. Cell migration and invasion were assessed using wound healing and transwell assays. Dual‐luciferase reporter and RIP assays were performed to confirm NCL as a direct target of miR‐206. Western blotting and qRT‐PCR were used to evaluate protein and miRNA expression. Animal experiments were conducted to explore the inhibitory effect of curcumol on invasion and migration in vivo. Curcumol significantly inhibited the invasion and migration of breast cancer cells, while potentiating the inhibitory effects of NCL knockdown on these processes. NCL was identified as a downstream target of miR‐206, and curcumol inhibits the invasion and migration of breast cancer cells via downregulating NCL expression and is associated with increased miR‐206 levels. Moreover, curcumol inhibits the invasion and migration of breast cancer cells by downregulating the NCL/ERα36 interaction. The anti‐breast cancer effect of curcumol is related to its regulation of the expression of miR‐206 and NCL and inhibits the invasion and migration of breast cancer via the miR‐206/NCL/ERα36 axis.

Keywords: breast cancer, curcumol, invasion and migration, miR‐206/NCL/ERα36

Mechanism of curcumol inhibiting the invasion and migration of breast cancer via miR‐206/NCL/ERα36.

graphic file with name PTR-40-5566-g008.webp

Abbreviations

ANOVA

analysis of variance

BC

breast cancer

Cur

curcumol

DMEM

Dulbecco's modified eagle's medium

ERα36

estrogen receptor‐alpha 36

FBS

fetal bovine serum

GAPDH

glyceraldehyde‐3‐phosphate dehydrogenase

H&E

hematoxylin and eosin

HER2

human epidermal growth factor receptor 2

lv

lentiviral vector (context: ERα36 lv)

MEGM

mammary epithelium growth medium

miRNA

microRNA

MMP9

matrix metalloproteinase 9

MTDH

metadherin

MUT

mutant

NC

negative control

NCCN

National Comprehensive Cancer Network

NCL

nucleolin

PBS

phosphate‐buffered saline

qRT‐PCR

quantitative reverse transcriptase polymerase chain reactions

RIP

RNA immunoprecipitation

RIPA

radioimmunoprecipitation assay buffer

RPMI

Roswell Park Memorial Institute 1640 medium

SD

standard deviation

shRNA (sh)

short hairpin RNA

siRNA (si)

small interfering RNA

WB

western blotting

WT

wild type

β‐actin

beta‐actin

1. Introduction

Breast cancer is the second most common cause of cancer‐related death among women worldwide and is a highly heterogeneous malignancy. Over the past 40 years, the incidence rate of breast cancer has increased sharply, and breast cancer patients tend to be younger (Bertoli et al. 2015; Sabit et al. 2021; Ye et al. 2023). Research shows that 20% to 30% of breast cancer patients may experience metastasis after the diagnosis and initial treatment of the primary tumor. Metastasis represents a critical event in cancer progression and is the leading cause of mortality among breast cancer patients. Recent evidence further emphasizes the central role of metastatic spread, with studies indicating that up to 90% of breast cancer‐related deaths are attributable to metastatic disease (Wang and Shao 2025). Once distant metastasis occurs, the 5‐year survival rate drops significantly (Andorfer et al. 2011). The main clinical treatments for breast cancer include surgery and radiotherapy, alongside systemic therapies such as chemotherapy (alone or in combination), endocrine therapy for hormone receptor–positive disease, targeted therapy for HER2‐positive tumors, and—in selected settings—immunotherapy for triple‐negative disease (Wang and Shao 2025). However, their therapeutic efficacy is limited by drug resistance and adverse effects, particularly in ER‐negative subtypes (Jallah et al. 2023; Ma et al. 2023). As evidenced in the 2023 NCCN guidelines, treatment decisions now increasingly rely on tumor biology and biomarkers to guide systematic therapy selection (Gradishar et al. 2024). Therefore, identifying safer and more effective agents remains an urgent priority.

Curcumol is an important active ingredient in the volatile oil of the authentic Guangxi medicinal herb Curcumae kwangsiensis and has a wide range of biological activities, such as antitumor, antiviral, and anti‐inflammatory effects, with minimal adverse reactions. Recent studies have demonstrated that curcumol exerts anticancer activity through multiple mechanisms, such as inhibiting proliferation and inducing apoptosis via the PI3K/AKT and MAPK/NF‐κB pathways (Liu et al. 2023), as well as modulating microRNAs (miRNAs) such as miR‐21 and miR‐30a‐5p (Yu et al. 2021). These findings demonstrate that curcumol influences cancer progression through various mechanisms and exerts potential anticancer activity.

Our previous studies have suggested that curcumol can inhibit the proliferation of both MCF‐7 and MDA‐MB‐231 breast cancer cells and induce cell cycle arrest. Furthermore, the inhibitory effect of curcumol on growth is related to its impact on the expression of NCL (Wang et al. 2018; Zhou et al. 2023). Nucleolin (NCL) is a multifunctional RNA‐binding protein that is localized mainly in the nucleoplasm, cytoplasm, and cell membrane. These three main domains allow NCL to interact with different proteins and RNA sequences (Gu et al. 2017; Palmieri et al. 2015), including miRNAs. miRNAs, small noncoding RNAs approximately 20–25 nucleotides (nt) in length processed from lengthy primary precursor miRNAs (pri‐miRNAs), regulate gene expression post‐transcriptionally by targeting mRNAs in a sequence‐specific manner (Bahari Khasraghi et al. 2023; Pillai et al. 2007). Gongol and colleagues reported that NCL is the protein that binds the most miRNAs among 66 miRNA‐binding proteins, including miR‐206 (Gongol et al. 2019). miR‐206 has been identified as a tumor suppressor in many human malignancies, including breast cancer. Studies have shown that miR‐206 is frequently downregulated in breast cancer and is closely related to lymph node metastasis in breast cancer (Shen et al. 2013; Zhou et al. 2013). Increased expression of miR‐206 can promote apoptosis and reduce cell survival in breast cancer cells (Seiri et al. 2025). Bose et al. reported that upregulation of the expression of miR‐206 can downregulate the expression of NCL in breast cancer cells (Bose et al. 2016). These findings suggested that NCL may be a target gene of miR‐206 and is regulated by miR‐206.

NCL has been identified as a protein target of curcumol, and its antitumor effects were associated with NCL expression. NCL overexpression promotes malignant behaviors of tumor cells, including proliferation, invasion, angiogenesis, and metastasis, by activating oncogenic signaling pathways and inhibiting apoptosis (Jia et al. 2017). Recently, we found that NCL could directly interact with ERα36 coimmunoprecipitation assay (Zhou et al. 2023). ERα36, a novel variant of estrogen receptor‐alpha, mediates various biological effects and serves as an important promoter of triple‐negative breast cancer (Pelekanou et al. 2012). We previously reported that NCL and ERα36 expression levels correlate with tumor malignancy in breast cancer cells, and curcumol exerts its anti‐proliferative and cell cycle arrest effects by downregulating both NCL and ERα36 (Zhou et al. 2023). Accumulating evidence reveals that multiple miRNAs contribute to the antitumor effects of curcumol, such as miR‐21, miR‐152‐3p, miR‐9, and so on. (Liu et al. 2019; Ning et al. 2020; Sheng et al. 2021). However, whether curcumol modulates miR‐206 expression in breast cancer remains unknown. To our knowledge, this study is the first to explore the role of miR‐206 in mediating curcumol's effects on invasion and migration in breast cancer, and to demonstrate the regulatory relationship among miR‐206, NCL/ERα36.

2. Methods

2.1. Reagents and Cell Culture

Curcumol (purity > 98%) was purchased from Guizhou Di Technology Co. Ltd. (Guizhou, China). A stock solution was prepared by dissolving curcumol in anhydrous ethanol. The working concentration was selected based on previous studies. Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Grand Island, USA). Mammary epithelium growth medium (MEGM) was purchased from Lonza (Rockville, MD, USA). Penicillin–streptomycin was purchased from Solarbio Biotechnology Company (Solarbio, Beijing, China). Antibodies against β‐actin, NCL, N‐cadherin, MTDH, and β‐catenin were purchased from Abcam (Cambridge, MA, USA).

MCF‐7 and MDA‐MB‐231 breast cancer cell lines, as well as MCF‐10A normal breast epithelial cells, were originally obtained from the laboratory of Professor Zhaoyi Wang (Department of Immunology and Microbiology, Creighton University School of Medicine, USA) and maintained in our laboratory. MDA‐MB‐231 and MCF‐7 cells, as well as transiently stably transfected cells, were cultured in DMEM supplemented with 10% FBS (Grand Island, USA) and 1% penicillin–streptomycin (Solarbio, Beijing, China). MCF‐10A cells were cultured with the MEGM kit (Grand Island, USA), while MCF‐7 ERα36‐overexpressing cells were cultured in 1640 medium (Grand Island, USA) supplemented with 12% FBS.

2.2. Extraction of Virus Plasmids and Virus Packaging

The plasmid mixture kit was purchased from Tiangen Biotech Co. Ltd. (Beijing, China). The plasmid was extracted according to the instructions of the plasmid extraction kit, and its purity and concentration were measured via NanoDrop One (Thermo, USA). 293T cells in the logarithmic growth phase were placed into a six‐well plate, and the cell density reached approximately 70%, making them suitable for virus packaging.

2.3. Transfection

Small interfering RNAs designed for ERα36 (siERα36) and NCL (siNCL), the miR‐206 mimic and the negative control (NC) were all purchased from General Biological Co. Ltd. (Anhui, China). The miR‐206 inhibitors and controls were purchased from Shanghai Genechem Co. Ltd. (Shanghai, China). The ERα36 overexpression cell line was obtained from long‐term preservation in our laboratory. Polybrene (hexadimethrine bromide) was purchased from Biosharp (Hefei, China). Logarithmic MDA‐MB‐231 and MCF‐7 cells were selected and inoculated into six‐well plates. When the cell density reached approximately 40%–50%, transfection was performed via Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's manual.

2.4. Wound Healing Assay

Cells from each group were seeded in 6‐well plates. A sterile 200‐μL pipette tip was used to scratch straight lines in the cell layer to produce wounds, and detached cells were gently removed by washing with phosphate‐buffered saline (PBS). Fresh medium containing curcumol (50 μg/mL) was added to the treatment wells, while control wells received fresh medium without curcumol. Images of the wound area were captured at 0 and 48 h using an inverted microscope (Scale bar = 200 μm). The wound area was quantified using ImageJ software (version 1.8.0.112). The wound closure rate was calculated using the following formula: Wound closure rate (%) = (A 0 − A t)/A 0 × 100% where A 0 is the initial wound area and A t is the wound area at the indicated time point.

2.5. Transwell Assay

For the Transwell invasion assay, 24‐well chambers with 8‐μm pore size inserts (Corning) pre‐coated with Matrigel were used. MDA‐MB‐231 cells (3 × 105) and MCF‐7 (7 × 105) were seeded into the upper chambers in serum‐free medium. The lower chambers were filled with 600 μL of complete medium containing 10% FBS, with or without curcumol (50 μg/mL). After incubation for 48 h at 37°C, non‐invading cells on the upper surface of the membrane were gently removed with a cotton swab. The invaded cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet. Micrographs were taken under an inverted microscope (Scale bar = 100 μm) and cell numbers were quantified using ImageJ software.

2.6. Dual‐Luciferase Reporter Assay

MDA‐MB‐231 and MCF‐7 cells were seeded in 96‐well culture plates, cultured for 24 h, and then transfected. There were four transfection groups: the wild‐type NCL (NCLWT) + control group (FAM), wild‐type NCL (NCLWT) + miR‐206 overexpression (miR‐206 mimic), mutant NCL (NCLMUT) + control (FAM), and mutant NCL (NCLMUT) + miR‐206 overexpression (miR‐206 mimic) groups. After 48 h of transfection, luciferase activities were measured via the Dual‐Luciferase Reporter Assay reagent. Firefly luciferase activity was normalized to Renilla luciferase activity.

2.7. Western Blot

After transfection, breast cancer cells were harvested and lysed. Total protein was extracted from cells by using radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China). The protein concentration was subsequently determined with a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). The proteins were subjected to electrophoresis, membrane transfer, blocking, and antibody incubation. Primary antibodies against NCL, N‐cadherin, β‐catenin, β‐actin, MTDH, and MMP9 were purchased from Abcam (Cambridge, UK). The ERα36 antibody was generously provided by Professor Zhaoyi Wang (Department of Immunology and Microbiology, Creighton University School of Medicine, USA). HRP‐conjugated goat anti‐mouse and goat anti‐rabbit IgG antibodies were purchased from Beijing Yimabio Technology Co. Ltd. (Beijing, China). Finally, the target protein was detected using a Bio‐Rad imaging system (Bio‐Rad, Hercules, CA, USA). The intensity of each band was quantified using ImageJ software.

2.8. Quantitative Real‐Time PCR

Breast cancer cells were collected after transfection and treated with curcumol. Total RNA was isolated from the cells via TRIzol (TIANGEN, China), and its concentration was determined with a nucleic acid microdetector. The reverse transcription reaction system was then used to convert total RNA into cDNA (Biosharp, China). Quantitative real‐time (qRT) PCR analysis was performed using a 7500 Fast Real‐Time PCR System (ABI, Foster City, CA, USA). The relative expression of miR‐206 was calculated via the relative quantification method (2−ΔΔt).

2.9. RNA Immunoprecipitation (RIP) Assay

RIP was performed using the Magna RIP RNA‐Binding Protein Immunoprecipitation Kit (Millipore, USA) as previously described with minor modifications. Briefly, cells were lysed in RIP lysis buffer supplemented with protease and RNase inhibitors, and clarified lysates were incubated with magnetic beads conjugated to 3 μg anti‐NCL antibody or normal IgG (negative control) at 4°C for 3 h to overnight. After extensive washing, RNA–protein complexes were digested with a proteinase K‐containing buffer, and the co‐immunoprecipitated RNAs were purified by salt‐ and ethanol‐mediated precipitation with the aid of a precipitation enhancer. The isolated RNAs were subjected to qRT‐PCR, and enrichment was calculated relative to IgG controls.

2.10. In Vivo Experiment

Female BALB/c nude mice (4–5 weeks old) were obtained from SJA Experimental Animal Co. Ltd. (Changsha, Hunan). The xenograft experiments were performed on the same group of nude mice that was established for our previously published study. Each mouse was subcutaneously injected into the right axilla with 5 × 106 MCF‐7 ERα36‐overexpressing cells or MCF‐7 NC control cells suspended in 0.2 mL PBS. Based on our previous dose‐comparison experiments (20, 40, and 80 mg/kg) in tumor‐bearing mice, which showed that 80 mg/kg curcumol exerted the most pronounced antitumor effect and did not show significant loss in body weight, we selected 80 mg/kg as representative doses for the present study. Importantly, 40 mg/kg was chosen for the NC + Cur group rather than 80 mg/kg, since the higher dose almost completely suppressed tumor growth and would have precluded meaningful comparison with the untreated NC group. Accordingly, mice were further assigned into four groups: NC, NC + Cur (40 mg/kg), ERα36 lv, and ERα36 lv + Cur (80 mg/kg). Curcumol was administered once daily by gavage for 3 weeks, rather than oral feeding to ensure precise dosing, given its poor water solubility and variable oral bioavailability. While the respective control groups received a solvent (ethanol: propylene glycol: PBS, 2:2:1 for ERα36 groups; corn oil for NC groups). Tumor growth was monitored every 2 days, and treatment was discontinued once the tumor diameter reached approximately 1.5 cm. Mice were euthanized with pentobarbital sodium (50 mg/kg), and xenograft tumors were harvested for further analysis.

A separate cohort of female BALB/c nude mice (4–5 weeks old) was used to establish the lung metastasis model. Mice were injected via the tail vein with 5 × 106 MCF‐7 ERα36‐overexpressing cells suspended in 0.2 mL PBS. One week later, the mice were randomly divided into three groups: ERα36 lv, ERα36 + Cur (80 mg/kg), and NC. Body weight and general condition were monitored daily. At the end of 8 weeks, all mice were euthanized, and lung tissues were harvested for H&E staining to assess metastatic nodules. All animal procedures were approved by the Animal Ethics Committee of Guilin Medical University (certificate no. GLMC‐IACUC‐2023389).

2.11. Hematoxylin and Eosin (H&E) Staining

Animal tissues were fixed in paraformaldehyde and embedded in paraffin. The slices were dewaxed with xylene and gradient alcohol solutions. The sections were subsequently stained with hematoxylin for 5 min and rinsed with tap water. After differentiation with 1% alcohol and hydrochloric acid for 3 s, the tissues were placed in water for 4 min until a blue color appeared. The tissue was dyed with eosin solution for 1 min, followed by gradient alcohol dehydration and xylene transparency. Finally, the tissues were covered with a thin glass cover slip and observed via an inverted light microscope.

2.12. Statistical Analysis

The data were analyzed via GraphPad 8.0 (GraphPad Inc., San Diego, CA, USA) and SPSS version 21.0 (IBM., Chicago, CA, USA) statistical software. All the experiments were performed with no less than three replicates per sample, and all the data are expressed as the means ± standard deviations. Independent sample t‐tests were used for comparisons between two groups, whereas one‐way analysis of variance (ANOVA) was used for data comparisons among multiple groups. A probability (p) value < 0.05 was considered statistically significant.

3. Results

3.1. Curcumol Inhibits the Invasion and Migration of Breast Cancer Cells

As shown in Figure 1A, curcumol (50 μg/mL), a concentration previously reported as non‐toxic yet effective in tumor models (Zhou et al. 2023), significantly reduced the migration of MCF‐7 and MDA‐MB‐231 cells, as indicated by decreased wound closure after 48 h. The treatment time was chosen based on prior validation as optimal for capturing migration and invasion changes (Yu et al. 2021). In Figure 1B, Transwell assays demonstrated that curcumol also markedly suppressed the invasive capacity of both cell lines. Collectively, these findings indicate that curcumol effectively inhibits both migration and invasion in breast cancer cells.

FIGURE 1.

FIGURE 1

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Curcumol inhibited the invasion and migration of BC cells. (A) Wound healing assay was used to evaluate the migration of MCF‐7 and MDA‐MB‐231 cells treated with or without curcumol (50 μg/mL, 48 h; scale bar = 200 μm). (B) Transwell invasion assay was performed to evaluate the invasive ability of MCF‐7 and MDA‐MB‐231 cells treated with or without curcumol (scale bar = 100 μm). The differences among the experimental groups were calculated by two‐tailed t‐tests. Data are presented as mean ± SD (n = 3). Ctrl means untreated curcumol cells.

3.2. Curcumol Enhances the Inhibitory Effect of NCL Knockdown on the Invasion and Migration of Breast Cancer Cells

Our previous research demonstrated that NCL is the target protein of curcumol, which can interact with the estrogen receptor variant ERα36 (Wang et al. 2018; Zhou et al. 2023). As shown in Figure 2A, NCL protein levels in MCF‐7 and MDA‐MB‐231 breast cancer cells were significantly higher than those in normal MCF‐10A cells. We further compared NCL expression across three breast cancer cell lines (MCF‐7, MDA‐MB‐231, and T47D) and found that MDA‐MB‐231 cells showed the highest expression (Figure S1A). Silencing NCL by siRNA in MDA‐MB‐231 cells effectively reduced its expression (Figure 2B). Wound healing and transwell assays further showed that NCL knockdown markedly decreased the migration and invasion of both MCF‐7 and MDA‐MB‐231 cells compared with controls, and curcumol treatment enhanced these inhibitory effects (Figure 2C,D).

FIGURE 2.

FIGURE 2

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Curcumol enhanced the inhibitory effect of NCL knockdown on the invasion and migration of BC cells. (A) Western blot analysis of NCL expression in MCF‐7, MDA‐MB‐231, and MCF‐10A cells. (B) Western blot detection of NCL expression in MDA‐MB‐231 cells after NCL knockdown. (C) Wound healing assay was used to assess the migratory ability of MCF‐7 and MDA‐MB‐231 cells after NCL knockdown with or without curcumol treatment (50 μg/mL, 48 h; scale bar = 200 μm). (D) Transwell invasion assay was performed to detect the invasive ability of MCF‐7 and MDA‐MB‐231 cells after NCL knockdown with or without curcumol treatment (scale bar = 100 μm). (E) Western blot detection of N‐cadherin, NCL, β‐catenin, MTDH, and ERα36 expression in MDA‐MB‐231 cells after NCL knockdown with or without curcumol treatment (50 μg/mL, 48 h). Data are presented as mean ± SD (n = 3). p values and significance were determined using two‐tailed t‐tests (A) and One‐Way ANOVA (B–E). NC means scrambled siRNA negative control.

We next examined the expression of invasion and migration‐related proteins. NCL knockdown significantly decreased the expression of N‐cadherin, β‐catenin, and ERα36. While curcumol treatment strengthened these inhibitory effects, the level of MTDH in the siNCL + curcumol group did not show a substantial difference compared with curcumol alone (Figure 2E).

3.3. Overexpression of miR‐206 Enhances the Inhibitory Effect of Curcumol on the Invasion and Migration of Breast Cancer Cells

NCL, as an RNA‐binding protein, can bind to various miRNAs to exert its functions. Prediction analysis via the TargetScan tool revealed that miR‐206 can bind to positions 90–97 in the 3′ UTR of NCL mRNA (Figure 3A). Therefore, whether the inhibitory effect of curcumol on the invasion and migration of breast cancer cells is related to the RNA‐binding function of NCL attracted our attention. To verify the direct interaction between NCL and its binding site within miR‐206, a dual‐luciferase reporter assay was performed. We mutated the predicted site and constructed NCL mutant (mut‐NCL) and NCL wild‐type (WT‐NCL) vectors. The results revealed that the overexpression of miR‐206 in MCF‐7 and MDA‐MB‐231 cells decreased the luciferase activity of WT‐NCL without affecting the luciferase activity of MUT‐NCL (Figure 3B). Consistently, RIP assays demonstrated that NCL was significantly enriched in the miR‐206 group compared with the IgG control (Figure 3C), further confirming the direct binding between NCL and miR‐206. Taken together, these results suggest that NCL is a downstream target gene of miR‐206.

FIGURE 3.

FIGURE 3

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miR‐206 enhanced the inhibitory effect of curcumol on the invasion and migration of BC cells. (A and B) TargetScan prediction, wild‐type (WT) and mutant (MUT) NCL binding sites, and dual‐luciferase reporter assays confirming NCL as a direct target of miR‐206. (C) The interaction between miR‐206 and NCL was assessed by RIP assay. (D) The expression of miR‐206 in MCF‐10A, MCF‐7, and MDA‐MB‐231 cells was detected by qRT‐PCR. (E and F) qPCR analysis of miR‐206 expression after transfection with miR‐206 mimics with or without curcumol treatment (50 μg/mL, 48 h). (G) Wound healing assay of MCF‐7 and MDA‐MB‐231 cells after miR‐206 overexpression with or without curcumol treatment (50 μg/mL, 48 h; scale bar = 200 μm). (H) Transwell invasion assay of MCF‐7 and MDA‐MB‐231 cells after miR‐206 overexpression with or without curcumol treatment (50 μg/mL, 48 h; scale bar = 100 μm). (I) Western blot detection of N‐cadherin, NCL, β‐catenin, MMP9, MTDH, and ERα36 expression in MDA‐MB‐231 cells. Data are presented as mean ± SD (n = 3). p values and significance were determined using two‐tailed t‐tests (C and E) and One‐Way ANOVA (B, D, F–I). NC means vector negative control.

Then we detected miR‐206 expression in breast cancer cells and normal breast epithelial cells. The results showed that miR‐206 expression in MCF‐7 and MDA‐MB‐231 cells was significantly lower than that in MCF‐10A cells, with a more pronounced reduction observed in MDA‐MB‐231 cells (Figure 3D). miR‐206 mimics were transiently transfected into MCF‐7 and MDA‐MB‐231 (Figure 3E). After curcumol treatment, miR‐206 expression was significantly upregulated in both the control group and the miR‐206 overexpression group (Figure 3F).

To investigate the role of miR‐206 on the inhibitory effect of curcumol on tumor metastasis, we performed wound healing and transwell assays. These experiments demonstrated that miR‐206 overexpression significantly reduced the invasive and migratory capacities of MCF‐7 and MDA‐MB‐231 breast cancer cells compared to the control group (Figure 3G,H). Notably, curcumol can further inhibit the invasion and migration of breast cancer cells when miR‐206 is overexpressed.

Western blot analysis showed that both miR‐206 overexpression and curcumol treatment reduced the expression of invasion and migration‐related factors, like β‐catenin, MMP9, N‐cadherin, and MTDH in MDA‐MB‐231 cells. Additionally, miR‐206 overexpression led to a downregulation of NCL, confirming NCL as a target gene of miR‐206, and the expression of ERα36 also decreased correspondingly (Figure 3I). Notably, when combined with curcumol, miR‐206 overexpression did not further reduce but instead slightly increased the levels of NCL compared with curcumol alone.

3.4. miR‐206 Silencing Alters the Inhibitory Effects of Curcumol on the Invasion and Migration of Breast Cancer Cells

To further investigate the role of miR‐206 in the inhibitory effects of curcumol on the invasion and migration of breast cancer cells, a stable miR‐206‐silenced cell line was constructed via lentiviral vector transduction. Fluorescence microscopy confirmed successful transfection (Figure 4A). qRT‐PCR confirmed a significant suppression of miR‐206 expression (Figure 4B). Notably, NCL expression was upregulated in miR‐206‐silenced MCF‐7 cells (Figure 4C), corroborating that it is regulated by miR‐206.

FIGURE 4.

FIGURE 4

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miR‐206 silencing altered the inhibitory effect of curcumol on the invasion and migration of BC cells. (A) White‐light and fluorescence images of MCF‐7 cells after transfection with miR‐206 inhibitor or negative control (scale bar = 100 μm). (B and C) qPCR analysis of miR‐206 and NCL expression after miR‐206 inhibitor transfection. (D) Wound healing assay of MCF‐7 and MDA‐MB‐231 cells after miR‐206 knockdown with or without curcumol treatment (50 μg/mL, 48 h; scale bar = 200 μm). (E) Transwell invasion assay of MCF‐7 and MDA‐MB‐231 cells after miR‐206 knockdown with or without curcumol treatment (50 μg/mL, 48 h; scale bar = 100 μm). (F) Western blot detection of N‐cadherin, NCL, and β‐catenin expression in MCF‐7 cells. Data are presented as mean ± SD (n = 3). p values and significance were determined using two‐tailed t‐tests (C) and One‐Way ANOVA (B, D–F). NC means vector negative control.

In both MCF‐7 and MDA‐MB‐231 cells, miR‐206 silencing substantially enhanced the invasion and migration abilities of breast cancer cells (Figure 4D,E). This phenotypic alteration correlated with upregulated expression of N‐cadherin and β‐catenin, and the downstream gene NCL. Interestingly, when combined with curcumol, miR‐206 silencing did not fully reverse the inhibitory effects of curcumol; instead, it further downregulated NCL expression compared with curcumol alone (Figure 4F). In previous studies demonstrating that NCL can regulate ERα36 expression at the post‐transcriptional level, we hypothesized that the miR‐206/NCL axis may modulate ERα36 to influence metastatic behavior. To test this, we examined whether ERα36 overexpression could reverse curcumol's effects, similar to what we observed with miR‐206 inhibition.

3.5. ERα36 Overexpression Reverses the Inhibitory Effect of Curcumol on the Invasion and Migration of Breast Cancer Cells

Our above RIP data confirming direct binding between miR‐206 and NCL, while we have reported that ERα36 and NCL interact with each other and verified that ERα36 overexpression is involved in the effect of curcumol on cell cycle arrest in breast cancer, which indicated ERα36 may play an important role in curcumol's anti‐cancer effects. To explore the effect of ERα36 on the invasion and migration of breast cancer, we constructed an ERα36 overexpression vector and transfected it into MCF‐7 cells. As shown in Figure 5A, ERα36 overexpression induced obvious morphological changes to a more aggressive phenotype, including cell aggregation, elongation, and the extension of long, thin processes. These findings suggest that high levels of ERα36 significantly increase the degree of malignancy of cells and may promote the invasion and migration of cancer cells. Western blot analysis subsequently verified that ERα36 was successfully overexpressed in MCF‐7 cells (Figure 5B). Then, transwell assay results revealed that ERα36 overexpression significantly increased the invasive ability of MCF‐7 cells and reversed the inhibition of cell invasion by curcumol (Figure 5C).

FIGURE 5.

FIGURE 5

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ERα36 overexpression reversed the inhibitory effect of curcumol on the invasion and migration of BC cells. (A) White‐light and fluorescence images of MCF‐7 cells after ERα36 overexpression or negative control plasmid transfection (scale bar = 100 μm). (B) Transwell invasion assay was performed to detect the invasive ability of MCF‐7 cells with ERα36 overexpression treated with or without curcumol (50 μg/mL, 48 h; scale bar = 100 μm). Data are presented as mean ± SD (n = 3). p values and significance were determined using One‐Way ANOVA (B and C), NC means Negative Control (Plasmid).

3.6. ERα36 Knockdown Enhances the Inhibitory Effect of Curcumol on the Invasion and Migration of Breast Cancer Cells

To confirm the role of ERα36 in the inhibitory effect of curcumol on invasion and migration, we used siRNA interference to downregulate ERα36 expression in MCF‐7 cells. Three siRNA fragments targeting ERα36 were constructed, and Western blot analysis indicated that the third fragment exerted the most pronounced knockdown effect (Figure 6A). Consequently, subsequent experiments were conducted using this third siRNA fragment. Therefore, this siRNA fragment was selected for subsequent experiments. As shown in Figure 6B, siERα36 combined with curcumol significantly reduced migration compared with the NC + Cur group, whereas in Figure 6C, the inhibitory effect on invasion was not markedly different. This differential response suggests that ERα36 may play a more prominent role in regulating migratory behavior than invasive capacity under curcumol treatment.

FIGURE 6.

FIGURE 6

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ERα36 knockdown enhanced the inhibitory effect of curcumol on the invasion and migration of BC cells. (A) Western blot detection of ERα36 expression in MCF‐7 cells after knockdown with three different siRNAs. (B) Wound healing assay of MCF‐7 cells after ERα36 knockdown with or without curcumol treatment (50 μg/mL, 48 h; scale bar = 200 μm). (C) Transwell invasion assay of MCF‐7 cells after ERα36 knockdown with or without curcumol treatment (50 μg/mL, 48 h; scale bar = 100 μm). Data are presented as mean ± SD (n = 3). p values and significance were determined using One‐Way ANOVA (A–C). NC means scrambled siRNA negative control.

3.7. Curcumol Inhibits the Invasion and Migration of Breast Cancer Cells Induced by ERα36 Overexpression In Vivo

To investigate whether ERα36 expression affects the inhibitory effect of curcumol on the invasion and migration of breast cancer cells in vivo, MCF‐7 cells overexpressing ERα36 were subcutaneously injected into the right axilla of nude mice. Comparison of tumor sizes before and after ERα36 overexpression and curcumol treatment revealed that ERα36 promoted the growth of MCF‐7 tumors in vivo (Figure 7A). The organ weight data from this cohort are now provided as Figure S1B. After being sacrificed, tumor tissues were removed for further analysis. Western blot analysis revealed that ERα36 overexpression upregulated the protein expression levels of NCL, ERα36, and invasion and migration‐related proteins (N‐cadherin, β‐catenin, and MTDH) in tumors. Notably, curcumol treatment inhibited the increase in these protein levels induced by ERα36 overexpression (Figure 7B). RNA extracted from tumor tissues was subjected to qRT‐PCR, which revealed that curcumol markedly upregulated miR‐206 expression in control (NC) cells. However, this upregulation was attenuated in ERα36‐overexpressing cells (Figure 7C).

FIGURE 7.

FIGURE 7

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Curcumol reversed the promoting effect of ERα36 on tumor growth in vivo. (A) Representative images of nude mice and xenograft tumors after ERα36 overexpression with or without curcumol treatment (40 mg/kg or 80 mg/kg). (B) Western blot detection of N‐cadherin, NCL, β‐catenin, MTDH, and ERα36 expression in xenograft tumor tissues. (C) qPCR analysis of miR‐206 expression in tumor tissues with or without curcumol treatment (80 mg/kg, 3 weeks). (D) Average body weights of mice during treatment. (E) H&E staining of lung tissues showing metastatic nodules (n = 5). Data are presented as mean ± SD (n = 3). p values and significance were determined using One‐Way ANOVA (B and C). NC means Negative Control (Plasmid).

To further assess metastatic potential, mice received tail vein intravenous injection of 5 × 106 MCF‐7 cells, which were either stable overexpressing ERα36 or transfected with an empty vector control, each group consisted of five mice. One week later, the ERα36‐overexpressing group of mice was intraperitoneally injected with curcumol (80 mg/kg) every other day. After 8 weeks, the mice were sacrificed, the lungs were removed, fixed in 4% paraformaldehyde, and embedded in paraffin. H&E staining indicated that curcumol treatment significantly reduced lung metastasis in breast cancer (Figure 7E). No visible metastases were found in other organs, such as the liver and brain (Figure S1C). As shown in Figure 7D, mice in the ERα36 overexpression group lost more body weight than the control group. After treatment with curcumol, there was some recovery. Overall, these findings demonstrate that curcumol effectively inhibits the promoting effects of ERα36 overexpression on the invasion and migration of breast cancer cells.

4. Discussion

Breast cancer is one of the most common cancers worldwide and remains the leading cause of cancer‐related death among women (Harbeck and Gnant 2017). Although the mortality rate has decreased over recent decades due to advances in early detection and treatment, metastasis continues to drive recurrence and poor prognosis (Yuan et al. 2024). Therefore, the inhibition of cancer cell migration and invasion by pharmacological agents represents a potential strategy for cancer treatment (Drappatz et al. 2009; Hanahan and Weinberg 2011; Saha et al. 2021).

Curcumol, a sesquiterpenoid component derived from the volatile oil of Curcuma zedoaria , has shown significant antitumor effects. These effects are associated with the inhibition of tumor growth and the prevention of tumor cell metastasis (Li et al. 2018; Liu et al. 2019; Wei et al. 2019). Previous studies have reported that curcumol regulates tumor progression mainly via PI3K/AKT, MAPK, or Hippo pathways, or by targeting other miRNAs (e.g., miR‐21, miR‐30a‐5p) (Liu et al. 2019; Yu et al. 2021) and NCL was first found as the target protein of curcumol in nasopharyngeal carcinoma by Juan Wang in 2018. In this study, we provide new insights about the mechanism of curcumol antitumor, which is related to its target protein NCL. We found that curcumol effectively inhibits the migration and invasion of breast cancer cells through regulation of the miR‐206/NCL/ERα36 axis. NCL, identified as a protein target of curcumol, plays a critical role in mediating its cell cycle arrest effects in breast cancer cells (Jia et al. 2017; Wang et al. 2018; Zhou et al. 2023). Notably, NCL is highly expressed in breast cancer cell lines, and its upregulation promotes tumor progression, metastasis and drug resistance (Pichiorri et al. 2013). In our study, we found that NCL knockdown markedly suppressed migration and invasion in MCF‐7 and MDA‐MB‐231 cells, supporting its role in tumor promotion. It is noteworthy that although MDA‐MB‐231 cells exhibited higher NCL expression compared to MCF‐7 cells, which showed greater sensitivity to curcumol treatment in terms of migration suppression. This apparent discrepancy may be explained by several context‐dependent factors, including differential basal miR‐206 expression levels between these breast cancer subtypes (Luminal A vs. triple‐negative), variation in ERα36 expression patterns. Future studies comparing comprehensive molecular profiles across multiple breast cancer cell lines will help clarify these context‐dependent responses.

It has been reported that NCL participates in various processes related to tumor occurrence and development, which is related to its function as a multifunctional RNA‐binding protein (Jia et al. 2017). When combined with multiple miRNAs, it can affect the malignant behavior of tumor cells. Studies have shown that the expression of NCL is correlated with that of NCL‐dependent miRNAs. The miRNAs that negatively regulate tumor growth can target and inhibit the expression of NCL, thus exerting an antitumor effect on breast cancer cells (Pichiorri et al. 2013). In the present study, we found that miR‐206 is an upstream miRNA of NCL, and it negatively regulates the expression of NCL. miR‐206 functions as a tumor suppressor by inhibiting the growth, invasion and migration of tumor cells (Kondo et al. 2008; Wang et al. 2011). In breast cancer, studies have shown that the overexpression of miR‐206 suppresses CALR expression and inhibits the proliferation and metastasis of breast cancer stem cells, ultimately leading to apoptosis (Sun et al. 2022). Additionally, it has been reported that miR‐206 inhibits the stemness and metastasis of breast cancer cells by targeting the MKL1/IL11 pathway (Samaeekia et al. 2017). Consistent with previous reports, we found that the overexpression of miR‐206 significantly inhibited the invasion and migration abilities of breast cancer cells and downregulated NCL expression, while silencing miR‐206 yielded the opposite results. Furthermore, miR‐206 overexpression enhanced the inhibitory effect of curcumol on cell invasion and migration, which was reduced in the miR‐206‐silenced cells. Therefore, based on NCL's RNA‐binding effect, we proposed that curcumol's antitumor effects were associated with increased miR‐206 levels and downregulation of NCL, which may be related to NCL's binding with miR‐206. When curcumol treated cancer cells, it can inhibit the target protein NCL's expression, causing the binding RNA with NCL decreased, then the free RNA in the cells increased, including miR‐206.

Besides the effect on binding with RNA, NCL also interacts with several proteins involved in cell proliferation, apoptosis angiogenesis to influence cancer development. Our previous study revealed that NCL interacts with ERα36 and affects curcumol's anti‐tumor activity (Zhou et al. 2023). Numerous studies found that ERα36 is highly expressed in breast cancer cells with high migratory ability (Gu et al. 2017; Park et al. 2022). We previously found that ERα36 expression was upregulated in NCL‐overexpressing cells and downregulated when NCL was silenced. Notably, when ERα36 was over‐expressed in MCF‐7 cells, it led a significant changes in cell morphology and showed a more aggressive phenotype. Both in vitro and in vivo experiments revealed that the overexpression of ERα36 attenuated the inhibitory effect of curcumol on the invasion and migration of breast cancer cells, whereas the silencing of ERα36 enhanced the inhibitory effect of curcumol on these processes. So, combining the effect of miR‐206 on NCL, our study demonstrated that curcumol inhibits NCL/ERα36 was associated with increased miR‐206 levels and affects the development of breast cancer. Although our study focused on TNBC, ERα36 is expressed across various breast cancer subtypes, included ERα‐positive and HER2‐positive cancers. However, subtype‐specific differences in hormone receptor status may influence therapeutic efficacy. Future studies across diverse subtypes are warranted to define curcumol's full therapeutic potential.

This study explored the effects of Chinese botanical drugs acting on miRNAs and their influence on the function of the target protein. By increasing the levels of miR‐206 and regulating the expression of NCL/ERα36 using curcumol, the invasion and migration of BC cells can be effectively inhibited. These findings provide a foundation for the further development of curcumol as an innovative therapeutic agent for breast cancer while also providing new insights into the modern development of Chinese medicinal materials. However, more cell lines, the probable mechanisms of miR‐206 expression upregulated by curcumol, its inhibitory effect on NCL/ERα36 or other pathways, structural or functional studies to elucidate the nature and significance of NCL and ERα36 interaction, rescue experiments, and more in vivo experiments were need further research.

5. Conclusions

In summary, this study explores how curcumol interferes with the RNA‐binding function of NCL to exert its inhibitory effect on migration and invasion in ER‐negative breast cancer cells. The effect of curcumol on the RNA‐binding function of NCL is related to its regulation of miR‐206/NCL. This study further revealed that curcumol negatively regulates NCL and is associated with increased miR‐206 levels and indirectly downregulates the expression of the NCL‐interacting protein ERα36. This research provides a detailed analysis of the impact of miR‐206/NCL/ERα36 on the anticancer effects of curcumol on breast cancer invasion and migration, and provides a new anticancer mechanism of curcumol.

Author Contributions

Xiaojuan Li: data curation, visualization, formal analysis, writing – review and editing. Muzhao Tian: conceptualization, data curation, formal analysis, investigation, methodology, writing – original draft. Luwei Zhou: investigation. Hongfang Ding: methodology. Riqiu Zhang: software, visualization. Xu Chen: funding acquisition, project administration, resources, supervision. Juan Wang: conceptualization, resources, supervision, validation, writing – review and editing.

Funding

This research was funded by the National Natural Science Foundation of China (82002822 and 81760443) and the Natural Science Foundation of Guangxi (2023GXNSFBA026271), the China Postdoctoral Science Foundation (2020M683630XB), and the Project of Guangxi Special Fund. Project for Innovation‐Driven Development (GuikeAA23023035).

Disclosure

This study was conducted in compliance with key recommendations from recent guidelines for best practices in pharmacological research on natural products (Heinrich et al. 2020; Wang et al. 2024).

Ethics Statement

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Guilin Medical University and approved by the Animal Ethics Committee of Guilin Medical University (Guilin, China).

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: (A) Western blot analysis of NCL expression in MDA‐MB‐231, MCF‐7, and T47D breast cancer cells. (B) Organ weights (liver, spleen, lung, kidney, brain) from the NC, NC + Cur, ERα36 lv, and ERα36 lv + Cur groups. (C) Representative H&E staining images of liver, brain, and kidney tissues from mice in the NC, ERα36 lv, and ERα36 lv + Cur groups (Scale bar = 100 μm). (D) Representative sequencing chromatograms of the NCL 3′UTR wild‐type (WT) and mutant (MUT) reporter constructs, confirming correct mutagenesis. Data are presented as mean ± SD (n = 3). p values and significance were determined using One‐Way ANOVA (A). NC means Negative Control (Plasmid).

PTR-40-5566-s001.zip (11.1MB, zip)

Acknowledgments

We thank Professor Zhaoyi Wang for providing the MDA‐MB‐231 and MCF‐7 breast cancer cell lines used in this study. We thank Dr. Shiqin Jiang for her valuable suggestions and editorial contributions to this manuscript.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: (A) Western blot analysis of NCL expression in MDA‐MB‐231, MCF‐7, and T47D breast cancer cells. (B) Organ weights (liver, spleen, lung, kidney, brain) from the NC, NC + Cur, ERα36 lv, and ERα36 lv + Cur groups. (C) Representative H&E staining images of liver, brain, and kidney tissues from mice in the NC, ERα36 lv, and ERα36 lv + Cur groups (Scale bar = 100 μm). (D) Representative sequencing chromatograms of the NCL 3′UTR wild‐type (WT) and mutant (MUT) reporter constructs, confirming correct mutagenesis. Data are presented as mean ± SD (n = 3). p values and significance were determined using One‐Way ANOVA (A). NC means Negative Control (Plasmid).

PTR-40-5566-s001.zip (11.1MB, zip)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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