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The H19 long non-coding RNA is involved in the development of tamoxifen resistance in breast cancer. However, the relationship between H19 and the metastatic potential and treatment options for tamoxifen-resistant (TAMR) breast cancer is not completely understood. Curcumin inhibits cellular proliferation, migration and invasiveness in several cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia. The present study aimed to investigate the role of H19 in MCF-7/TAMR cell epithelial-mesenchymal transition (EMT), migration and invasiveness, and to assess the ability of curcumin to inhibit H19-mediated effects. Reverse transcription-quantitative PCR and western blot analysis were conducted to detect the gene or protein expression. Cell Counting Kit-8, wound healing and Transwell invasion assays were performed to estimate the capabilities of cell viability, invasion and migration. H19 overexpression enhanced MCF-7/TAMR cell EMT, invasion and migration by upregulating Snail. Furthermore, curcumin notably decreased the expression levels of epithelial marker E-cadherin and markedly increased the expression levels of mesenchymal marker N-cadherin in MCF-7/TAMR cells compared with the control group. In addition, following treatment with curcumin for 48 h, H19 expression was decreased in a dose-dependent manner. Moreover, curcumin treatment for 48 h significantly attenuated H19-induced alterations in N-cadherin and E-cadherin expression levels. Curcumin also prevented H19-induced invasion and migration. The present study indicated that H19 may serve as a promoting factor of EMT, invasion and migration in MCF-7/TAMR cells, suggesting that curcumin may prevent H19-associated metastasis. Therefore, curcumin may serve as a promising therapeutic drug for patients with TAMR breast cancer.
Breast cancer is the most common malignancy and the leading cause of cancer-associated death in women worldwide (
Several mechanisms influencing resistance to endocrine therapy have been identified. The identified mechanisms are complex and primarily focused on the epigenetic regulation, mutation, truncation and fusion events of the ER1 gene (
Curcumin, a natural compound derived from turmeric, has been reported to possess antitumor effects, including the prevention of metastasis and progression in multiple cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia (
The MCF-7 human breast cancer cell line was obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. MCF-7/TAMR cells were established by treating MCF-7 cells at 37°C with 1 µM 4-hydroxytamoxifen (Sigma-Aldrich; Merck KGaA) for 3 weeks and then 100 nM 4-hydroxytamoxifen for 6 months, as previously described (
MCF-7/TAMR cells were seeded (5×103 cells/well) into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Cells were treated with different concentrations of curcumin (0, 5, 10, 20, 30 and 40 µM) for 48 h at 37°C with 5% CO2. Subsequently, 10 µl Cell Counting Kit-8 solution (Sigma-Aldrich; Merck KGaA) was added to each well and incubated at 37°C for 1 h. DMSO (0.1%) was added to the control wells. The optical density was determined at a wavelength of 450 nm using the iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories, Inc.).
H19 pcDNA3.1 expression vector (H19-epv), empty vector negative control (H19-epv-NC), scrambled small interfering (si)RNA negative control (siNC cat. no. siN0000001-1-5) and H19 siRNA (5′-CCTGTAACCAAAAGTGACCG-3′) were obtained from Guangzhou RiboBio Co., Ltd. Briely, 2×104 MCF-7/TAMR cells were plated in phenol red-free medium containing 10% FBS in 6-well plates, then transfected with 100 nM siRNA (siRNAH19 or siNC) or 1 µg of H19 expression plasmid (H19-epv or empty vector) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as previously described (
MCF7/TAMR cells were seeded (5×105 cells/well) into 6-well plates. Following transfection, cells were treated with curcumin for 48 h at 37°C. At 90–100% confluence, a sterile pipette tip was used to form a single scratch across the cell monolayer. After washing with PBS, cells were incubated in RPMI-1640 supplemented with 1% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO2. The width of the wound in each group was examined at 0, 24 and 48 h using a light microscope (magnification, ×40; Olympus Corporation), then analyzed using Image J software (version 1.8.0; National Institutes of Health). The migration rate was calculated as: Migration rate = (Width of the wound at 0 h - Width of the wound at 24 or 48 h) / Width of the wound at 0 h.
Following transfection and curcumin treatment, cell invasion was assessed using modified Boyden chambers (pore size, 8.0 µm; Costar; Corning, Inc.). The Transwell inserts were pre-coated with Matrigel overnight at 37°C with 5% CO2. A total of 2×104 transfected cells were resuspended in 200 µl serum-free medium and plated into the upper chambers of the Transwell inserts in a 24-well plate. Subsequently, 600 µl RPMI-1640 supplemented with 20% FBS was added to the lower chambers. Following incubation at 37°C for 24 h, non-invading cells were removed with cotton swabs and the filters were rinsed with PBS. Invading cells were fixed with methanol for 20 min at room temperature, stained with 1% crystal violet for 30 min at room temperature. Stained cells were counted in eight random microscopic fields using a light microscope (magnification, ×200; Olympus Corporation).
Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (1 µg) was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. Subsequently, qPCR was performed using iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Inc.) and the iQ™5 real-time detection system (Bio-Rad Laboratories, Inc.). The following primers were used for qPCR: H19 forward, 5′-GTCCGGCCTTCCTGAACACCTT-3′ and reverse, 5′-GCTTCACCTTCCAGAGCCGAT-3′; E-cadherin forward, 5′-CAACAAAGACAAAGAAGGCAAGG-3′ and reverse, 5′-TGAGAGAAGAGAGTGTATGTGGC-3′; vimentin forward, 5′-GGAGGAGATGCTTCAGAGAGAG-3′ and reverse, 5′-GGATTTCCTCTTCGTGGAGTTTC-3′; Snail forward, 5′-AGGACCACAGTGGCTCAGAAAGGAPDH-3′ and reverse, 5′-TGATGACCCTTTTGGCTCCC-3′; and GAPDH forward, 5′-GGAAGCTTGTCATCAATGGAAATC-3′ and reverse, 5′-TGATGACCCTTTTGGCTCCC-3′. The following thermocycling conditions were used for qPCR: 95°C for 5 min; followed by 40 cycles at 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. mRNA expression levels were quantified using the 2−ΔΔCq method (
Total protein was extracted from cells using RIPA buffer (Sigma-Aldrich; Merck KGaA) containing 1% PMSF, 0.3% protease inhibitor and 0.1% phosphorylated proteinase inhibitor. Total protein was quantified using the BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Proteins (20 µg) were separated via 12% SDS-PAGE and transferred to nitrocellulose membranes, which were blocked in blocking buffer [5% non-fat dry milk in TBS with 0.5% Tween, (TBS-T)] at room temperature for 2 h. After washing with TBS-T, the membranes were incubated overnight at 4°C with primary antibodies targeted against: E-cadherin (1:300; Abcam; cat. no. ab40772), N-cadherin (1:300; Abcam; cat. no. ab76011), vimentin (1:300; Abcam; cat. no. ab16700), Snail (1:300; Abcam; cat. no. ab216347) and GAPDH (1:1,000; cat. no. G9545; Sigma-Aldrich; Merck KGaA). Following primary incubation, the membranes were incubated for 2 h at room temperature with a HRP-conjugated Affinipure goat anti-rabbit secondary antibody (1:500; Abcam; cat. no. ab6721). Protein bands were visualized using Immobilon Western Chemilum HRP Substrate (cat. no. WBKLS0100; EMD Millipore) and the expression levels of each protein were analyzed using Image Lab software version 4.1 (Bio-Rad Laboratories, Inc.).
Cells were plated onto confocal laser small dishes at a density of 5×104, and treated with curcumin as aforementioned. The cells on chamber slides were washed with PBS for 15 min, fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After three washes with PBS (5 min each), non-specific binding was blocked with 3% BSA (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Subsequently, cells were incubated with primary antibodies targeted against E-cadherin (1:100 in PBS with 1% BSA) and N-cadherin (1:100 in PBS with 1% BSA) for 2 h at room temperature. Following washing with PBS, cells were incubated with Alexa Fluor 488 goat anti-rabbbit IgG (Proteintech, cat. no. SA00006-2) for E-cadherin and Alex Fluor 594 goat anti-rabbbit IgG (Proteintech; cat. no. SA00006-4) secondary antibody for 1 h at room temperature. After three 5-min washes with PBS, cell nuclei were stained with 10 µg/ml Hoechst 33258 for 10 min at room temperature. Following washing with PBS, cells were visualized using a fluorescence microscope (magnification, ×200).
Data are presented as the mean ± SEM. Comparisons among multiple groups were analyzed using one-way ANOVA following by the SNK or Tukey's post hoc test. Comparisons between two groups were analyzed using an unpaired Student's t-test. Statistical analyses were performed using SPSS software (version 21.0; IBM Corp.). Each experiment was performed at least three times. P<0.05 was considered to indicate a statistically significant difference.
To determine the effects of H19 on MCF-7/TAMR cell EMT, H19 was overexpressed and knocked down using HPV-epv and siRNAH19, respectively. The results indicated that following transfection with siRNAH19 for 24 h, H19 expression was significantly decreased by 40-fold, vimentin expression was notably downregulated and E-cadherin expression was markedly upregulated compared with the siRNAH19-NC group (
Wound healing and Transwell assays were performed to determine the influence of H19 expression on MCF-7/TAMR cell migration and invasion. Following transfection with H19-epv or siRNAH19 for 24 h, the results demonstrated that H19 overexpression promoted wound closure, whereas H19 knockdown inhibited wound closure compared with the H19-epv-NC and siRNAH19-NC groups, respectively (
Snail is a key regulator of the EMT process (
The impact of curcumin on MCF-7/TAMR cells proliferation was investigated. As demonstrated in
Subsequently, the effects of different concentrations of curcumin on the expression of H19 in MCF-7/TAMR cells were assessed. H19 expression was significantly decreased in a dose-dependent manner by concentrations of curcumin between 5 and 20 µM for 48 h (
To verify the influence of curcumin on H19-induced migration and invasion, MCF-7/TAMR cells were transfected with H19-epv for 24 h, and then incubated in the presence or absence of curcumin for 48 h. The results demonstrated that cell migration in the H19-epv group was significantly increased compared with H19-epv-NC. However, treatment with curcumin for 48 h significantly decreased wound closure in H19-overexpression cells (
Despite its wide use for the treatment of breast cancer, a third of patients with breast cancer develop resistance to tamoxifen, which results in tumor progression (
Previous studies have indicated that H19 is upregulated in a variety of cancer cell types, including breast cancer, gastric cancer and glioma cells, and that H19 knockdown inhibits tumor growth, migration and invasion (
Accumulating evidence has demonstrated that curcumin exerts anticancer effects via multiple signaling pathways. Hu
In conclusion, the present study suggested that H19 promoted EMT, migration and invasion in MCF-7/TAMR cells, whereas curcumin inhibited H19-induced effects. Therefore, the use of curcumin to inhibit the H19/Snail/E-cadherin axis may serve as a promising therapeutic option for patients with TAMR breast cancer.
Not applicable.
The present study was supported by the Fujian Science and Technology Innovation Joint Fund Project (grant no. 2017Y9067), the Medical Science Research Project (grant no. BJBQEKYJJ-B19001CS), the Young and Middle-Aged Backbone Talents Project (grant no. 2019-ZQN-35), the Science and Technology Department of Fujian Province (grant no. 2017Y9098), the High-Level Hospital Foster Grants from Fujian Provincial Hospital (grant no. 2019HSJJ06), the Fujian Natural Science Foundation Project (grant no. 2019J01177) and the Startup Fund for Scientific Research, Fujian Medical University (grant nos. 2016QH106 and 2017XQ1024).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JC and JZ conceptualized the study. JC, BZ and HS designed the study. JC, HS, MX and GZ carried out the experiments and curated the data. JC, CX and XH analyzed the data. JC and HS wrote the manuscript. JZ reviewed and edited the manuscript. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
H19 induces MCF-7/tamoxifen-resistant cell epithelial-mesenchymal transition. Transfection efficiency of (A) siRNAH19 and (B) H19-epv. The effects of (C) siRNAH19 and (D) H19-epv on E-cadherin and vimentin mRNA expression levels were determined via reverse transcription-quantitative PCR. (E) The effects of siRNAH19 and H19-epv on E-cadherin and vimentin protein expression levels were determined via western blotting. **P<0.05 vs. siRNAH19-NC or H19-epv-NC. E-cad, E-cadherin; siRNA, small interfering RNA; epv, expression vector; NC, negative control.
H19 induces MCF-7/tamoxifen-resistant cell migration and invasion. Cells were transfected with H19-epv or siRNAH19 for 24 h. Cell (A) migration (magnification, ×40) and (B) invasion (Magnification, ×200) were assessed by performing wound healing and Transwell invasion assays, respectively. **P<0.05 vs. siRNAH19-NC, ##P<0.01 vs. H19-epv-NC. NC, negative control; epv, expression vector; siRNA, small interfering RNA.
H19 promotes MCF-7/tamoxifen-resistant cell epithelial-mesenchymal transition via regulating Snail. Cells were transfected with H19-epv or siRNAH19 for 24 h. Snail (A) protein and (B) mRNA expression levels were measured via western blotting and reverse transcription-quantitative PCR, respectively. **P<0.05 vs. siRNAH19-NC, ##P<0.05 vs. H19-epv-NC. NC, negative control; epv, expression vector; siRNA, small interfering RNA.
Curcumin influences MCF-7/tamoxifen-resistant cell viability and epithelial-mesenchymal transition. (A) The effect of curcumin on cell viability was determined by performing a Cell Counting Kit-8 assay. *P<0.05 and **P<0.01 vs. 0 µM curcumin groups. E-cadherin and N-cadherin (B) protein and (C) mRNA expression levels were determined via western blotting and reverse transcription-quantitative PCR, respectively. **P<0.01 vs. Con. Con, control; E-cad, E-cadherin; N-cad, N-cadherin.
Curcumin inhibits H19 mRNA expression in MCF-7/tamoxifen-resistant cells. Cells were treated with various concentrations of curcumin for 48 h. Subsequently, H19 mRNA expression levels were determined via reverse transcription-quantitative PCR. **P<0.05, ***P<0.01 vs. 0 µM curcumin.
Curcumin attenuates H19-induced epithelial-mesenchymal transition in MCF-7/tamoxifen-resistant cells. Following transfection with H19-epv, cells were treated with or without curcumin for 48 h. (A) E-cadherin and N-cadherin protein expression levels were determined via western blotting. **P<0.01 vs. H19-epv-NC; ##P<0.01 vs. H19-epv. (B) Cytolocalization of E-cadherin and N-cadherin in MCF-7/TAMR cells was detected by performing immunofluorescence staining. Magnification, ×200. E-cad, E-cadherin; N-cad, N-cadherin; epv, expression vector; NC, negative control.
Curcumin inhibits H19-induced MCF-7/tamoxifen-resistant cell migration and invasion. Following transfection with H19-epv for 24 h, cells were treated with or without curcumin for 48 h. Cell (A) migration (magnification, ×40) and (B) invasion (magnification, ×200) were determined by performing wound healing and Transwell invasion assays, respectively. **P<0.05 vs. H19-epv-NC; ##P<0.01 vs. 19-epv. NC, negative control; epv, expression vector.