The MCF-7 and T47D breast cancer cell lines were purchased from the American Type Culture Collection. Cells were maintained in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), at 37°C with 5% CO₂.

MCF-7 and T47D cells were analyzed for mycoplasma to ensure that they were not contaminated with mycoplasma using the Mycoplasma Detection kit (Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's instructions. Lutein was purchased from the Agri-Food Canada Research Centre. Lutein was dissolved in different concentrations of DMSO (Beijing Solarbio Science & Technology Co., Ltd., 0.00, 6.25, 12.50, 25.00 and 50.00 µg/ml).

Total RNA was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A PrimeScript™ RT reagent kit (cat. no. RR047A; Takara Biotechnology Co., Ltd.) was used to reverse transcribe the extracted RNA from MCF-7 and T47D cells into cDNA. RT was performed at 37°C for 60 min. qPCR was subsequently performed using SYBR Green (cat. no. DRR041A; Takara Biotechnology Co., Ltd.). The following primer sequences were used for qPCR: miR-590-3p forward, 5′-TAATTTTATGTATAAGCTAGT-3′ and reverse, 5′-GCAGGGTCCGAGGTATTC-3′; Cancer Susceptibility 9 (CASC9) forward, 5′-CAGGTAATCTCAGCAGTCAT-3′ and reverse, 5′-ACATCCACAGGTCTCCAA-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; and GAPDH forward, 5′-GGAGTCCACTGGCGTCTT-3′ and reverse, 5′-ATCTTGAGGCTGTTGTCATAC-3′. The following thermocycling conditions were used for qPCR: Initial denaturation for 15 min at 95°C, followed by 40 cycles of denaturation for 10 sec at 95°C, annealing for 30 sec at 60°C and extension for 20 sec at 72°C. Relative expression levels were calculated using the 2^−ΔΔCq method (20) and normalized to the internal reference genes GAPDH and U6.

MCF7 cells were divided into two groups as follows: One group was treated with 50.00 µg/ml lutein, whereas the other group was treated with solvent alone (control group). The cells were incubated for 24 h and total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA samples were purified using the RNasey Mini kit (Qiagen, Inc.), amplified and labeled using the Quick Amp Labeling kit, One-Color (Agilent Technologies, Inc.), according to the manufacturer's protocol. An equal amount of labeled cRNA from each sample was hybridized using the Agilent Gene Expression Hybridization kit (Agilent Technologies, Inc.), followed by image acquisition and data analysis using the Agilent Feature Extraction software (version 11.0.1.1; Agilent Technologies, Inc.).

The sequences of CASC9 small interfering RNA (siRNA) (si-CASC9), scrambled siRNA [si-negative control (NC)], miR-590-3p mimic and the NC mimic were purchased from Shanghai GenePharma Co., Ltd. The sequences were as follows: si-CASC9^1#, 5′-AUGAACAUCCACAAACACCAA-3′; si-CASC9^2#, 5′-UAAUAUUUCUUGAUAGUGCCA-3′; si-CASC9 NC, 5′-GAAUCCUACUUUCACAGCCAU-3′; miRNA-590-3p mimic, 5′-TAATTTTATGTATAAGCTAGT-3′; mimic NC, 5′-CTAGTCACTATATAGGAGCTG-3′. MCF-7 and T47D cells were cultured until they reached 70–80% confluence and subsequently each well was transfected with si-CASC9 (45 nM), si-NC (45 nM), miR-590-3p mimic (40 nM) and NC mimic (40 nM) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions, at 37°C for 48 h. Subsequent experiments were performed 48 h post-transfection.

The DIANA TOOLS database (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2/index) was used to determine the molecular mechanism by which CASC9 regulates miR-590-3p.

The CASC9 fragments containing the predicted miR-590-3p binding sites were separately amplified via RT-qPCR analysis and cloned into a pmirGLO dual luciferase miRNA target expression vector (Invitrogen; Thermo Fisher Scientific, Inc.) to create a wild-type CASC9 reporter vector. Subsequently, the putative mutant miR-590-3p binding sites were constructed in CASC9 by altering the sequences and replacing them to form a CASC9-mutated-type. The miRNAs (miR-590-3p mimics or miR-NC) and recombinant plasmids were co-transfected into 293T cells (American Type Culture Collection), using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h at 37°C, luciferase activities were detected using a dual luciferase reporter gene assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Following transfection, MCF-7 and T47D cells were seeded into 96-well microplates at a density of 5×10⁴ cells/ml (100 µl/well) and treated with different concentrations of lutein (0.00, 6.25, 12.50, 25.00 and 50.00 µg/ml), at 37°C for 24, 48 and 72 h. Subsequently, cells were incubated with 20 µl MTT solution (KGA312; Nanjing KeyGen Biotech Co., Ltd.) for 4–6 h at 37°C. The MTT solvent included in the assay kit was used to dissolve the purple formazan. The absorbance was measured at a wavelength of 570 nm, using a microplate reader (SpectraMax M5; Molecular Devices, LLC).

Statistical analysis was performed using SPSS 22.0 software (IBM Corp.). All experiments were performed in triplicate and data are presented as the mean ± standard deviation. Unpaired Student's t-test was used to compare differences between two groups, while one-way ANOVA followed by Tukey's post hoc test were used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Our previous study demonstrated that lutein exhibits antitumor effects in breast cancer cells (10). In the present study, to identify whether specific lncRNAs are involved in the antiproliferative effects of lutein, their expression levels were detected in MCF7 cells with or without lutein treatment (50.00 µg/ml). A total of 1,083 lncRNAs exhibited significant changes in their expression levels following treatment of cells with lutein. In the present study, 11 lncRNAs (fold change ≥20; P≤0.05; Fig. 1A) were identified with significantly upregulated expression levels and 10 demonstrated the most significantly downregulated expression levels (fold change ≤20; P≤0.05; Fig. 1B). Among these, LINC00240 was the most highly upregulated lncRNA (fold change=65.65; P=0.024), whereas CASC9 exhibited the highest downregulation compared with the expression levels of the remaining lncRNAs (fold change=103.119800; P=0.033) (Fig. 1A and B). These effects were noted in MCF7 cells treated with lutein. Thus, RT-qPCR analysis was performed to detect the expression levels of CASC9 following treatment of MCF-7 and T47D cells with different concentrations of lutein for 24 h. The results demonstrated that CASC9 expression exhibited a negative association with increasing concentrations of lutein (Fig. 1C and D). In addition, CASC9 expression was the lowest when the cells were treated with 50.00 µg/ml lutein. Thus, the present study further investigated whether CASC9 expression levels decreased in a time-dependent manner following treatment of cells with 50.00 µg/ml lutein. The results demonstrated that CASC9 expression decreased in MCF-7 and T47D cells treated with 50.00 µg/ml lutein in a time-dependent manner (Fig. 1E and F). Taken together, these results suggest that lutein downregulates CASC9 expression in breast cancer cells.

To assess the potential functional roles of CASC9 on the antiproliferative effects of lutein, CASC9 expression was knocked down using the siRNA1 and siRNA2 sequences. RT-qPCR analysis demonstrated that transfection with both siRNAs into the cells was successful (Fig. 2A and B). The knockdown efficiency of siRNA2 was higher, and thus this sequence was selected for subsequent experimentation. Subsequently, the effect of CASC9 knockdown on cell proliferation was assessed. The results demonstrated that CASC9 knockdown inhibited cell proliferation (Fig. 2C and D). The cells were treated with different concentrations of lutein and concomitantly transfected with the siRNA sequences to assess the effect of CASC9 on lutein-mediated tumor suppression. The results demonstrated that transfection with si-CASC9 sequences significantly increased the tumor-inhibitory effects of lutein (Fig. 2E and F). Collectively, these results suggest that CASC9 knockdown can potentiate the suppressive role of lutein on breast cancer.

Recent studies have reported that the lncRNA/miRNA/mRNA regulatory axis plays important roles in tumor development (21,22). Thus, the present study aimed to identify the potential targets of CASC9. Bioinformatic analysis revealed that miR-590-3p is a target gene of CASC9 (Fig. 3A). The dual-luciferase reporter assay was performed to determine whether CASC9 possesses putative binding sites of miR-590-3p. Following co-transfection with miR-590-3p and si-CASC9, the luciferase activity of the wild-type CASC9 reporter was attenuated (Fig. 3B). Notably, this was not observed in the mutant CASC9 reporter or in the transfected NC-miR 293T cells, suggesting that miR-590-3p can bind to CASC9. RT-qPCR analysis was performed to detect miR-590-3p expression following treatment of MCF-7 and T47D cells with different concentrations of lutein for 24 h. The results demonstrated that miR-590-3p expression was positively associated with increasing concentrations of lutein treatment (Fig. 3C and D). miR-590-3p mimics or NC mimics were transfected into MCF-7 and T47D cells to assess the potential functional roles of miR-590-3p on lutein-mediated tumor suppression. RT-qPCR analysis demonstrated that transfection was successful (Fig. 3E). Subsequently, the cells were treated with different concentrations of lutein and concomitantly transfected with miR-590-3p mimics to assess the effect of miR-590-3p on lutein-mediated tumor suppression. The results demonstrated that following 24 h treatment of the cells with lutein, miR-590-3p mimic caused a significant increase in the tumor-inhibitory effect of this compound (Fig. 3F and G). Taken together, these results suggest that overexpression of miR-590-3p can enhance the suppressive role of lutein on breast cancer.

Taken together, the results of the present study suggest that CASC9 and miR-590-3p are both involved in the tumor suppressive role of lutein on breast cancer. Thus, subsequent experiments were performed to investigate the interaction between the CASC9/miR-590-3p axis and lutein. MCF-7 and T47D cells were treated with different concentrations of lutein and transfected concomitantly with miR-590-3p mimics and si-CASC9. Transfection of the cells with miR-590-3p mimics or si-CASC9 alone led to a significant increase in the tumor-inhibitory effects of lutein (Fig. 4A and B). Notably, the simultaneous transfection of miR-590-3p mimics and si-CASC9 enhanced the antiproliferative activity of lutein on breast cancer cells, suggesting that the CASC9/miR-590-3p axis participates in the antitumor action of this compound.