Quercetin suppresses cell survival and invasion in oral squamous cell carcinoma via the miR-1254/CD36 cascade in vitro

Abstract

Objective:

This study aimed to investigate the effects of quercetin on the proliferation and invasion in oral squamous cell carcinoma (OSCC) and examine its effect on the activation of the miR-1254/CD36 signaling pathway.

Methods:

Proliferation and invasion experiments were performed in the OSCC cell line CAL-27 in which miR-1254 was overexpressed or inhibited. The levels of miR-1254 and CD36 were determined using quantitative real-time polymerase chain reaction and Western blotting assays.

Results:

Quercetin significantly suppressed the proliferation and invasion of CAL-27 cells in a dose-dependent manner, while up-regulating miR-1254 and down-regulating CD36. The overexpression of miR-1254 also considerably down-regulated CD36 and enhanced the ability of quercetin to inhibit CAL-27 cell survival and invasion. Conversely, the inhibition of miR-1254 significantly up-regulated CD36 and antagonized the inhibitory effects of quercetin.

Conclusion:

Our study suggests that quercetin might suppress the progression of OSCC by activating the miR-1254/CD36 signaling pathway, indicating its potential as a treatment against OSCC.

Keywords

Quercetin miR-1254/CD36 pathway oral squamous cell carcinoma proliferation invasion

Introduction

Although the incidence of oral tumors has slightly decreased in recent years, the incidence of tongue cancer has increased,^1,2 and extensive studies have shown that about 90% of oral tumors are oral squamous cell carcinoma (OSCC).³ OSCC accounts for approximately 90% of head and neck tumors.^4,5 Given that the vast majority of patients with OSCC are diagnosed at an advanced stage due to poor clinical symptoms,⁶ identifying drug targets and developing prevention or treatment strategies for OSCC are urgently needed.

MicroRNAs (miRNAs) are endogenous non-coding RNA molecules consisting of 17–25 nucleotides that can inhibit gene expression at the post-transcriptional level by binding to target mRNAs, leading to their degradation and translational repression.^7,8 Additional studies have also shown that miRNAs regulate survival, differentiation, and invasion of cancer cells,^9,10 and abnormal miRNA expression has been associated with the development or progression of tumors such as OSCC.¹¹ A recent study revealed that miR-1254 is down-regulated in OSCC tissues and cells and suppresses the tumor by directly targeting the CD36 protein, which is closely related to the occurrence and growth of tumors¹²; CD36 up-regulation may promote tumor cell proliferation and migration in OSCC.^13,14 Therefore, the development of a drug that could suppress the survival of OSCC cells by regulating the miR-1254/CD36 signaling pathway could significantly improve the treatment of OSCC.

To date, several studies have focused on the role of natural bioactive products in the prevention and therapy of various cancer types. Quercetin is a polyphenolic and natural compound found in various fruits, vegetables, leaves, and grains.¹⁵ It is almost insoluble in water, soluble in ethanol. Recent literature has reported its anticancer activity towards various tumors,^16–18 as well as its ability to inhibit proliferation, migration, and invasion of OSCC cells in vitro.^19,20 Extensive investigations have reported that quercetin could target Notch-1 pathway to enhances the radiosensitivity of colon cancer cells,²¹ and it also inhibits cell viability, migration and invasion by regulating microRNA-22/WNT1/β-catenin axis in oral cancer.²² It is well known that quercetin can effectively inhibit the production of free radicals, as well as enhance the pharmacological role of anticancer drugs. However, whether quercetin inhibit OSCC progression is still unclear. Therefore, in this study, we aimed to investigate the anti-tumor activity of quercetin in OSCC cells and to elucidate its mechanism of action.

Materials and methods

Cell culture and treatment

Human OSCC CAL-27 cells were purchased from Wuhan Bafeier Biological Co., Ltd. (Wuhan, China) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; HyClone) including 10% fetal bovine serum (Invitrogen, Carlsbad, California), 1% penicillin G, and streptomycin (Invitrogen) in a 5% CO₂ incubator at 37°C. Then quercetin (Sigma–Aldrich, St. Louis, MO) was dissolved in DMSO and added to cultures at final concentrations of 10, 20, 40, 80, or 160 μM, and the cells were incubated for 12, 24, and 48 h.

Cell transfection

The control mimic, miR-1254 mimic #1, miR-1254 mimic #2, control inhibitor, and miR-1254 inhibitor were obtained from Wuhan Bafeier Biological. CAL-27 cells were transfected with 100 nM of miR-1254 mimic #1, miR-1254 mimic #2, and control mimic or with 50 nM of miR-1254 inhibitor or control inhibitor using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Cell counting kit-8 (CCK-8) assay

The principle of the assay is that the reagent can be reduced to a highly aqueous orange-yellow formazon by dehydrogenase in mitochondria in the presence of electron-coupling reagent, and the color depth is proportional to the cell proliferation.²³ A CCK-8 assay experiment was performed to assess the cytotoxicity of quercetin in CAL-27 cells. Specifically, CAL-27 cells (1 × 10⁴ cells per well) were grown in 96-well plates and incubated with DMEM. After stabilization, the medium was replaced by a medium containing different quercetin concentrations (10, 20, 40, 80, or 160 μM) for 12, 24, and 48 h. Then, the medium was replaced with 10 µL of CCK-8 reagent and the cells were incubated at 37°C for 2 h. The plate absorbance was measured at 450 nm using a microplate reader (Bio-Rad, USA).

Cell count

The miR-1254-overexpressing and miR-1254-silenced CAL-27 cells were grown in 12-well plates (1 × 10⁵ cells) and treated with quercetin for 24 h. The number of viable cells was determined with a Vi-CELL XR cell counter (Beckman Coulter, Fullerton, CA, USA).

AlamarBlue assay

The alamarBlue assay was applied to measure quantitatively the proliferation of tumor cells. Cells (3000 per well) were grown in 96-well plates overnight, treated as described for the CCK-8 assay, and 10 mL (10%, v/v) of alamarBlue dye (Invitrogen) was added into each well. After incubation for 4 h, the absorbance was measured at 570 nm using a microplate reader (Bio-Rad, USA).²⁴

Cell invasion

The invasion ability of the CAL-27 cells was assessed using transwell matrigel invasion chambers (Invitrogen, Carlsbad, California).²⁵ The cancer cells were added into the upper chamber in serum-free DMEM, while the lower chamber was filled with 500 µL of complete culture medium. After incubation with quercetin for 24 h, the remaining CAL-27 cells in the upper chamber were removed. Afterward, the invaded cells were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet for another 30 min, and counted using a fluorescence microscope (Beckman Coulter).

Western blotting assay

CAL-27 cells were lysed on ice for 30 min, and the lysates were centrifuged at 13,000 g and 4°C for 6 min. The protein concentration was determined using a commercial kit (Bio-Rad).²⁶ The protein lysates were then denatured at 100°C for 5 min after mixing with a loading buffer. Protein (50 μg) was fractionated by sodium dodecyl sulphate–polyacrylamide electrophoresis for 4 h (70 V) and transferred to a nitrocellulose membrane for 70 min (120 mA). After blocking with 5% non-fat milk, the strips were treated with rabbit anti-CD36 (ab1209843, Abcam, UK) and anti-β-actin (ab1143099, Abcam, UK) antibodies at 4°C for 12 h. After washing three times with TBS containing 2% Tween-20 (TBST), the strips were incubated with a secondary anti-rabbit IgG antibody (ab2098543, Abcam, UK) at room temperature for 1.5 h. Strips were washed with TBST for three times, then treated with ECL-Plus Western blotting kit (Amersham Biosciences, USA) for 3 min. Lastly, the signals were measured using BioRad ChemiDoc MP. The protein gray values were calculated using Image J software and the relative expression levels of proteins were normalized to β-action.

Quantitative real-time polymerase chain reaction (qRT-PCR) assay

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and cDNA was synthesized using PrimeScript RT reagent (Invitrogen).²⁷ Levels of miR-1254 and CD36 mRNAs were measured using the SYBR Green Mastermix (Takara Biotech Corporation) on an ABI Prism7500 RT-PCR system (Applied Biosystems, Foster City, CA, USA). U6 and β-actin were used as internal controls for miR-1254 and CD36, respectively. The following primers were used: miR-1254 forward, 5′-CTGGCTGACACGTAA-3′; miR-1254 reverse, 5′-AATTGGCTGAATCGTA-3′; CD36 forward, and; CD36 F: 5′-TGTGCAAAATCCACACGAATTTGCGT-3′; and CD36 reverse, 5′-GCCACGCCAGTTGACTGGCCATGCTA-3′.

Statistical analysis

The results of this study were analyzed using SPSS 20.0 software (IBM, Chicago, IL, USA). The numerical data were shown as mean ± standard deviation. If data are normally distributed, and the differences among multiple groups were assessed for significance using one-way analysis of variance (ANOVA), followed by a Bonferroni post-test. If data are not normally distributed, and the differences were analyzed by nonparametric test. Differences associated with p < 0.05 were considered statistically significant.

Results

Quercetin suppressed the proliferation of CAL-27 cells

The CCK-8 assay revealed that the cell optical density was significantly reduced in the quercetin-treated CAL-27 cells compared to the control cells (Figure 1(a)), and the reduction was dose-dependent (Figure 1(b)). The IC₅₀ values of quercetin treatment for 12, 24, and 48 h are 124 μM, 82 μM, and 56 μM, respectively. The alamarBlue assay experiments demonstrated that the relative absorbance was significantly lower in quercetin-treated cells than in the control cells (Figure 1(c)). Furthermore, the Matrigel invasion assay indicated that the cell invasion decreased significantly with quercetin dose (Figure 1(d)).

Figure 1.

Quercetin suppressed cell proliferation and invasion in CAL-27 cells. (a) to (c) Cells were treated with different quercetin concentrations for the indicated times. (a) Change in the cell optical density (OD) of control and quercetin-treated CAL-27 cells, as determined in the CCK-8 assay. The differences of different time points and dose points were compared using ANOVA. (b) Change in the number of control and quercetin-treated CAL-27 cells, as determined in the cell counting assay. (c) Relative absorbance of control and quercetin-treated CAL-27 cells, as was detected in the alamarBlue assay. (d) Cells were treated with 0–160 μM quercetin for 24 h in a Matrigel invasion assay, and relative cell invasion was quantified. *p < 0.05 compared to control cells.

Quercetin activated the miR-1254/CD36 pathway in OSCC cells

In OSCC, miR-1254 acts as a tumor suppressor by inhibiting CD36 expression.²⁸ Therefore we examined whether quercetin may exert its anti-tumor effects by activating the miR-1254/CD36 signaling pathway in CAL-27 cells. As shown in Figure 2(a) and (b), quercetin concentrations of up to 80 μM significantly reduced levels of CD36 protein while also increasing levels of miR-1254 (Figure 2(c)).

Figure 2.

Quercetin activated the miR-1254/CD36 signaling pathway in CAL-27 cells. Cells were treated for 24 h with the indicated concentrations of quercetin (0, 20, 40, and 80 μM), then levels of miR-1254 and CD36 were measured, respectively, using qRT-PCR and Western blotting. (a) A representative Western blot. (b) Relative level of CD36. (c) Relative level of miR-1254 by qRT-PCR. *p < 0.05 compared to control cells.

miR-1254 down-regulated the expression of CD36 mRNA

Cells transfected with miR-1254 mimic #1 or mimic #2 showed significantly higher levels of miR-1254 (Figure 3(a)) and significantly lower levels of CD36 mRNA (Figure 3(b)) than cells transfected with control miR-1254 mimic. Conversely, miR-1254 inhibitor led to significantly lower levels of miR-1254 (Figure 3(c)) and higher levels of CD36 mRNA (Figure 3(d)).

Figure 3.

Impact of miR-1254 up- or down-regulation on the expression of CD36 in OSCC cells. (a) Relative expression of miR-1254 in CAL-27 cells transfected with miR-1254 mimic #1 or mimic #2. (b) Effect of miR-1254 up-regulation on CD36 mRNA levels in CAL-27 cells. (c) Relative expression of miR-1254 expression in CAL-27 cells transfected with the miR-1254 inhibitor. (d) Effect of miR-1254 down-regulation on CD36 mRNA levels in CAL-27 cells. *p < 0.05 compared to control cells.

Quercetin suppressed the CD36 pathway by inducing miR-1254 expression

In order to investigate whether quercetin affects the CD36 pathway by regulating miR-1254 expression, CAL-27 cells transfected with miR-1254 mimic #2 or miR-1254 inhibitor were treated with 80 μM quercetin for 24 h and compared with control cells and CAL-27 cells treated only with quercetin. Cells also treated with the mimic showed significantly lower CD36 mRNA level (Figure 4(a)) and significantly higher CD36 mRNA level (Figure 4(b)), suggesting that quercetin inhibited the CD36 signaling pathway by inducing miR-1254 expression.

Figure 4.

Quercetin suppressed the CD36 pathway in CAL-27 cells by inducing the expression of miR-1254. (a) Relative CD36 mRNA level and (b) relative CD36 mRNA level in cells treated with quercetin (Qu) in the presence or absence of miR-1254 mimic #2-treated cells. *p < 0.05 compared to the control cells; ^#p < 0.05 compared to the Qu group.

miR-1254 overexpression enhanced the ability of quercetin to inhibit proliferation of OSCC cells

To examine whether the regulation of miR-1254 expression by quercetin can explain its suppressive effects on cancer cell proliferation, CAL-27 cells were transfected or not with miR-1254 mimic #2, then treated with quercetin, and the effects on proliferation and invasion were examined. Proliferation and invasion were significantly lower in the presence of the mimic (Figure 5(a) to (d)). These results indicate that miR-1254 overexpression enhanced the inhibitory effects of quercetin on OSCC cell proliferation.

Figure 5.

Up-regulation of miR-154 enhanced the ability of quercetin to inhibit the proliferation and invasion of CAL-27 cells. Cells were transfected or not with miR-1254 mimic #2, then treated with 80 μM of quercetin (Qu) for 24 h. (a) Cell optical density (OD), as determined in the CCK-8 assay. (b) Number of CAL-27 cells, as measured in the cell counting assay. (c) Relative absorbance of CAL-27 cells, as detected in the alamarBlue assay. (d) Relative invasion, as determined in the Matrigel invasion assay. *p < 0.05 compared to control cells; ^#p < 0.05 compared to the Qu group.

To confirm that the observed effects of quercetin on OSCC cell survival were related to miR-1254 up-regulation, CAL-27 cells were transfected or not with the miR-1254 inhibitor, then treated with quercetin. Proliferation and invasion were greater in the presence of the miR-1254 inhibitor (Figure 6(a) to (d)). Thus, inhibition of miR-1254 expression impaired the anti-tumor activity of quercetin.

Figure 6.

Inhibition of miR-1254 partially reversed quercetin-mediated suppression of OSCC cell proliferation and invasion. CAL-27 cells were transfected or not with the miR-1254 inhibitor, then treated with 80 μM of quercetin for 24 h. (a) Cell optical density (OD), as determined in the CCK-8 assay. (b) Number of CAL-27 cells, as measured in the cell counting assay. (c) Relative absorbance of the CAL-27 cells, as detected in the alamarBlue assay. (d) Relative invasion, as determined in the Matrigel invasion assay. *p < 0.05 compared to control cells; ^#p < 0.05 compared to the Qu group.

Discussion

The inhibition of cancer cell survival is considered a potential strategy against tumor progression. Several studies have described natural products that exert anti-tumor activity by promoting cell apoptosis, suppressing cell growth, and limiting the invasion abilities of cancer cells.^29,30 Quercetin has been reported to suppress survival of various cancer cell types by regulating the apoptosis, proliferation, migration, and invasion of malignant cells³¹; it has been shown to inhibit proliferation of OSCC cells by up-regulating apoptosis.³² However, the molecular pathways by which quercetin exerts anti-tumor effects remain unclear. The present study shows that in one OSCC cancer cell line, quercetin appears to exert anti-tumor effects on proliferation and invasion by inducing miR-1254 expression, which in turn down-regulates CD36. Although the molecular mechanism behind the induction of miR-1254/CD36 signaling pathway by the quercetin is not observed, we speculate that quercetin may directly act the miR-1254 promoter and induce its expression.

Our results are consistent with a previous report that overexpression or down-regulation of miR-1254 reduce or increase, respectively, survival and invasion of OSCC cell lines,¹² suggesting that miR-1254 may suppress OSCC tumors. That study further showed that miR-1254 expression is low in OSCC tissues and increases with more advanced tumor stage.

CD36 is a glycosylated transmembrane protein of the family of β-class scavenger receptors,³³ and its expression positively correlates with occurrence and progression of cancer.^34,35 High CD36 expression in OSCC cells is associated with greater viability and migratory ability,¹⁴ suggesting its contribution to the progression of OSCC. Our results that miR-1254 levels in CAL-27 cells were negatively associated with levels of CD36 agree with the recent finding that CD36 is a direct target of miR-1254,¹² which suggests that miR-1254 might act as a cancer suppressor in OSCC by inhibiting the CD36 expression. Therefore, we conclude that quercetin can effectively suppress the survival and invasion of OSCC cells by inducing the miR-1254/CD36 signaling pathway. This conclusion is supported by our findings that overexpression of miR-1254 in CAL-27 cells potentiated the anti-tumor effects of quercetin, while inhibition of miR-1254 antagonized them. The IC₅₀ value of quercetin was identified to be approximately 80 μM, which was consistent with the results of previous studies.^36,37

Intriguingly, quercetin has already been shown to inhibit CD36 expression in the context of atherosclerosis: this inhibition, in fact, protected against cell proliferation, migration and invasion.³⁸ That work and our present study suggest promising therapeutic potential for quercetin and the miR-1254/CD36 pathway in cancer and potentially other diseases.

Conclusions

Taken together, quercetin might suppress the progression of OSCC by activating the miR-1254/CD36 signaling pathway. Furthermore, more experiments using different cell lines and experimental animals should be performed to observe its potential as a treatment against OSCC.

Footnotes

Acknowledgment

The authors thank the Department of Pharmacy, Wuhan Third Hospital (Tongren Hospital of Wuhan University).

Author contributions

LC, J-SX, and J-HW conceived and designed the research. Y-GC conducted the experiments. C-JQ contributed to the new reagents and analytical kits. LC, J-SX, and J-HW analyzed and interpreted the data. LC drafted the manuscript. C-JQ read and approved the final manuscript. All authors read and approved the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

C-J Qiu

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