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Introduction

Non-small-cell lung cancer (NSCLC) is one of the most fatal cancer types worldwide, accounting for ~80% of all primary lung cancer types (1). Statistically, 70–80% of patients with NSCLC will progress to an advanced disease state once diagnosed (2). With regards to the current treatment options, intensive chemotherapy combined with aggressive surgical techniques have demonstrated significant successes; however, the prognosis of patients with NSCLC remains disappointing due to the chemoresistance, invasion and metastasis of the tumors (3,4). Thus, there is an urgent requirement to develop novel chemotherapies for the treatment of NSCLC.

Curcumin is a naturally active phenolic compound extracted from the rhizome of the plant Curcuma longa, which is used as a traditional Chinese medicine (5). Curcumin has become an increasingly popular compound of interest, both in research and clinically, among different types of cancer, including lung adenocarcinoma, a type of NSCLC (6–8). It is reported to have anticancer properties that are exerted by affecting multiple cellular processes, such as cell proliferation, invasion, metastasis and chemoresistance (9); however, the mechanisms of action of curcumin in NSCLC remain largely unknown.

MicroRNAs (miRNAs/miRs) are a class of endogenous small non-coding RNAs of ~22 nucleotides in length that interact with mRNAs, which leads to mRNA degradation or the inhibition of translation (10). Functionally, miRNAs are involved in diverse cellular functions, such as growth, proliferation, apoptosis and migration (11). Accumulating evidence has demonstrated that curcumin may represent a novel strategy for cancer treatment by serving as an epigenetic regulator; initially, curcumin was identified to exert epigenetic activity over miRNAs in pancreatic cancer (12), and since this, other studies have also reported an association between the pharmacological effect of curcumin and miRNAs in lung cancer (10). For example, Ye et al (13) discovered that miR-192-5p and miR-215 were involved in the proapoptotic effects of curcumin, whereas Jin et al (14) also suggested that the antiproliferative role of curcumin in human NSCLCs depended on the increased expression levels of miR-192-5p and the inactivation of the PI3K/AKT signaling pathway. However, apart from these studies, to the best of our knowledge, there are very few studies into the role of miR-192-5p in NSCLC progression, especially in processes such as migration and invasion. In addition, the underlying regulatory mechanism of curcumin in NSCLC remains to be fully investigated.

The Wnt/β-catenin signaling pathway is one of the main and most frequently dysregulated pathways in several types of tumor, and it is suggested that altered cell proliferation, invasive behaviors and cell resistance are all attributed to the dysregulation of this signaling pathway (15,16). Notably, several studies have reported that curcumin affects cell proliferation through the Wnt/β-catenin signaling pathway in multiple different types of cancer, including both colon cancer and medulloblastoma (17,18). In fact, in lung cancer, this signaling pathway has been demonstrated to participate in the anticancer role of curcumin in tumor cell growth, invasion, epithelial-mesenchymal transition and cancer stem cells traits (15,16,19,20). In addition, the involvement of miR-192-5p and the Wnt/β-catenin signaling pathway is noted in several types of tumor (21–23), excluding lung cancer; however, little is known regarding the interaction between curcumin, miR-192-5p and the Wnt/β-catenin signaling pathway in NSCLC.

In the present study, the expression levels of miR-192-5p and c-Myc, an important oncogene, were investigated in NSCLC cells following curcumin treatment. In addition, functional and mechanistic assays were used to investigate the effect of miR-192-5p dysregulation on curcumin-mediated cell proliferation, invasion, migration and the Wnt/β-catenin signaling pathway in NSCLC cells. In short, this study suggested a novel molecular mechanism for curcumin in NSCLC.

Materials and methods

Cell culture and reagents

The human NSCLC cell lines, A427 and A549, and the human embryonic kidney cell line 293T were purchased from the American Type Culture Collection. The NSCLC cells were cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences) and 293T cells were maintained in DMEM (HyClone; GE Healthcare Life Sciences). Both mediums were supplemented with 10% FBS (HyClone; GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) and cells were then maintained in a humidified atmosphere of 5% CO2 at 37°C.

Curcumin treatment

Curcumin (100 mM in DMSO) was purchased from Sigma-Aldrich (Merck KGaA). The working concentration of curcumin was 10, 20 or 40 µM diluted in culture medium. Cells were cultured in 6-well or 96-well plates (Corning, Inc.) and exposed to 10–40 µM curcumin for 48 h at 37°C. The control group was treated with 0 µM of curcumin diluted in 0.1% DMSO (24). The 20 µM curcumin concentration was selected to treat A427 and A549 cells for further analysis.

Cell transfection

The hsa-miR-192-5p mimic (5′-CUGACCUAUGAAUUGACAGCC-3′) and miR-negative control (NC) mimic (5′-UUCUCCGAACGUGUCACGUTT-3′) were obtained from Shanghai GenePharma Co., Ltd. The pcDNA3.1 (pcDNA; Invitrogen; Thermo Fisher Scientific, Inc.) plasmid was used to construct the c-Myc overexpression vector (pcDNA-c-Myc). Small interfering (si)RNA against c-Myc (si-c-Myc; 5′-CCTGAGACAGATCAGCAACAA-3′) and its NC (si-NC; 5′-TTCTCCGAACGTGTCACGT-3′), and the hsa-miR-192-5p inhibitor (in-miR-192-5p; 5′-GGCUGUCAAUUCAUAGGUCAG-3′) and miR-NC inhibitor (in-miR-NC; 5′-CAGUACUUUUGUGUAGUACAA-3′) were purchased from Shanghai GenePharma Co., Ltd. Cells at 70% confluency were cultured in 6-well plates (Corning, Inc.) and the transfection of plasmids (2 µg) and oligonucleotides (40 nM) was performed using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cells were transfected for 48 h at 37°C prior to further studies.

MTT staining for cell viability

Cell viability was determined using an MTT reagent (Sangon Biotech Co., Ltd.). Following curcumin treatment, 5×10−3 mg/ml MTT was added to each well and incubated for 4 h at 37°C. The culture medium containing MTT was discarded with micropipettor and formazan crystals were dissolved in 100 µl dimethyl sulfoxide. The optical density (OD) was measured at 490 nm using a SpectraMax M4 microplate reader (Molecular Devices, LLC). Cell viability (%) was calculated using the following formula: OD value of treated group/OD value of control group ×100. All experiments were performed in quadruplicate.

Cell Counting Kit-8 (CCK-8) assay for cell proliferation

The proliferative ability of the NSCLC cells was determined using a CCK-8 assay (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Briefly, 1×104 A427 and A549 cells/well were plated into 96-well plates (Corning, Inc.) for 24 h at 37°C. Subsequently, 20 µl CCK-8 solution (5 mg/ml) diluted in PBS was added to each well for another 1 h and the absorbance was measured at 450 nm using a SpectraMax M4 microplate reader (Molecular Devices, LLC). Results were expressed as a percentage relative to the control group (defined as 100%). Data are from ≥5 independent experiments.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using a PrimeScript™ RT reagent kit (Takara Bio, Inc.) according to the manufacturer's instuctions. The temperature conditions were as followed: 25°C for 10 min, 42°C for 15 min and 85°C for 5 min. qPCR was subsequently performed using a SYBR Green PCR Master mix (Takara Bio, Inc.) and a TaqMan miRNA assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol to amplify mRNAs and miRNAs, respectively, on an ABI PRISM 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation was 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec, and the melting curve generation condition was 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. The following primer pairs were used for the qPCR: c-Myc forward, 5′-GGCTCCTGGCAAAAGGTCA-3′ and reverse, 5′-CTGCGTAGTTGTGCTGATGT-3′; miR-192-5p forward, 5′-GGACTTTCTTCATTCACACCG-3′ and reverse, 5′-GACCACTGAGGTTAGAGCCA-3′; GAPDH forward, 5′-CGAGCCACATCGCTCAGACA-3′ and reverse, 5′-GTGGTGAAGACGCCAGTGGA-3′; and U6 forward, 5′-TCGCTTCGGCAGCACATATACT-3′ and reverse, 5′-ACGCTTCACGAATTTGCGTGTC-3′. Expression levels were quantified using the 2−ΔΔCq method (25) and miRNA was normalized to U6 (miRNA) and GAPDH (mRNA). The reactions were performed in quadruplicate for each sample, with ≥3 independent runs.

Western blotting

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified with Bradford method and 20 µg protein/lane was separated via 10–12% SDS-PAGE, and then transferred onto PVDF membranes. After blocking with 5% skimmed milk for 2 h at 25°C, the membranes were incubated with the following primary antibodies (all Abcam): Anti-c-Myc (1:1,000; cat. no. ab32072), anti-β-catenin (1:4,000; cat. no. ab6302), anti-cyclin D1 (1:5,000; cat. no. ab40754) and anti-β-actin (1:5,000; cat. no. ab8227) for overnight at 4°C. After incubation with horseradish peroxidase-conjugated secondary antibody anti-Rabbit (1:50,000; Abcam; cat. no. ab205718) for 1 h at 25°C, the protein bands were visualized using an ECL reagent (EMD Millipore). Protein expression was normalized to the loading control β-actin on Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

Computational prediction

TargetScanHuman 7.1 database (http://www.targetscan.org/ENST00000377970.2) was used to predict the potential miRNAs targeting Myc gene on the 3′untranslated region (c-Myc 3′UTR). The in silico data provided a potential binding site between miR-192-5p and c-Myc 3′UTR on position 738–744.

Dual-luciferase reporter assay

The pMIR-REPORT Luciferase miRNA Expression Reporter Vector and pMIR-REPORT β-galactosidase Reporter Control Vector, were purchased from Invitrogen; Thermo Fisher Scientific, Inc. The putative binding site of miR-192-5p in c-Myc 3′UTR was mutated. The wild-type (WT) of c-Myc 3′UTR fragment (c-Myc-3′UTR-WT) and the mutant type (MUT) of c-Myc 3′UTR fragment (c-Myc-3′UTR-MUT) were cloned into the pMIR-REPORT miRNA Expression Reporter Vector, respectively. 293T cells at 60% confluency were co-transfected with 100 ng pMIR-REPORT-c-Myc-WT/MUT vectors, pMIR-REPORT β-galactosidase vectors and 50 nM miR-192-5p mimic/NC mimic using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following incubation for 48 h at 37°C, transfected 293T cells were collected and the firefly luciferase and β-galactosidase activities were detected using a Dual-Luciferase Reporter assay system (Promega Corporation) according to the manufacturer's protocol. Firefly luciferase activity was normalized to β-galactosidase expression levels. Data are presented as the mean of ≥3 independent transfections.

RNA immunoprecipitation (RIP)

Following the transfection of miR-192-5p mimic or miR-NC, RIP was performed in cell extracts in RIPA lysis buffer (Beyotime Institute of Biotechnology). A Magna RIP™ RNA-binding protein immunoprecipitation kit (EMD Millipore) was used according to the manufacturer's protocol to obtain the RIP that bound to argonaute 2 (Ago2; Abcam; 1:20; cat. no. ab32381) or IgG (1:50; Abcam; cat. no. ab2410) antibody. Then, RT-qPCR was performed to detect the expression levels of c-Myc mRNA in RIP.

Transwell assays

For the migration and invasion assays, A427 and A549 cells (5×105) were suspended in 200 µl RPMI-1640 medium without FBS and plated in the upper chambers of Transwell plates (Corning, Inc.) without or with Matrigel (Corning, Inc.) for migration and invasion, respectively. The Matrigel membrane was established using Matrigel (1:8) after air drying for 16 h at 4°C. RPMI-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was plated in the lower chambers. Following incubation at 37°C for 48 h, the migratory and invasive cells in the lower chambers were stained with 0.2% crystal violet for 15 min at 25°C. Absorbance was measured at a wavelength of 570 nm using a microplate reader and the migratory/invasive ability was presented as a percentage of the control group.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, Inc.) and data are presented as the mean ± SD. Statistical differences between groups were determined using a one-way ANOVA with Tukey's post hoc analysis. P<0.05 was considered to indicate a statistically significant result.

Results

Curcumin treatment suppresses NSCLC cell progression in vitro

Following the exposure of NSCLC A427 and A549 cells to different concentrations of curcumin (10, 20 and 40 µM), the tumor-suppressive role of curcumin in NSCLC in vitro was investigated. Cellular proliferation was significantly decreased in both cell lines following 10, 20 and 40 µM curcumin treatment compared with the control cells (Fig. 1A and B), which was consistent with the cell viability status (Fig. 1G and H). In addition, the migratory ability of NSCLC cells was determined; the migratory ability of both cell lines was significantly attenuated by 10, 20 and 40 µM of curcumin compared with the control (Fig. 1C and D). Curcumin was also found to significantly reduce the invasive ability of both cell lines at all concentrations (10, 20 and 40 µM) compared with the control (Fig. 1E and F). Of note, the effects of curcumin on NSCLCs were all observed to occur in a dose-dependent manner. These results suggested that curcumin treatment may promote NSCLC cell cytotoxicity and suppress cell migration and invasion, leading to the antitumor effect in NSCLC in vitro.

Figure 1. Effects of curcumin on non-small-cell lung cancer cells. A427 and A549 cells were treated with 10, 20 or 40 µM curcumin for 48 h. Cell proliferation rates in (A) A427 and (B) A549 cells were determined using a Cell Counting Kit-8 assay. Cell migration in (C) A427 and (D) A549 cells was determined using a Transwell assay. Cell invasion in (E) A427 and (F) A549 cells was determined using a Matrigel assay. Cell viability of (G) A427 and (H) A549 cells exposed to curcumin was measured using an MTT assay. *P<0.05 vs. control (0 µM).

Expression levels of miR-192-5p and c-Myc are altered following curcumin treatment in NSCLC

To determine the potential association between miR-192-5p and c-Myc in NSCLC, RT-qPCR and western blotting were used to quantify their expression levels in both A427 and A549 cells exposed to different concentrations of curcumin (10, 20 and 40 µM). Curcumin treatment led to a significant concentration-dependent increase in miR-192-5p expression levels compared with the control (Fig. 2A and B), whereas the expression levels of c-Myc at both the mRNA (Fig. 2C and D) and protein level (Fig. 2E and F) were significantly decreased in a dose-dependent manner in both cell lines compared with the control. These findings suggested that miR-192-5p expression was increased and c-Myc expression was decreased in NSCLC cells following curcumin exposure, which may contribute to curcumin-induced NSCLC cell cytotoxicity and suppression of cell migration and invasion. Subsequently, the role of miR-192-5p and c-Myc in the role of curcumin in NSCLC cells were further investigated, and 20 µM of curcumin was used (Figs. 1 and 2).

Figure 2. Effects of curcumin on the expression levels of miR-192-5p and c-Myc in non-small-cell lung cancer. A427 and A549 cells were treated with 10, 20 or 40 µM curcumin for 48 h. Effects of curcumin treatment on miR-192-5p expression levels in (A) A427 and (B) A549 cells were analyzed using RT-qPCR. Effects of curcumin treatment on c-Myc expression levels in (C) A427 and (D) A549 cells were analyzed using RT-qPCR. Effects of curcumin on c-Myc protein expression levels in (E) A427 and (F) A549 cells were determined using western blotting. *P<0.05 vs. control (0 µM). miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.

miR-192-5p knockdown reverses the inhibitory effects of curcumin on NSCLC cell proliferation, migration and invasion

The dysregulation of miR-192-5p expression following curcumin treatment was confirmed in the human NSCLC cell lines, A427 and A549. Thus, the functions of miR-192-5p following its knockdown by transfection in curcumin-induced anticancer activity were further investigated. Following the transient transfection of the in-miR-192-5p into A427 and A549 cells, a significant decrease was observed in miR-192-5p expression levels in A427 and A549 cells compared with the in-miR-NC-transfected cells, as indicated by RT-qPCR (Fig. S1A). The increased miR-192-5p expression levels induced by curcumin were impaired following the exogenous administration of in-miR-192-5p (Fig. 3A). In addition, following 20 µM curcumin treatment, in-miR-192-5p-transfected cells demonstrated significantly increased proliferation rates in A427 and A549 cells compared with the curcumin + in-miR-NC group (Fig. 3B). The curcumin induced a decrease in cell migratory and invasive abilities in A427 and A549 cells, which was significantly reversed by miR-192-5p inhibition (Fig. 3C and D). These results indicated that the beneficial effects of curcumin were partially abolished by miR-192-5p knockdown, suggesting that miR-192-5p upregulation may be behind the underlying anticancer role of curcumin in NSCLC.

Figure 3. Influence of miR-192-5p knockdown on curcumin-induced NSCLC progression. A427 and A549 cells were transfected with in-miR-192-5p or miR-NC and the effect of the in-miR-192-5p on NSCLC cells treated with 20 µM curcumin was investigated. (A) Expression levels of miR-192-5p in each group were detected in A427 and A549 cells using reverse transcription-quantitative PCR. (B) Cell proliferation of each group was determined in A427 and A549 cells using a Cell Counting Kit-8 assay. (C) Cell migratory ability in each group was determined in A427 and A549 cells using a Transwell assay. (D) Invasive ability in each group was determined in A427 and A549 cells using a Matrigel assay. *P<0.05. miR, microRNA; NSCLC, non-small-cell lung cancer; NC, negative control; in, inhibitor.

c-Myc is a direct target of miR-192-5p

Accumulating evidence has reported that c-Myc undertakes important roles in the numerous biological functions of miRNAs in NSCLC (26–28); however, the regulatory effect of miR-192-5p on c-Myc remains largely unknown. In this study, a potential binding site between miR-192-5p and c-Myc was identified using computational prediction on the TargetScanHuman database (Fig. 4A). Then, c-Myc-WT and c-Myc-MUT 3′UTRs were cloned into pMIR vectors, and a dual-luciferase reporter assay was used to demonstrate that the relative luciferase activity of c-Myc-3′UTR-WT in 293T cells was significantly reduced following co-transfection with miR-192-5p mimic compared with the miR-NC group (Fig. 4B), whereas the relative luciferase activity was significantly increased following co-transfection with in-miR-192-5p compared with the in-miR-NC group (Fig. 4C). The miR-192-5p mimic transfection was also validated; a significant increase in miR-192-5p expression levels was observed in A427 and A549 cells in the miR-192-5p mimic-transfected cells compared with the miR-NC group, as indicated by RT-qPCR (Fig. S1B). However, there were no significant differences in c-Myc-3′UTR-MUT-transfected cells between the NCs- and inhibitor/mimic-transfected cells (Fig. 4B and C). A RIP assay further validated the existence of a binding site between miR-192-5p and c-Myc (Fig. 4D), and western blotting demonstrated that c-Myc expression was significantly inhibited by the miR-192-5p mimic in A427 and A549 cells compared with the miR-NC group (Fig. 4E). These results suggested that miR-192-5p may negatively regulate c-Myc expression through target binding.

Figure 4. Experimental validation of c-Myc as a target gene of miR-192-5p. (A) Putative target binding site (red) of miR-192-5p in c-Myc was determined using computational predictions on the TargetScanHuman database. (B and C) Dual-luciferase reporter assay was used to determine the relative luciferase activities of the c-Myc-3′UTR-WT/MUT in 293T cells following the co-transfection with the (B) miR-192-5p mimic or miR-NC or (C) in-miR-192-5p or in-miR-NC. (D) RNA immunoprecipitation assay was performed to further identify c-Myc expression levels in 293T cells transfected with miR-192-5p mimic/NC. (E) Western blotting was used to analyze the effect of miR-192-5p expression on the expression levels of c-Myc in A427 and A549 cells. *P<0.05 vs. miR-NC or in-miR-NC. miR, microRNA; UTR, untranslated region; WT, wild-type; MUT, mutant; NC, negative control; in, inhibitor; Ago2, argonaute 2.

Curcumin exerts its anticancer role through the miR-192-5p/c- Myc axis in NSCLC in vitro

Rescue experiments were used to clarify the role of c-Myc in mediating the biological action of miR-192-5p in A427 and A549 cells treated with curcumin. The transfection of in-miR-192-5p in both cell lines under curcumin treatment significantly increased the expression levels of c-Myc compared with the in-miR-NC group (Fig. 5A), whereas the co-transfection of in-miR-192-5p and si-c-Myc resulted in a significant decrease in c-Myc expression levels compared with the in-miR-192-5p + si-NC group. In addition, in cells transfected with in-miR-192-5p, cell proliferation was significantly increased compared with the in-miR-NC group (Fig. 5B); however, in combination with si-c-Myc, this increase was subsequently blocked (Fig. 5B). The downregulation of c-Myc expression in in-miR-192-5p-transfected cells also partly abolished the promotive effects of in-miR-192-5p transfection on cell migration and invasion (Fig. 5C and D). The transfection efficiency of si-c-Myc was assessed using RT-qPCR and western blotting (Fig. S1C and D). Moreover, pcDNA-c-Myc resulted in the overexpression of c-Myc (Fig. S1E and F), and this transfection also counteracted the suppressive effect of miR-192-5p overexpression on c-Myc expression and the abilities of cell proliferation, migration and invasion in curcumin-treated cells (Fig. S2A-D). These results suggested that the miR-192-5p/c-Myc axis may mediate the anticancer effects of curcumin in NSCLC.

Figure 5. Effect of c-Myc genetic knockdown on the curcumin/miR-192-5p axis in non-small-cell lung cancer progression. A427 and A549 cells were transfected with in-miR-192-5p or in-miR-NC and co-transfected with si-c-Myc or si-NC. (A) Expression levels of c-Myc were determined in A427 and A549 cells using reverse transcription-quantitative PCR. (B) Cell proliferation in A427 and A549 cells using a Cell Counting Kit-8 assay. Rate of (C) cell migration and (D) invasion in A427 and A549 cells were determined using a Transwell assay or Matrigel assay, respectively. *P<0.05. miR, microRNA; in, inhibitor; NC, negative control; si, small interfering RNA.

Curcumin inactivates the Wnt/β-catenin signaling pathway by upregulating miR-192-5p expression in NSCLC cells in vitro

To determine whether curcumin affected the Wnt/β-catenin signaling pathway through miR-192-5p, the expression of Wnt/β-catenin signaling pathway-related proteins was detected in A427 and A549 cells transfected with in-miR-192-5p followed by treatment with 20 µM curcumin for 48 h using western blotting. The expression levels of β-catenin, cyclin D1 and c-Myc were significantly reduced by curcumin treatment alone compared with the control group (Fig. 6A and B); however, these decreased expression levels were subsequently significantly reversed in cells treated with curcumin and transfected with in-miR-192-5p compared with the curcumin + in-miR-NC group. These findings suggested that the curcumin-induced inactivation of the Wnt/β-catenin signaling pathway in NSCLC may depend on the high expression levels of miR-192-5p.

Figure 6. Role of miR-192-5p in the Wnt/β-catenin signaling pathway in non-small-cell lung cancer following curcumin treatment. A427 and A549 cells were transfected with in-miR-192-5p and the effect of miR-192-5p knockdown on the Wnt/β-catenin signaling pathway-related proteins were determined following 20 µM curcumin treatment. Expression levels of β-catenin, cyclin D1 and c-Myc were detected in (A) A427 and (B) A549 cells using western blotting. *P<0.05. miR, microRNA; in, inhibitor; NC, negative control.

Discussion

Curcumin, isolated from the rhizomes of the plant Curcuma longa, has been reported to exhibit various anti-inflammatory, antiangiogenic, antiproliferative and antioxidant properties (29,30). It has been suggested that curcumin may induce apoptosis in malignant cells, and therefore, it demonstrates great potential as a cancer treatment (31). In addition, several studies have observed that curcumin exerts growth suppressive effects in both small cell lung cancer and NSCLC cells (32,33); however, the effect of curcumin in NSCLC, in addition to its associated mechanism of action, remain poorly understood. The present study confirmed the cytotoxicity of curcumin in human NSCLC cell lines A427 and A549, and curcumin was found to inhibit cell proliferation, migration and invasion in a dose-dependent manner. Moreover, the expression levels of miR-192-5p and c-Myc were discovered to be increased and decreased, respectively, following curcumin exposure, which suggested that miR-192-5p upregulation may be an important event for the curcumin-mediated antiproliferative, antimigratory and anti-invasive effects.

The close interaction between miRNAs and the anti-lung cancer role of curcumin has been previously reported. For example, Zhang et al (34,35) demonstrated that curcumin promoted apoptosis in cisplatin-resistant A549 cells through miRNA signaling pathways, including the downregulation of miR-186 expression. Zhan et al (36) constructed a miRNA gene network that is attributed to the antimetastatic role of curcumin in A549 cells, such as let-7a-3p, miR-1262 and miR-330-5p. Similarly, Jiao et al (37) identified that curcumin induces metastasis inhibition depending on a miRNA/transcription factor/target gene network, including the miR-34a-5p/miR-34c-5p/miR-302b-3p/lymphoid enhancer-binding factor 1/cyclin D1/Wnt1/c-Myc axis. Moreover, curcumin has also been found to be effective in increasing paclitaxel sensitivity in cancer stem cells, which may occur by interacting with miRNAs (38,39). In the present study, the increased expression levels of miR-192-5p initiated the antiproliferative, antimigratory and anti-invasive roles of curcumin in A549 and A427 cells by targeting c-Myc expression. Taken together, these results suggested that curcumin could be an epigenetic agent that could provide a new therapeutic strategy for cancer treatment, and the miRNAs/genes network, including the miR-192-5p/c-Myc axis, may serve a vital role in the anticancer activity of curcumin in lung cancer.

Previous studies have shown that the increased expression levels of miR-192-5p contribute to curcumin-induced antitumor effects in NSCLC (13,14). To date, the tumor-suppressive activities of curcumin have mainly focused on its ability to promote cell apoptosis, and enhance chemo- and radiosensitization, in addition to its ability to inhibit cell proliferation and migration in lung cancer cells, and modulate miRNAs (11,40). For example, antagonizing miR-192-5p was found to attenuate curcumin-stimulated apoptosis promotion in H460, A427 and A549 cells through the p53/miR-192-5p/X-linked inhibitor of apoptosis protein axis (13), of which miR-192-5p was identified as one of the most responsive miRNAs following curcumin treatment. Another study reported that the ectopic expression of miR-192-5p enhanced the effects of curcumin on the inhibition of cell viability and the promotion of apoptosis in A549 cells through the PI3K/AKT signaling pathway (14). Nonetheless, to the best of our knowledge, numerous roles of miR-192-5p exist in NSCLC progression, which have not been investigated in detail, such as migration and invasion. The present study suggested that the upregulation of miR-192-5p contributed to the curcumin-induced antiproliferative effects in both A549 and A427 cells. Furthermore, the migratory and invasive abilities of the cells were also significantly inhibited by curcumin treatment, whereas miR-192-5p inhibition could partially reverse the curcumin-induced antimigration and anti-invasion effects through inactivation of the Wnt/β-catenin signaling pathway by decreasing the expression levels of β-catenin, cyclin D1 and c-Myc.

As a well-documented oncogene, c-Myc has been known to be extensively involved in cell cycle progression and apoptosis (41). The dysregulation of this gene has been associated with multiple types of cancer, including NSCLC (42): For example, it was reported that c-Myc was a major target for miR-145, which negatively mediated eukaryotic translation initiation factor 4E, a downstream target of c-Myc, in NSCLC (25); epidermal growth factor receptor was discovered to promote lung tumorigenesis through activating c-Myc, which in turn stimulated miR-7 expression (43); and increased expression levels of c-Myc were demonstrated to reverse the inhibitory effect of miR-449c on NSCLC tumor growth in vivo (44). In addition, cell proliferation and invasion in A549 cells were also altered by the miR-376a/c-Myc axis in a previous study (45). Therefore, to investigate whether c-Myc was a direct target gene of miR-192-5p, a dual-luciferase reporter assay and RIP were performed; it was observed that c-Myc was negatively regulated by miR-192-5p, and that the overexpression of c-Myc relieved the inhibition of miR-192-5p on A427 and A549 cell proliferation, migration and invasion. However, the anticancer effect of miR-192-5p induction through c-Myc knockdown was only investigated in the human NSCLC cell lines, A427 and A549, in the present study, and to the best of our knowledge, there is currently no published xenograft experiments that have focused on the increased expression levels of miR-192-5p as the underlying mechanism of the curcumin-mediated tumor-suppressive role (11,13,14).

The Wnt pathway, especially the Wnt/β-catenin dependent pathway is the main target for therapeutic interventions in cancer types, including lung cancer (46,47). Activating the Wnt/β-catenin leads to the accumulation of β-catenin and further promotes oncogenes, including c-Myc and Cyclin D1 (48). Curcumin can inhibit the Wnt/β-catenin pathway in lung cancer stem cells and lung cancer cells (15,16). Moreover, miR-192-5p is also associated with the Wnt/β-catenin pathway in malignant tumors (49,50). However, the relationship between curcumin, miR-192-5p and the Wnt/β-catenin pathway have not been previously investigated. Thus, the present results suggested that miR-192-5p upregulation mediated the antitumor role of curcumin in NSCLC by activating the Wnt/β-catenin pathway and targeting c-Myc.

In conclusion, the present study demonstrated that curcumin exhibited antiproliferative and antimigratory and anti-invasive properties in NSCLC in vitro, which were largely mediated through the increased expression levels of miR-192-5p by targeting c-Myc and inhibiting the Wnt/β-catenin signaling pathway. These findings suggested a novel mechanism for the anticancer function of curcumin and suggested that the restoration of miR-192-5p could be used as a novel therapeutic approach for NSCLC treatment.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

YP and CZ conceptualized and designed the experiments, analyzed the data, and prepared the original draft of the manuscript. YS and ZL performed the experiments, collected and validated the data, and revised it critically for important intellectual content prior to reviewing and editing. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References