Res was purchased from Tocris Bioscience (Bristol, UK). High-glucose Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, penicillin/streptomycin, Trypsin-EDTA and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Dimethyl sulfoxide (DMSO; cat. no. D2650), Aza (cat. no. A3656), and antibodies against ZFP36 (cat. no. T5327) and β-actin (cat. no. A5441) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Antibodies against DNMT1 (cat. no. Ab19905), VEGFA (cat. no. Ab46154) and MYC (cat. no. Ab32072) were purchased from Abcam (Cambridge, MA, USA). The antibody against Cyclin D1 (CCND1; cat. no. 2978S) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

A549 and H23 human lung cancer cells line was obtained from American Type Culture Collection (Manassas, VA, USA). A549 cells were grown in high-glucose DMEM and H23 cells were grown in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured at 37°C in a 5% CO₂ humidified incubator.

A549 and H23 cells were seeded in 100-mm dished at 1×10⁶ cells/dishes. Cells were allowed to adhere overnight at 37°C and then were treated with the appropriate concentration of control [0.1% DMSO (0 µM)] or Aza (5 µM) for 72 h at 37°C.

A549 cells were seeded in 100-mm dished at 1×10⁶ cells/dishes. Cells were allowed to adherent overnight at 37°C and then treated with the appropriate concentration of control [0.1% DMSO (0 µM)] or Res (20, 50 and 100 µM) for 72 h at 37°C.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was added to cell lysates, according to the manufacturer's protocols. The RT-PCR was performed using a Moloney murine leukemia virus reverse transcriptase kit (cat. no. 30201; Beams Biotechnology Co., Ltd., Seognam, Korea). Complementary DNA synthesis using an iCycler thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was performed by incubating at 37°C for 90 min. RT-qPCR analysis was performed using TOPreal™ SYBR^®-Green (Enzynomics, Daejeon, Korea) and the following program in a Real-Time PCR Instrument Light Cycle^® 480 (Roche Applied Science, Madison, WI, USA): 95°C for 5 min, followed by 45 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. The 2^−ΔΔCq method was used to calculate the relative levels of target mRNAs and GAPDH was used as a reference gene (21). The primer sequences were used as follows: ZFP36, forward, 5′-TGGGATCCGACCCTGATGAA-3′ and reverse, 5′-AAAACTCCCGCCTCGAAGAC-3′; MYC, forward, 5′-AGAGTTTCATCTGCGACCCG-3′ and reverse, 5′-AAGCCGCTCCACATACAGTC-3′; CCND1, forward, 5′-TGCCAACCTCCTCAACGAC-3′ and reverse, 5′-TTTGAAGTAGGACACCGAGGG-3′; VEGFA, forward, 5′-AGGGAAAGGGGCAAAAACGA-3′ and reverse, 5′-GAGGCTCCAGGGCATTAGAC-3′; DNMT1, forward, 5′-TACCTGGACGACCCTGACCTC-3′ and reverse, 5′-TACCTGGACGACCCTGACCTC-3′; GAPDH, forward, 5′-ACGCCACAGTTTCCCGGAGG-3′ and reverse, 5′-GCACCCCTGGCCAAGGTCAT-3′.

Cells were harvested, centrifuged at 890 × g for 1 min at 4°C and lysed with radioimmunoprecipitation buffer [25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS; Thermo Fisher Scientific, Inc.] containing a protease inhibitor cocktail [4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride, aprotinin, bestatin hydrochloride-aminopeptidases, N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide, EDTA and leupeptin hemisulfate salt; Sigma-Aldrich; Merck KGaA]. Total protein concentration was determined using a Pierce Bicinchoninic Acid Assay kit (Thermo Fisher Scientific, Inc.). Equivalent quantities of total protein (20–30 µg) were separated by SDS-PAGE on 8–10% polyacrylamide gels, and then transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Little Chalfont, UK) using a Trans-Blot^® SD Semi-Dry Electrophoretic Transfer Cell (cat. no. 170-3940; Bio-Rad Laboratories, Inc.) submerged in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine and 20% methanol). The membrane was blocked with 5% skimmed milk in 0.1% Tween-20/Tris-buffered saline (TBST) for 2 h at room temperature, and then incubated with primary antibodies for ZFP36, DNMT1, VEGFA, MYC, CCND1 (1:1,000) and mouse monoclonal anti-β-actin (1:10,000) at 4°C overnight. Subsequently, the blots were washed three times in TBST and incubated with goat anti-rabbit horseradish peroxidase (HRP) conjugate (cat. no. 31430; Thermo Fisher Scientific, Inc.) and goat anti-mouse HRP conjugate (cat. no. 31460; Thermo Fisher Scientific, Inc.) IgG secondary antibodies (1:10,000; Thermo Fisher Scientific, Inc.) for 50 min at room temperature. The specific antibody signals were detected by chemiluminescence (Advansta, Inc., San Jose, CA, USA) using a LAS 3000 instrument (Fujifilm, Tokyo, Japan).

Cells were seeded in 100-mm dishes at 1×10⁶ cells/dishes. Cells were allowed to adhere overnight at 37°C and were then treated with the appropriate concentration of control [0.1% DMSO (0 µM)], Res (100 µM) or Aza (5 µM) for 72 h 37°C. Following treatment, cells were lysed in buffer containing 10 mM Tris-HCl (pH 8), 100 mM NaCl, 10 mM EDTA (pH 8), 10% Triton X-100 and proteinase K overnight at 56°C. Subsequently, NaCl (6 M) was added to the cells, and then lysates were cleared by centrifugation at 4,700 × g for 10 min at 4°C. A total of 600 µl isopropanol was used for extraction of DNA from the supernatant. To analyze CpG methylation of the ZFP36 promoter, the region (+63 to −894) was selected due to this region containing numerous transcription factor binding sites (5,6). Among 46 CpG islands, MS1-3 and MS4 were selected for Acc II and Hap II restriction enzymes, respectively. Subsequently, 1 µg genomic DNA was digested with 10 U Acc II (cat. no. 1002A, Takara Bio, Inc., Osaka, Japan) or Hap II (cat. no. 1053A, Takara Bio, Inc.) overnight at 37°C prior to qPCR (21). The primer sequences used were as follows: Methylation Site 1 (MS1), forward, 5′-GAAGGGAACCAGTCCAGGG-3′ and reverse, 5′-CCGAGAGCCGGCTACTTATAG-3′; MS2, forward, 5′-CTCGGTCACGGCTGTCC-3′ and reverse, 5′-CTGGACTGGTTCCCTTCCG-3′; MS3, forward, 5′-CCCCATCCGTCTGTGTCG-3′ and reverse, 5′-TGTAGAAGGAAACTGGGGCG-3′; and MS4, forward, 5′-CTCAGGTAATCCACCTGCCTC-3′ and reverse, 5′-TACAGGTGTGAGCCACCAAG-3′.

The migration assay was conducted using a culture insert (IBIDI, LLC, Verona, WI, USA), according to the manufacturer's protocols. Briefly, cells were seeded in culture-insert 4 well at 7×10⁴ cells/well. Cells were allowed to adhere for 24 h at 37°C and then the culture insert was removed, leaving gaps in the sheets of cells. Different concentrations of control [0.1% DMSO (0 µM)] and Res (20, 50, and 100 µM) were used to treat cells for 48 h 37°C. After 48 h, the area destitute of cells was analyzed using a Carl Zeiss microscope (Olympus TH4 200; Carl Zeiss AG, Oberkochen, Germany).

For the MTS assay, cells were seeded in 96-well plates at 3×10³ cells/well and then cells were allowed to adhere overnight at 37°C. Different concentrations of control [0.1% DMSO (0 µM)] and Res (20, 50 and 100 µM) were used to treat cells for 48 h at 37°C. Following the indicated treatments, a total of 20 µl CellTiter 96^® AQueous One Solution cell proliferation assay reagent (Promega Corporation, Madison, WI, USA) was added to each well, according to the manufacturer's protocols, and absorbance was measured at 490 nm using an Infinite M200 Pro microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland).

Data are presented as the mean ± standard error of the mean. Statistical significance was determined using the Student's t-test or one-way analysis of variance was performed using Dunnett's post test (GraphPad Prism; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To examine the methylation of ZFP36 promoter regions, methylated CpG island site were predicted and MS1-4 regions were designated up to restriction enzyme availabilities (Fig. 1). To determine the effects of ZFP36 demethylation, A549 and H23 cells were firstly treated with Aza, and it was determined that Aza significantly increased ZFP36 mRNA and protein, compared with the control group (P<0.001; Fig. 2A and B). Increased ZFP36 expression following Aza treatment indicated that ZFP36 expression is epigenetically regulated in A549 and H23 lung cancer cells. Using enzyme restriction sensitive methylation analysis with Acc II and Hap II, which recognizes and cleaves CGCG and CCGG sequences, respectively, ZFP36 promoter methylation was examined. Epigenetic analysis demonstrated that Aza significantly reduces methylation at MS1-4 in A549 cells, and MS2 and 4 in H23 cells, compared with the control group (P<0.05 and P<0.001, respectively; Fig. 2C and D).

Recently, Res was demonstrated to induce ZFP36 expression in U87MG human glioblastoma cells (17). To determine whether these effects also occur in lung cancer cells, ZFP36 expression was analyzed in A549 cells following treatment with Res, and it was determined that ZFP36 mRNA levels and protein expression were significantly increased, compared with the control group (P<0.001; Fig. 3A and B). To analyze whether Res demethylates CpG islands in the ZFP36 promoter, A549 lung cancer cells were treated with Res and genomic DNA was isolated to determine its methylation status. Notably, it was determined that Res significantly reduced methylation at MS1, 3 and 4 of the ZFP36 promoter region, compared with the control group (P<0.001; Fig. 3C). These data indicate that Res activates ZFP36 mRNA expression by demethylating CpG islands in the promoter region.

To investigate whether Res-induced ZFP36 expression affects downstream ARE-mRNA decay, the expression of ZFP36 target gene was examined following Res treatment. A549 lung cancer cells were treated with Res, and the transcript and protein expression of CCND1, MYC and VEGFA were determined by RT-qPCR and western blotting, respectively. As expected, it was determined that Res treatment significantly decreased mRNA and protein expression of these ZFP36 targets, compared with the control group (P<0.001; Fig. 4A and B).

To investigate whether DNMT1 is involved in regulation of ZFP36 methylation by Res, RT-qPCR and western blotting were performed to detect DNMT1 mRNA and protein levels in A549 lung cancer cells, respectively. It was determined that Res treatment significantly reduced mRNA and protein levels, compared with the control group (P<0.001; Fig. 5A and B) of DNMT1, indicating that Res regulates the downregulation of DNMT1 expression in A549 lung cancer cells.

To investigate the migration and anticancer activity of Res on A549 and H23 lung cancer cells, cell migration and MTS assays were performed following Res treatment. It was determined that Res significantly reduced the migration of A549 and H23 cells, compared with the control group (P<0.001), as assessed with a wound-healing assay. Additionally, further examination demonstrated Res significantly inhibited the proliferation of A549 and H23 lung cancer cells in a concentration-dependent, compared with the control group (P<0.001; Fig. 6A and B).