Effects of Zebularine on Invasion Activity and Intracellular Expression Level of let-7b in Colorectal Cancer Cells

Abstract

The effects of zebularine, a DNA methyltransferase inhibitor, on the invasion activity as well as intracellular expression level of let-7b, tumor suppressor microRNA, were examined in three human colorectal cancer (CRC) cell lines: SW480, SW620, and oxaliplatin-resistant SW620 (SW620/OxR). Zebularine suppressed the invasion activity of SW620 and SW620/OxR cells. The intracellular expression level of let-7b was up-regulated by zebularine in SW620 and SW620/OxR cells. The overexpression of let-7b by the transfection of let-7b mimic suppressed invasion activity in SW620 and SW620/OxR cells. These results suggest that zebularine may inhibit invasion activity by up-regulating the intracellular expression level of let-7b in high-invasive CRC cells.

DNA methylation is a major epigenetic mechanism of gene expression level. Zebularine (Zeb) has been shown to inhibit DNA methyltransferase (DNMT). Furthermore, Zeb and other DNMT inhibitors, such as decitabine (DAC) and azacitidine (AC), exert differential effects on gene expression levels.¹⁾ Flotho et al. reported that Zeb did not regulate the expression levels of a large number of mRNAs or markers for genome-wide methylation.¹⁾ We previously showed that DAC suppressed invasion activity with the up-regulation of epithelial cell marker expression levels not only in parental SW620 cells, high-invasive colorectal cancer (CRC) cells, but also in oxaliplatin (L-OHP)-resistant SW620 (SW620/OxR) cells.²⁾ Pakneshan et al. demonstrated that AC enhanced invasion activity by up-regulating the urokinase plasminogen activator in prostate cancer.³⁾ However, information on the effects of Zeb on invasion activity is limited.

MicroRNAs (miRNAs), small non-coding RNAs that suppress target mRNA, play important roles in cancer metastasis. Members of the let-7 family (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i in addition to miR-98 and miR-202) are tumor suppressor miRNAs.⁴⁾ Kang et al. previously reported that let-7b suppressed invasion activity in gastric cancer.⁵⁾ In addition, Kahlert et al. showed that let-7b expression level in CRC tumor invasion front was lower than that in CRC tumor center.⁶⁾ The expression levels of let-7 family members are also regulated by DNA methylation. The location of let-7b is in the 3′-untranslated region of MIRLET7BHG as a long intergenic noncoding RNA.⁷⁾ Nishi et al. found that intracellular expression level of let-7b may be regulated by the DNA methylation status of CpG islands near transcription start site (TSS) of MIRLET7BHG.⁷⁾ Kobayashi et al. showed that let-7 family members were more abundant in exosomes, small membrane vesicles of approximately 100 nm in diameter, from high-invasive ovarian cancer cells than in those from low-invasive ovarian cancer cells.⁸⁾ In addition, Wong et al. reported that the expression levels of let-7 family members were up-regulated by Zeb in nasopharyngeal carcinoma cells.⁹⁾

SW480 cells (low-invasive CRC cells) and SW620 cells (high-invasive CRC cells) were derived from primary CRC and lymph node metastasis, respectively, in the same patient.¹⁰⁾ We previously demonstrated that the invasion activity of SW620/OxR cells was higher than that of SW620 cells.¹¹⁾ In the present study, we examined the effects of Zeb on invasion activities and the intracellular and exosomal expression levels of let-7 family members in these three CRC cell lines.

MATERIALS AND METHODS

Materials

Zeb was purchased from Tokyo Chemical Industry (Tokyo, Japan). L-OHP, DAC, and AC were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The primers used for the PCR in this study were purchased from Invitrogen (Carlsbad, CA, U.S.A.). Monoclonal mouse anti-heat shock protein 70 (HSP70), monoclonal mouse anti-β-actin, goat anti-rabbit immunoglobulin G-horseradish peroxidase (IgG-HRP), and goat anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Monoclonal rabbit anti-E-cadherin and monoclonal rabbit anti-vimentin were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.).

Cell Culture

The human CRC cell lines SW480 (DS Pharmabiomedical, Osaka, Japan), SW620 (American Tissue Culture Collection, Manassas, VA, U.S.A.), and SW620/OxR developed from SW620 cells as previously reported¹¹⁾ were maintained in Leibovitz 15 medium (Life Technologies Corp., Carlsbad, CA, U.S.A.) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, U.S.A.), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C with 100% air. SW620/OxR cells were maintained in the presence of 80 µM L-OHP and were cultured without L-OHP for 1 passage before experiments.¹¹⁾

Drug Treatment

DNMT inhibitors were previously reported to induce the demethylation of epigenetically silenced genes at less toxic concentrations, causing a decrease of approximately 25% in cell proliferation, in various cancer cell lines.¹⁾ In our preliminary study, the effects of DNMT inhibitors on cell proliferation were evaluated according to the sigmoid inhibitory effect model (data not shown). The proliferation of SW480, SW620, and SW620/OxR cells was decreased by 10-30% following a treatment with 150 µM Zeb, 2 µM DAC, or 3 µM AC for 72 h (Supplementary Fig. S1). Therefore, these concentrations of DNMT inhibitors were used in the present study.

Real-Time RT-PCR for mRNAs

Total RNA was isolated from cultured cells using RNeasy Mini kit (QIAGEN, Valencia, CA, U.S.A.). Real-time RT-PCR using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan) and SYBR Green was performed as previously described.¹¹⁾ The expression level of each mRNA was normalized to that of Ribosomal protein 27 (RPL27) using the 2^−ΔΔCt method. The sequences of the primers used for mRNA expression levels in this study are listed in Supplementary Table S1.

Invasion Assay

A transwell invasion assay using 8.0-µm Transwell inserts coated with Matrigel (Corning, New York, NY, U.S.A.) was performed as previously described.¹¹⁾ Invasion cells present on the lower surface of the insert were stained with Diff-Quik reagent (Sysmex, Kobe, Japan) and counted at ×200 magnification. Number of cells in 5 different fields of each membrane were counted and averaged.

Real-Time RT-PCR for Intracellular miRNAs

Total RNA (<200 nucleotides) was isolated using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan). Real-time RT-PCR using Mir-X miRNA First Strand Synthesis Kit (TaKaRa Bio Inc., Otsu, Japan) was performed as previously described.¹¹⁾ The expression level of each miRNA was normalized to that of U6 snRNA using the 2^−ΔΔCt method. The sequences of the primers used for miRNA expression levels in this study are listed in Supplementary Table S2. The U6 primer was included in the Mir-X miRNA First Strand Synthesis Kit.

Real-Time RT-PCR for Exosomal miRNAs

After a stepwise centrifugation-ultracentrifugation method, as described in our previous study,²⁾ exosomal RNA was isolated using Total Exosome RNA & Protein Isolation Kit (Invitrogen, Carlsbad, CA, U.S.A.). The presence of the HSP70 protein, an exosomal marker, was confirmed by a Western blot analysis of the pellet after ultracentrifugation (Supplementary Fig. S2). The maximum volume of exosomal RNA was reverse-transcribed into cDNA using miScript Reverse Transcription Kit (QIAGEN). cDNA was amplified using miScript SYBR Green Kit (QIAGEN). The expression level of each miRNA was normalized to that of RNU6B snRNA using the 2^−ΔΔCt method. The expression level of RNU6B was analyzed by Human RNU6B miScript Primer Assay (QIAGEN). Supplementary Table S2 shows the primer sequences used in this study.

miRNA Mimic Transfection

SW620 or SW620/OxR cells were transfected with hsa-let-7b-5p mimic (let-7b mimic-transfected cells) or miRNA mimic Negative Control #1 (negative control-transfected cells) (Bioneer, Seoul, Korea) as a control at a final concentration of 10 nM. Transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s protocol. After 24 h, cells were used in subsequent experiments.

Western Blot Analysis

Whole cell proteins were lysed by M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, Waltham, MA, U.S.A.). A Western blot analysis using 10% or 4–12% NuPAGE Bis-Tris gel for HSP70 and β-actin or E-cadherin and vimentin, respectively, iBlot (Invitrogen), and Blocking One (Nacalai Tesque) was performed as previously described.¹¹⁾ Bound antibodies were visualized by Chemi-Lumi One Super (Nacalai Tesque). Density of each band was quantified with Image J, version 1.47, and was normalized to that of β-actin. The relative protein expression levels in treated cells, which were compared with that in control cells, were presented.

Statistical Analysis

Results are expressed as the mean±standard error of the mean (S.E.M.). Differences between two groups were analyzed by the unpaired Student’s t-test. A one-way ANOVA followed by the Student–Newman–Keuls test was used to compare more than two groups. Correlations between parameters were examined by Pearson’s correlation test.

RESULTS

Effects of DNMT Inhibitors on a DNA Genome-Wide Demethylation Marker

The mRNA expression level of Melanoma-associated antigen 1 (MAGEA1), a genome-wide DNA demethylation marker,¹²⁾ was not detected in control or Zeb-treated SW480, SW620, and SW620/OxR cells. In contrast, the mRNA expression level of MAGEA1 was detected in the three DAC or AC-treated CRC cell lines (Fig. 1).

Fig. 1. Effects of DNMT Inhibitors on MAGEA1 mRNA Expression Levels in CRC Cells

The mRNA expression level of MAGEA1 from cultured cells after a 72-h treatment with 150 µM Zeb, 2 µM DAC, or 3 µM AC was analyzed. The expression level of each mRNA was normalized to that of RPL27 using the 2^−ΔΔCt method. Each column represents the mean±S.E.M. of three independent experiments. ND: not detected.

Suppression of Invasion Activity by Zeb

The invasion activity of SW620/OxR cells was higher than that of SW620 cells, while the invasion activity of Zeb or DAC-treated SW620 and SW620/OxR cells was lower than that of control SW620 and SW620/OxR cells (Table 1). In SW620 and SW620/OxR cells, invasion activity was significantly enhanced by AC (Table 1). Since epithelial–mesenchymal transition (EMT) is associated with CRC metastasis, we examined the expression levels of EMT-related molecules. Consistent with previous findings,²⁾ DAC increased the expression levels of the epithelial cell markers (E-cadherin and miR-200 family members¹³⁾) in SW620 and SW620/OxR cells (Supplementary Fig. S3). However, Zeb significantly decreased the expression level of E-cadherin and did not significantly change the expression levels of the mesenchymal cell marker (vimentin) and miR-200 family in SW620 cells. Zeb did not regulate the expression levels of E-cadherin, vimentin, and miR-141 in SW620/OxR cells, although Zeb significantly increased the expression level of miR-200c in SW620/OxR cells (Supplementary Fig. S3). From these results, Zeb might suppress the invasion activity of SW620 and SW620/OxR cells without inhibitory effect on EMT.

Table 1. Effects of DNMT Inhibitors on Invasion Activity of CRC Cells

Cell line	Invasion cells/high-power field (×200)
	Control	Zeb	DAC	AC
SW480	8.3±0.5††	5.1±1.0	2.9±0.3	6.9±0.3
SW620	61.6±4.1	27.5±1.5**	41.5±2.0**	85.7±2.9**
SW620/OxR	87.7±11.1††	30.9±2.1**	40.0±2.8**	122.7±9.1**

Each value represents the mean±S.E.M. of three independent experiments (Student–Newman–Keuls test, ** p<0.01 significantly different from control SW620 or SW620/OxR cells, ^†† p<0.01 significantly different from control SW620 cells).

Effects of Zeb on Intracellular Expression Levels of let-7 Family Members

Zeb up-regulated the intracellular expression level of let-7b in SW620 and SW620/OxR cells, but not in SW480 cells (Fig. 2A). In addition, Zeb up-regulated the intracellular expression levels of let-7c and let-7g in SW620 and SW620/OxR cells, respectively (Fig. 2A). The intracellular expression levels of let-7c and let-7g, as well as let-7b, also showed a negative correlation with invasion activities in the three cell lines treated with or without Zeb (Fig. 2B and Supplementary Fig. S4). The DNA methylation status at the upstream of let-7b coding region (CpG island near TSS of MIRLET7BHG⁷⁾) was not distinctly changed by the treatment of Zeb in the three cell lines (Supplementary Fig. S5). In addition, the band of methylated DNA in control SW620/OxR cells was strong compared with that in control SW480 and SW620 cells (Supplementary Fig. S5).

Fig. 2. Effects of Zeb on Intracellular Expression Levels of let-7 Family Members in CRC Cells (A) and Correlation between Intracellular let-7b Expression and Invasion Activity (B)

(A) The intracellular expression levels of let-7a, let-7b, let-7c, and let-7g from cultured cells after a 72-h treatment with 150 µM Zeb. The expression level of each miRNA was normalized to that of U6 snRNA using the 2^−ΔΔCt method. Each column represents the mean±S.E.M. of three independent experiments (unpaired Student’s t-test, * p<0.05, ** p<0.01 significantly different from control SW620 or SW620/OxR cells). (B) A correlation analysis was performed using the data shown in Fig. 2A and Table 1. Each point represents the mean±S.E.M. of three independent experiments. r: Pearson’s correlation coefficient.

Effects of Zeb on Exosomal Expression Levels of let-7 Family Members

The exosomal expression levels of let-7a, let-7b, and let-7g in SW620 cells showed significantly higher than in SW480 or SW620/OxR cells (Fig. 3). These results suggest that the exosomal expression level of let-7b is associated with invasion activity in SW480 and SW620 cells, consistent with previous findings.⁸⁾ Furthermore, Zeb decreased the exosomal expression levels of let-7b and let-7g in SW620 cells (Fig. 3). The exosomal expression levels of let-7 family members did not correlate with invasion activities in the three cell lines including L-OHP-resistant cell (Supplementary Fig. S6). These results suggested that the intracellular expression levels of let-7 family members were more important for invasion activities than the exosomal expression levels of let-7 family members.

Fig. 3. Effects of Zeb on Exosomal Expression Levels of let-7 Family Members in CRC Cells

The exosomal expression levels of let-7a, let-7b, let-7c, and let-7g from cultured cells after a 72-h treatment with 150 µM Zeb was analyzed. The expression level of each miRNA was normalized to that of RNU6B snRNA using the 2^-ΔΔCt method. Each column represents the mean±S.E.M. of three to four independent experiments (Student–Newman–Keuls test, ** p<0.01 significantly different from control SW620 or SW620/OxR cells, ^†† p<0.01 significantly different from control SW620 cells). Open columns indicate control cells and closed columns indicate Zeb-treated cells.

Effects of let-7b Overexpression on Invasion Activity

Since intracellular expression level of let-7b was up-regulated by Zeb in both SW620 and SW620/OxR cells, we focused on the relationship between invasion activity and intracellular let-7b. The intracellular expression level of let-7b was markedly increased in SW620 and SW620/OxR cells by transfection of the let-7b mimic (Fig. 4A). Invasion activity was lower with the let-7b mimic than the negative control (Fig. 4B). In addition, the let-7b mimic did not significantly increase the expression levels of the epithelial cell markers (E-cadherin, miR-200c, and miR-141) and did not significantly decrease the mesenchymal cell marker (vimentin) in SW620 and SW620/OxR cells (Figs. 4C, D). These results suggest that the up-regulation of intracellular let-7b expression levels may be associated with the inhibition of invasion activity by Zeb. In addition, we thought that intracellular let-7b overexpression might not suppress EMT in SW620 and SW620/OxR cells.

Fig. 4. Effects of let-7b Mimic on Invasion Activity and Intracellular EMT-Related Molecule Expression Levels in CRC Cells

SW620 and SW620/OxR cells were transfected with 10 nM of the let-7b mimic or negative control. (A) Intracellular let-7b expression levels. (B) Invasion activity. (C) EMT-related protein expression levels. Density of each band was quantified with Image J, version 1.47, and was normalized to that of β-actin. (D) Intracellular miR-200 family expression levels. The expression level of each miRNA was normalized to that of U6 snRNA using the 2^−ΔΔCt method. The data were presented relative value which was calculated by dividing each value in let-7b mimic-transfected cells by that in negative control-transfected cells. Each result represents the mean±S.E.M. of three independent experiments (unpaired Student’s t-test, ** p<0.01 significantly different from negative control-transfected SW620 or SW620/OxR cells). Open columns indicate negative control-transfected cells and closed columns indicate let-7b mimic-transfected cells.

DISCUSSION

We herein demonstrated that Zeb suppressed invasion activity and increased the intracellular expression level of let-7b in SW620 and SW620/OxR cells. These results suggest that Zeb inhibited invasion activity by different mechanisms from those of DAC in SW620 and SW620/OxR cells.²⁾ Kang et al. previously reported that let-7b suppressed invasion by silencing AKT signaling.⁵⁾ The targets of other let-7 family members, such as matrix metalloproteinase,¹⁴⁾ may have suppressed invasion activities in SW620 and SW620/OxR cells treated with Zeb in this study.

A previous study suggested that Zeb did not demethylate genome-wide DNA.¹⁾ Similarly, the result of genome-wide DNA demethylation marker expression analysis in this study suggested that Zeb might not demethylate genome-wide DNA. In addition, we investigated the DNA methylation status at the upstream of let-7b coding region (CpG island near TSS of MIRLET7BHG). As shown in Supplementary Fig. S5, the DNA methylation level in SW620/OxR cells was high compared to that in SW480 and SW620 cells. The DNA methylation status at the upstream of let-7b coding region might be involved in the difference of the intracellular let-7b expression level among three cell lines studied. However, since Zeb did not change the DNA methylation status in this study (Supplementary Fig. S5), Zeb might not specifically demethylate the upstream of let-7b coding region. These results suggested that Zeb did not increase the expression level of let-7b by demethylation of genome-wide DNA or the upstream of let-7b coding region in SW620 and SW620/OxR cells. Therefore, Zeb may up-regulate the intracellular expression level of let-7b by other mechanism, such as the specific demethylation of other genes.

Kahlert et al. reported that low expression level of let-7b was observed in invasion front of CRC tissue.⁶⁾ Therefore, let-7b may play an important role in CRC metastasis. The intracellular expression levels of let-7c and let-7g have been associated with the metastasis of colorectal and breast cancers, respectively.^14,15) As shown in Fig. 2A and Supplementary Fig. S4, the intracellular expression levels of let-7c and let-7g were up-regulated by Zeb in SW620 and SW620/OxR cells, respectively, and correlated with invasion activities. These results suggested that intracellular expression levels of let-7c and let-7g, as well as let-7b, may cause suppression of invasion activities in Zeb-treated SW620 and SW620/OxR cells, respectively. In addition, the correlation between the intracellular expression levels of let-7c or let-7g and invasion activity was higher than that of let-7b. We intend to investigate the effects of let-7c mimic or let-7g mimic on invasion activity in our future study.

In SW620 cells, Zeb suppressed the expression level of E-cadherin, which was different from the result of experiments using let-7b mimic in this study. Zeb may up-regulate E-cadherin suppressor, such as miR-9 which is regulated by DNA methylation.^16,17) The down-regulated expression level of E-cadherin causes the acquisition of metastasis phenotype.¹⁸⁾ However, the invasion activity of SW620 cells was suppressed by Zeb. On the other hand, in SW620/OxR cells, Zeb increased the expression level of miR-200c. The increased expression level of miR-200c suppresses EMT by down-regulation of the E-cadherin transcription repressors, ZEB1 and ZEB2.¹⁸⁾ Therefore, the suppression of invasion activity in Zeb-treated cells might be little affected by the change of E-cadherin or miR-200c expression level in this study. Also, we consider that the down-regulation of E-cadherin expression and the up-regulation of miR-200c expression did not mediate EMT in Zeb-treated SW620 and Zeb-treated SW620/OxR cells.

The exosomal expression levels of let-7a, let-7b, and let-7g were higher in SW620 cells than in SW480 cells. The exosomal expression levels of let-7 family members were higher in high-invasive ovarian cancer cells than in low-invasive ovarian cancer cells.⁸⁾ The exosomal expression levels of let-7a, let-7b, and let-7g were markedly higher in SW620 cells than in SW620/OxR cells. However, we previously reported that the invasion activity of SW620/OxR cells was higher than that of SW620 cells,¹¹⁾ as also observed in the present study. These results suggest that the acquisition of L-OHP resistance may inhibit the incorporation of let-7a, let-7b, and let-7g into exosomes. Koppers-Lalic et al. indicated that 3′-end uridylated miRNA are overexpressed in exosomes.¹⁹⁾ Since let-7 family members are 3′-end uridylated miRNA, this mechanism may be linked to the incorporation of let-7a, let-7b, and let-7g into exosomes. The suppression of this mechanism may be produced by acquisition of L-OHP resistance and may cause lower expression levels of exosomal let-7a, let-7b, and let-7g in SW620/OxR cells than those in SW620 cells.

In the present study, Zeb inhibited invasion activity and up-regulated let-7b expression levels. The overexpression of let-7b by the transfection of the let-7b mimic inhibited invasion activity in SW620 and SW620/OxR cells. These results suggest that Zeb may inhibit invasion activity by up-regulating the intracellular expression levels of let-7b in high-invasive CRC cells.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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