MiRNA-203 suppresses tumor cell proliferation, migration and invasion by targeting Slug in gastric cancer

Dear Editor, 
 
Snail, a family of zinc finger transcription factors, plays an important role in morphogenesis and embryogenesis. Snail zinc finger family 2 (SNAI2 or Slug) has been demonstrated to regulate carcinogenesis of several human cancers including breast, prostate, head, neck, pancreas and endometrial carcinomas (Zhang et al., 2011; Markiewicz et al., 2012; Behnsawy et al., 2013; Smith et al., 2013; Tanaka et al., 2013), and it contributes to various tumorigenesis processes ranging from tumor cell invasion and metastasis to cell survival and proliferation (Phillips and Kuperwasser, 2014; Shi et al., 2015). However, its participation in the carcinogenesis of GC has only a few studies. Moreover, the molecular mechanisms of upstream and downstream regulation of Slug are largely unknown. Recently, Shi et al. confirmed miR-203 suppression in GC promotes Slug-mediated cancer metastasis (Shi et al., 2015). In this study, we explored the relationship of Slug and miRNAs using bioinformatics analysis, and demonstrated the roles of candidate miRNA, miR-203, in the carcinogenesis of gastric cancer cells. 
 
We first examined Slug expression in gastric cancer tissues by Western blotting. As shown in Fig. 1A, the Slug protein levels were significantly increased in the gastric cancer tissues compared to the corresponding adjacent normal tissues, suggesting that Slug may serve as an oncogene in the gastric cancer. In contrast, the Slug mRNA levels did not significantly differ between cancerous and noncancerous tissues (Fig. 1B). The disparity between Slug protein and mRNA expression in gastric cancer vs. adjacent normal tissues suggests that Slug may be regulated through a post-transcriptional mechanism. 
 
 
 
Figure 1 
 
Upregulation of Slug protein but not mRNA expression and downregulation of miR-203 in human gastric cancer tissues. (A) Western blotting analysis of the expression levels of Slug protein in 6 pairs of GCT and NCT samples. (B) Quantitative RT-PCR analysis ... 
 
 
 
MiRNAs mediate post-transcriptional regulation by repressing mRNA transcription. We used three computational algorithms, including TargetScan (Lewis et al., 2003), miRanda (John et al., 2004) and PicTar (Krek et al., 2005), to investigate miRNAs that can potentially target Slug, and miR-203 was identified as the candidate regulator of Slug. The predicted interaction between miR-203 and the target sites in the Slug 3′-UTR are illustrated in Fig. 1C. One predicted hybridization was identified between miR-203 and the 3′-UTR of Slug. The minimum free energy values of the two hybridizations were −24.3 kcal·mol−1, and the value was well within the range of genuine miRNA-target pairs. Moreover, the miR-203 binding sequences in the Slug 3′-UTR are highly conserved across species. 
 
MiRNAs and their targets are usually thought to express in an opposite pattern in tissues. We next investigated whether miR-203 was inversely correlated with Slug in gastric cancer tissues. We thus examined the miR-203 levels in the same six pairs of gastric cancer tissues and noncancerous tissues, and observed that miR-203 levels were indeed decreased in gastric cancer tissues as compared to those in adjacent normal tissues (Fig. 1D). These results implied a miR-203-mediated post-transcriptional regulatory mechanism in Slug repression. 
 
The correlation between miR-203 and Slug was further examined after evaluating Slug expression in the human gastric carcinoma cell line MKN-45 after the overexpression or knockdown of miR-203. Overexpression was achieved after transfecting the cells with miR-203 mimic, a synthetic RNA oligonucleotide that mimics the miR-203 precursor, and knockdown was achieved after transfecting cells with miR-203 inhibitor, a chemically modified antisense oligonucleotide designed to specifically target mature miR-203. The transfection efficiency was presented in Fig. 2A. As anticipated, overexpressing miR-203 significantly increased miR-203 levels and suppressed the Slug protein levels in MKN-45 cells, whereas miR-203 knockdown had the opposite effect on MKN-45 expression in these cells (Fig. 2B). We also examined the expression of the Slug mRNA levels, and found overexpression or knockdown of miR-203 did not affect Slug mRNA levels (Fig. 2C). Furthermore, we found that, overexpression of miR-203 also significantly decreased Slug protein levels in AGS cells (Fig. S1). 
 
 
 
Figure 2 
 
miR-203 might inhibit cell proliferation, migration and invasion through silencing Slug. (A) Quantitative RT-PCR analysis of miR-203 levels in MKN-45 cells transfected with miR-203 mimic or inhibitor. (B) Western blotting analysis of Slug protein levels ... 
 
 
 
To determine whether the negative regulatory effects of miR-203 on Slug expression were mediated through binding of miR-203 to the presumed sites in the 3′-UTR of the Slug mRNA, the full-length 3′-UTR of Slug was placed downstream of the firefly luciferase gene in a reporter plasmid. The resulting plasmid was transfected into MKN-45 cells along with miR-203 mimic, miR-203 inhibitor or scrambled negative control RNAs. Luciferase activity was markedly decreased in cells transfected with miR-203 mimic and increased in the cells transfected with miR-203 inhibitor (Fig. 2D). Moreover, we introduced point mutations into the corresponding complementary sites in the 3′-UTR of Slug to eliminate the predicted miR-203 binding sites. This mutated luciferase reporter was unaffected through either the overexpression or knockdown of miR-203 (Fig. 2D). This finding suggested that the binding sites contribute to the interaction between miR-203 and Slug mRNA. In conclusion, the results demonstrate that miR-203 inhibits Slug expression by binding to the 3′-UTR of Slug mRNA transcript and inhibits Slug translation. 
 
Slug has been reported to involve in a diverse number of processes ranging from tumor cell invasion and metastasis to cell survival and proliferation (Phillips and Kuperwasser, 2014). To investigate the cellular phenotypes triggered by the miR-203 mediated downregulation of Slug, MKN-45 cells were transfected with either miR-203 mimic, miR-203 inhibitor or si-Slug and analyzed for changes in cell proliferation, migration and invasion. 
 
Firstly, we investigated the role of Slug in proliferation, migration and invasion of gastric cancer cell lines. To knock down Slug, a siRNA targeting Slug was designed and transfected into MKN-45 cells. The efficient knockdown of Slug in MKN-45 cells is shown in Fig. S2A and S2B. MKN-45 cells transfected with Slug siRNA showed decreased cell proliferation, migration and invasion (Fig. S2C–G). Subsequently, we evaluated the effects of miR-203 on the cell proliferation, migration and invasion of MKN-45 cells by repressing Slug. As expected, MKN-45 cells transfected with miR-203 showed decreased proliferation, migration and invasion (Fig. 2E–G); in contrast, knocking down miR-203 with miR-203 inhibitor enhanced cell proliferation, migration and invasion (Fig. 2H–J). 
 
Taken together, we confirmed that Slug expression is up-regulated in gastric cancer tissues and plays a key function in gastric cancer cells proliferation, migration and invasion. We also identified Slug as a novel target of miR-203 in gastric cancer. These data suggest that Slug and miR-203 expression might be useful to predict invasiveness of gastric cancer, or be used as a prognostic factor in gastric cancer patients. The potential of Slug or miR-203 as novel molecular targets for gastric cancer therapy requires further investigations.

using fixed threshold settings, and the mean C T of the triplicate PCRs was determined. A comparative C T method was used to compare each condition to the controls. The relative levels of the miRNAs in cells and tissues were normalized to U6. The amount of miRNA relative to the internal control U6 was calculated using the 2 -△△CT equation, in which △△C T = (C T miRNA -C T U6)target -(C T miRNA -C T U6)control. To quantify Slug mRNA, 1 µg of total RNA was reverse-transcribed to cDNA using oligo dT and Thermoscript (TaKaRa) in the reaction, which was performed with the following conditions: 16°C for 30 min , 42°C for 30 min and 85°C for 5 min. Next, real-time PCR was performed using the RT product, SYBER Green Dye (Invitrogen), and specific primers for Slug and β-actin. The sequences of the primers were as follows: Slug (R): 5'-GTGTTTGCAAGATCTGCGGC-3'; Slug (F): 5'-GAGCCCTCAGATTTGACCTGT -3'; β-actin (R): 5'-ACTCGTCATACTCCT GCT-3'; And β-actin (F): 5'-ACAGGATGCAGAAGG AGATAC-3'. The reactions were incubated at 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. After the reactions were completed, the C T values were determined by setting a fixed threshold. The relative amount of Slug mRNA was normalized to β-actin.

Protein extraction and Western blotting
Protein was extracted from the cultured cells and human tissues using RIPA Lysis buffer (Beyotime, Shanghai, China) according to the manufacturer's instructions. Briefly, the cells and tissues were lysed in RIPA Lysis buffer (Beyotime, Shanghai, China) supplemented with a Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific 78440) on ice for 30 min and then centrifuged for 10 min (12,000 x g, 4°C). The supernatant was collected, and the protein concentration was calculated with a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The Slug protein levels were analyzed by Western blotting with a monoclonal anti-human Slug antibody (9585S,CST) .The protein levels were normalized by probing the same blots with β-actin antibody (Mab1445,MULTI). The anti-Slug and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (CA, USA).

Luciferase reporter assay
To test the direct binding of miR-203 to the target gene Slug, a luciferase reporter assay was performed according to the manufacturer's instructions. A sequence containing the presumed miR-203 binding site was designed from the human Slug 3'-untranslated region (3'-UTR). The sequence was inserted into the p-MIR-reporter plasmid (Ambion). The insertion was confirmed to be correct by sequencing. To test the binding specificity, the sequences that interacted with the miR-203 seed sequence were mutated, and the mutant Slug 3'-UTR was inserted into an equivalent luciferase reporter. For the luciferase reporter assays, MKN-45 cells were cultured in 24-well plates, and each well was transfected with 0.4µg of firefly luciferase reporter plasmid, 0.4µg of a β-galactosidase (β-gal) expression plasmid (Ambion), and equal amounts (20 pmol) of pre-miR-203, anti-miR-203, or the scrambled negative control RNAs using Lipofectamine 2000 (Invitrogen). The β-gal plasmid was used as a transfection control. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit (Promega, Madison, WI, USA).

Cell proliferation assay
MKN-45 cells were seeded at 7×10 3 cells per well on 96-well plates and then incubated overnight in RPMI-1640 supplemented with 10% FBS. The cells were collected at 12, 24, 36, or 48h post-transfection. After transfection, 10 μL of Cell Counting Dojindo) was added to the corresponding tested wells and incubated for 2h. The absorbance was measured at a wavelength of 450 nm. All experiments were performed in triplicate.

Cell migration and invasion assay
The migration and invasion ability of MKN-45 cells transfected with miR-203 mimic, miR-203 inhibitor or the Slug siRNA was tested in a Transwell Boyden Chamber (8 mm, Costar, USA). The polycarbonate membranes on the bottom of the upper compartment of the Transwell were coated with 1% human fibronectin (R&D systems 1918-FN, USA). The cells were harvested 12h after transfection and suspended in fetal bovine serum (FBS)-free 1640 culture medium. Then, cells were added to the upper chamber (6×10 4 cells/well). At the same time, 0.6 ml of 1640 with 20% FBS was added to the lower compartment, and the Transwell-containing plates were incubated for 24h in a 5% CO 2 atmosphere saturated with H 2 O. After incubation, cells that had entered the lower surface of the filter membrane were fixed with 4% paraformaldehyde for 25 min at room temperature, washed 3 times with distilled water and stained with 0.5% crystal violet at room temperature. Cells remaining on the upper surface of the filter membrane were scraped off gently with a cotton swab. The lower surfaces were captured by a photomicroscope (five fields per chamber) (BX51 Olympus,Japan), and the cells were counted carefully.

Statistical analysis
All Western blot images are representative of at least three independent experiments. Quantitative RT-PCR, luciferase reporter assays, and cell apoptosis assays were performed in triplicate, and each experiment was repeated several times. The data are shown as the means ± SE of at least three independent experiments. The differences were considered statistically significant at p < 0.05 using Student's t-test.