[ARTICLE WITHDRAWN] miR-630 Inhibits Epithelial-to-Mesenchymal Transition (EMT) by Regulating the Wnt/β-Catenin Pathway in Gastric Cancer Cells.

THIS ARTICLE WAS WITHDRAWN BY THE PUBLISHERS IN NOVEMBER 2020.

INTRODUCTION

Currently, gastric cancer is considered to be the fifth most common cancer1–3 and the second highest cause of cancer-related deaths3 in the world. The incidence of gastric cancer shows obvious geographical variation, with China and Japan having a higher number of gastric cancer patients compared to Western countries4,5. The presence of gastric cancer is usually considered as a grave prognostic sign, and one of the most common contributing factors includes the late diagnosis at an advanced stage. The overall 5-year survival rate ranges from 40% (when diagnosed at an early stage) to 5% (when diagnosed at an advanced stage)4–9. Therefore, effective means of early diagnosis and treatment for gastric cancer are urgent needs in these countries with high morbidity and mortality.

MicroRNAs (miRNAs) are a special class of highly conserved small noncoding RNAs consisting of about 22 nucleotides and are responsible for regulation of gene expression especially at the posttranscriptional stage through either translational repression or cleavage of messenger RNA (mRNA)10,11. Various studies have unequivocally established the pivotal role of various miRNAs in different biological processes including cellular development, cell proliferation, cell differentiation, apoptosis, and cellular metabolism12. Functional dysregulation of miRNAs is also related with tumorigenesis and metastasis13; some of the miRNAs act as tumor suppressors, whereas some act as oncogenes14. miRNA dysregulation has been shown to be associated with cancer in different organs such as the stomach (gastric cancer), breast, prostate, lung, pancreas, and blood (chronic lymphocytic leukemia)15–17. Aberrant expressions of miRNAs such as miR-21, miR-22, miR-23, miR-127, miR-148, miR-202, and miR-433 have been implicated in the pathogenesis of gastric cancer18–21. The study of the molecular mechanism of gastric cancer will contribute to the early diagnosis and treatment of gastric cancer. Therefore, the study of miRNAs in gastric cancer might provide a new therapeutic strategy for the diagnosis and treatment of gastric cancer.

Epithelial-to-mesenchymal transition (EMT) is characterized by differentiation of epithelial cells into mesenchymal cells. It is considered one of the pivotal biological events associated with both physiological and pathological conditions including development, wound healing, regulation of stem cell function, and metastasis22. EMT is common and well conserved and is associated with different conditions in vivo, but minor differences exist depending on the tissue type23. The major events of EMT include dissolution of epithelial cell-to-cell junctions and the change in basal–apical polarity to front–rear polarity22–24. These occur due to the decreased expression levels of epithelial-related genes and increased expression levels of mesenchymal-related genes. Loss of E-cadherin, which is a marker of epithelial characteristics, and expression of N-cadherin, a marker of mesenchymal characteristics, are the hallmarks of EMT24. Studies have demonstrated that miRNAs target EMT or promoted EMT in multiple cancers, such as epithelial cancer cell lines25 and gastric carcinoma cells26.

Wnt/β-catenin is a highly conserved signaling pathway that regulates different biological functions like cellular proliferation, differentiation, and others27. For EMT, as mentioned earlier, the Wnt/β-catenin pathway also takes part in both physiological processes, such as the development of the embryo28, and pathological processes including cancers such as breast cancer, colon cancer, liver cancer, melanoma, non-small cell lung cancer, and ovarian cancer29–34. As a potent EMT inducer, transforming growth factor-β (TGF-β) has already been known to be present in the tumor microenvironment and also plays an important role in regulating biological processes such as cell-to-cell adhesion and determination of cellular polarity35,36. Therefore, studying the relationship between these three and miRNAs in gastric cancer might open up a new direction for the study of the pathogenesis and molecular mechanism of gastric cancer. In this study we have explored the role of miR-630 in the pathogenesis of gastric cancer as well as the possible underlying mechanism.

MATERIALS AND METHODS

Cell Culture

The normal gastric epithelium cell line GES-1 and human gastric cancer cell lines SGC-7901, BGC-823, MGC803, and AGS were obtained from the China General Microbiological Culture Collection Center (Beijing, P.R. China) and cultured in minimal Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco BRL, Gaithersburg, MD, USA) that contained 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic antimycotic (Gibco) in an atmosphere with 5% CO2 and 95% air at 37°C37.

Cell Transfection

The miR-630 mimic (5′-AGUAUUCUGUACCAGGGAAGGU-′3/5′-CUUCCCUGGUACAGAAUACUUU-3′) and scramble (negative control: 5′-UUCUCCGAACGUGUCACGUTT-3′/5′-ACGUGACACGUUCGGAGAATT-3′) were synthesized by GenePharma Co. (Shanghai, P.R. China). Cell transfections were performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. After 48 h of transfection, the cells were collected for further analysis. Both human gastric cancer cell lines SGC-7901 and BGC-823 were divided into three test groups according to the treatment condition, namely, control without transfection, cells transfected with miR-630 mimic (miR-630 mimic), and cells transfected with scramble (negative control).

Cell Viability Assay

Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) colorimetric assay according to standard methods. Briefly, after cells were transfected with miR-630 mimic or scramble, cells were seeded into 96-well plates and cultured for 1–4 days. MTT was added into each well and incubated for another 4 h at 37°C. Then dimethyl sulfoxide (DMSO) was added, and the plates were shaken for 10 min. The absorbance was detected at 450 nm. Each experiment was performed three times.

Scratch Assay

Cells were seeded in 24-well plates and cultured until confluence. Then cells were pretreated with 50 μM mytomicin C, and wounds were created by scratching the cell layer using a sterile 200-μl pipette tip. The culture medium was replaced with fresh medium containing either DMSO or icariin. The specific size of the scratched areas was determined by taking pictures using an inverted microscope (Leica, Germany) every 24 h. The widths of gaps were measured, and relative wound widths were calculated.

Migration Assay

Cell migration was determined using a modified two-chamber migration assay with a pore size of 8 μm. After transfection, the cells were resuspended in 200 μl of serum-free medium and seeded on the upper compartment of a 24-well Transwell culture chamber, and 600 μl of complete medium was added to the lower compartment. After 12 h of incubation at 37°C, the cells were fixed with methanol. Cells that had not traversed were removed carefully from the upper surface of the filter using a cotton swab. Traversed cells on the lower side of the filter were stained with crystal violet and counted under an optical microscope (DMI L; Leica Microsystems, Wetzlar, Germany).

Invasion Assay

The invasion behavior of the cells was determined using 24-well BD BioCoat™ Matrigel™ Invasion Chambers (8-μm pore size polycarbonate filters; BD Biosciences). Briefly, after the cells were treated for the indicated condition, 5 × 104 SKOV-3 cells in 200 μl of serum-free Dulbecco’s modified Eagle’s medium (DMEM) were plated onto the upper chambers, while complete medium containing 10% FBS was added to the lower chamber. After incubating the invasion chambers for 48 h at 37°C with 5% CO2, the noninvaded cells were removed with a cotton swab. The invaded cells were fixed in 100% methanol and then stained with crystal violet solution and counted under a microscope (DMI L; Leica Microsystems). The data are presented as the average number of cells attached to the bottom surface from five randomly chosen fields.

Quantitative Real-Time Reverse Transcription PCR (qRT-PCR)

Total RNA was extracted from the cells by TRIzol reagent (Invitrogen). For miR-630 detection, reverse-transcribed complementary DNA was synthesized by the PrimeScript RT Reagent Kit (TaKaRa, Dalian, P.R. China), and qRT-PCR was performed with SYBR Premix ExTaq (TaKaRa) using the Stratagene Mx3000P real-time PCR system (Agilent Technologies, Inc., Santa Clara, CA, USA). Expression levels were normalized against the endogenous short nuclear RNA (snRNA) U6 control. For mRNA analyses, cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR was performed with SYBR Premix ExTaq with the Stratagene Mx3000P real-time PCR system. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for mRNA quantification. The relative expression ratios were calculated by the 2−ΔΔCT method38. PCRs for each gene were repeated three times. Independent experiments were done in triplicate.

Western Blot

The proteins in the transfected cells that were used for Western blotting were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) supplemented with protease inhibitors (Roche, Guangzhou, P.R. China). The proteins were purified and quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Western blots were established using a Bio-Rad Bis-Tris Gel system according to the manufacturer’s instructions. Primary antibodies at a dilution of 1:1,000 were incubated with polyvinylidene difluoride (PVDF) membranes at 4°C overnight, followed by washing and then incubating with secondary antibodies tagged by horseradish peroxidase for 1 h at room temperature. After rinsing, the PVDF membranes and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS system, and 200 μl of Immobilon Western Chemiluminescent HRP Substrate (Millipore, Boston, MA, USA) was added to cover the membrane surface. The signals were captured, and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, P.R. China).

Statistical Analysis

All experiments in our study were performed with at least three independent experiments and repeated at least three times. The results of the multiple experiments were presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad statistical software Prism 6.0. The two-tailed Student’s t-test was used to evaluate the significance of the differences between two groups; one-way analysis of variance (ANOVA) was used to evaluate significance of the differences in mean values within and between multiple groups. The p values were calculated using two-way ANOVA since two factors are tested once during Western blot measurement including miR-630 and TGF-β1. A value of p < 0.05 was considered to be statistically significant.

RESULTS

Expression of miR-630 in Gastric Cancer Cell Lines

As displayed in Figure 1A, compared with the normal gastric epithelium cell line GES-1, the expression of miR-630 was decreased in tested gastric cancer cell lines, including SGC-7901 (p < 0.01), BGC-823 (p < 0.01), MGC803 (p < 0.05), and AGS (p < 0.05). The most obvious changes occurred on two cell lines, SGC-7901 and BGC-823. Thus, the SGC-7901 and BGC-823 cell lines were chosen for further studies.

Figure 1

Expression of microRNA-630 (miR-630) in gastric cancer cell lines SGC-7901 and BGC-823. miR-630 levels were detected in some gastric cancer cell lines. Cells were transfected with miR-630 mimic or scramble control, and transfection efficiency was evaluated. miR-630 expression levels were measured by quantitative real-time reverse transcription (qRT)-PCR. (A) Expression of miR-630 in several gastric cancer cell lines. (B) Expression of miR-630 in the gastric cancer cell line SGC-7901 after transfection assay. (C) Expression of miR-630 in the gastric cancer cell line BGC-823 after transfection assay. *p < 0.05, **p < 0.01, ***p < 0.001.

Expression of miR-630 in Transfected Gastric Cancer Cell Lines

The expressions of miR-630 in SGC-7901 and BGC-823 cell lines after transfection with miR-630 mimic were significantly (p < 0.001) promoted compared with scramble control cells or negative control (Fig. 1B and C). These results demonstrated that miR-630 mimic transfection effectively increased the expression of miR-630 in gastric cancer cells.

miR-630 Inhibited Cell Viability

For the groups of cells transfected with miR-630 mimic or scramble control in both the SGC-79010 and BGC-823 cell lines, cell viability assay results showed that cell viability of miR-630 mimic-transfected cells was compared to control cell viability (p < 0.05 or p < 0.01) (Fig. 2). This suggested that transfection of miR-630 mimic inhibited the cell viability of gastric cancer cells.

Figure 2

Inhibition of cell viability by miR-630 mimic transfection in gastric cancer cell lines SGC-7901 and BGC-823. (A) Relative cell viability after miR-630 mimic or scramble transfection in gastric cancer cell line SGC-7901. (B) Relative cell viability of miR-630 mimic or scramble-transfected gastric cancer cell line BGC-823. *p < 0.05.

miR-630 Suppressed Wound Healing

The results of the scratch assay showed that transfection of miR-630 mimic in both gastric cancer cell lines SGC-7901 and BGC-823 suppressed wound healing as evidenced by a minimal change in the relative wound width over a period of 5 days compared to the control and scramble test groups, although the differences were not statistically significant (p < 0.001) (Fig. 3A and B).

Figure 3

Transfection of miR-630 mimic suppressed wound healing, migration, and invasion in gastric cancer cell lines SGC-7901 and BGC-823. (A) miR-630 mimic transfection suppressed wound healing in the gastric cancer cell line SGC 7901. (B) miR-630 mimic transfection suppressed wound healing in the gastric cancer cell line BGC-823. (C) miR-630 mimic transfection suppressed migration of the gastric cancer cell line SGC-7901. (D) miR-630 mimic transfection suppressed migration of the gastric cancer cell line BGC-823. (E) miR-630 mimic transfection suppressed invasion of the gastric cancer cell line SGC-7901. (F) miR-630 mimic transfection suppressed invasion of the gastric cancer cell line BGC-823. *p < 0.05, **p < 0.01, ***p < 0.001.

miR-630 Suppressed Cell Migration and Invasion

SGC-7901 and BGC-823 gastric cancer cells were transfected with miR-630 mimic and showed significant suppression of migration and invasion (p < 0.05 or p < 0.01) in comparison to scramble-transfected groups (Fig. 3C–F). The data suggested that transfection of miR-630 mimic in gastric cancer cells might suppress cell migration and invasion.

miR-630 Inhibited TGF-β1-Mediated EMT

After the cells reached confluence, the culture medium was replaced with a culture medium containing TGF-β1, as the potent EMT inducer, and then the transfection was performed. With the presence of TGF-β1 in the cell culture medium, cells were transfected with miR-630 mimic, scramble control, or not. After 72 h, the expressions of different proteins related with EMT were measured including zinc finger protein SNAI1 (Snail), zinc finger protein SNAI2 (SLUG), E-cadherin (E-Cad), N-cadherin (N-Cad), and vimentin (Vim). As shown in Figure 4, mRNA and protein expression levels of E-Cad were suppressed compared with the control group without treatment (p < 0.05 or p < 0.01 for mRNA-level comparison), suggesting that EMT had occurred. Increased expression of all other factors at both the mRNA and protein levels indicated the induction of EMT (p < 0.05, p < 0.01, or p < 0.001 for mRNA-level comparisons). The cells transfected with miR-630 mimic in the presence of TGF-β1 (TGF-β1 + miR-630 mimic) showed increased expressions of E-Cad and decreased expressions of other proteins including Snail, SLUG, N-cadherin, and Vim compared to other treated cell groups, such as gastric cancer cells in the presence of TGF-β1, and scramble cells in the presence of TGF-β1 transfected with scramble control (TGF-β1 + scramble).

Figure 4

miR-630 mimic transfection inhibited transforming growth factor-β1 (TGF-β1)-mediated epithelial–mesenchymal transition (EMT) in gastric cancer cell lines SGC-7901 and BGC-823. (A) miR-630 mimic transfection inhibited TGF-β1-mediated EMT in the gastric cancer cell line SGC-7901. The expression of EMT-related factors in both mRNA and protein levels in the gastric cancer cell line SGC-7901. (B) miR-630 mimic transfection inhibited TGF-β1-mediated EMT in the gastric cancer cell line BGC-823. The expression of EMT-related factors in both mRNA and protein levels in the gastric cancer cell line BGC-823. *p < 0.05, **p < 0.01, ***p < 0.001 for all mRNA expression comparisons. The p values were calculated using two-way analysis of variance (ANOVA) for two factors as miR-630 and TGF-β1.

miR-630 Suppressed EMT Through Activation of the Wnt/β-Catenin Signaling Pathway

As shown in Figure 5, the cells transfected with miR-630 mimic in the presence of TGF-β1 (TGF-β1 + miR-630 mimic) showed increased expression of proteins associated with the Wnt/β-catenin signal pathway including Wnt3a, Wnt5a, and β-catenin (activation of the Wnt/β-catenin signal pathway) and at the same time reduced the expression of EMT-related proteins, as the results showed decreased expressions of Snail and Vim as well as increased expression of E-Cad, in comparison to the treated cell groups including gastric cancer cells in the presence of TGF-β1, and scramble-transfected cells in the presence of TGF-β1 (TGF-β1 + scramble). These findings were further supported by the fact that in the presence of Wnt/β-catenin inhibitor ICG001, all the above-mentioned changes were reversed in the TGF-β1 + miR-630 mimic test groups (p < 0.001) (Fig. 5).

Figure 5

miR-630 mimic transfection suppressed EMT through the Wnt/β-catenin signal pathway in gastric cancer cell lines SGC-7901 and BGC-823. (A) miR-630 mimic transfection suppressed EMT through regulating the activity of the Wnt/β-catenin signal pathway in the gastric cancer cell line SGC-7901. (B) miR-630 mimic transfection suppressed EMT through regulating the activity of the Wnt/β-catenin signal pathway in the gastric cancer cell line BGC-823. (C) Relative expression of proteins related with EMT and the activity of the Wnt/β-catenin signal pathway in the gastric cancer cell line SGC-7901. (D) Relative expression of proteins related with EMT and the activity of the Wnt/β-catenin signal pathway in the gastric cancer cell line BGC-823. ***p < 0.001.

DISCUSSION

Gastric cancer is one of the most common cancers worldwide with a poor prognosis. Hence, besides the currently available treatment modalities, newer drug targets are very urgently needed. Understanding of the molecular mechanisms that are responsible for suppressing gastric cancer progression is needed, and it would be helpful in exploring novel drug targets.

We found that transfection of miR-630 mimic in the two gastric cancer cell lines suppressed cell viability, migration, and invasion (Figs. 2 and 3C–F). These findings were consistent with the study conducted by Jin et al. in esophageal squamous cancer cells that upregulation of miR-630 resulted in the inhibition of proliferation, migration, and invasion of the cancerous cells, and inversely miR-630 suppression led to the opposite results39.

Also, miR-630 mimic transfection suppressed wound healing in two gastric cancer cell lines (BGC-823 and SGC-7901) (Fig. 3A and B). EMT is one of the most important events underlying the process of metastasis. Jin et al. also explored the correlation between miR-630 expression level and EMT expression in esophageal squamous cell carcinoma. The results suggested that miR-630 suppression resulted in the induction of EMT39. Chen et al. demonstrated that transfection with miR-630 mimic led to the attenuation of EMT phenotype in in vitro models35. Similar to these findings, our study demonstrated that transfection of miR-630 mimic led to the inhibition of TGF-β1-mediated EMT phenotype. Chen et al. further concluded that miR-630 suppressed EMT through Slug, which was further supported by the fact that knockdown of Slug led to the reversal of the miR-630 inhibitory effects on EMT phenotype35. Similar to the above findings, our study results suggested that in gastric cancer cells, transfection of miR-630 mimic even in the presence of TGF-β1 (TGF-β1 + miR-630 mimic) increased the expression of E-Cad and decreased the expression of Snail, Slug, N-Cad, and Vim in comparison to other test groups (Fig. 4). The present study aimed to investigate the effect of miR-630 on TGF-β-induced EMT of cells. To the best of our knowledge, there is no evidence that miR-630 alone (in the absence of TGF-β) is effective on EMT induction. Thus, the EMT of cells transfected with miR-630 alone was not evaluated in the present study.

Literature searches revealed that most of the studies discussed that suppressing the Wnt/β-catenin pathway in turn suppressed the EMT phenotype30,32,33. However, in our study, we found that gastric cancer cells transfected with miR-630 mimic in the presence of TGF-β1 also showed increased expression of proteins associated with the Wnt/β-catenin pathway, including Wnt3a, Wnt5a, and β-catenin. This study also demonstrated suppression of EMT phenotype as evidenced by decreased expression of Snail and Vim and increased expression of E-Cad (Fig. 5).

In a review article, Ghahhari et al. described the importance of the Wnt/β-catenin signaling pathway in the maintenance of the epithelial cellular phenotype40. They demonstrated that dysregulation of the different members in this pathway might lead to EMT and tumorigenesis. Meanwhile, several miRNAs regulated protein expressions in the Wnt/β-catenin signaling pathway by its upregulation or downregulation, suggesting that investigation of specific miRNAs as therapeutic targets might be beneficial for treatment of specific types of cancer. These findings were further supported by the addition of the Wnt/β-catenin inhibitor ICG001, which led to the decreased expression of all the proteins associated with the Wnt/β-catenin pathway, such as Wnt3a, Wnt5a, and β-catenin, and increased expression of Snail and Vim, as well as decreased expression of E-cad (Fig. 5). Thus, we concluded that miR-630 suppressed the EMT phenotype through activation of the Wnt/β-catenin pathway in gastric cancer cells.

It is undeniable that there were some limitations in this experiment. For example, during the Transwell assay, the relative results on the traversed cells were counted microscopically, which is clumsy. Considering that cells were fixed and stained, the valid and feasible counting method was limited. A scientific and effective method to replace the microscopic count method is needed, such as taking pictures of the stained cells and performing densitometry analysis using ImageJ software to replace microscopic counting. A more in-depth study of the interaction between these factors and miR-630 would contribute to a comprehensive understanding of the role of miR-630 in gastric cancer cells in vitro and in vivo.

In conclusion, we found that miR-630 expression was increased in gastric cancer cell lines after miR-630 mimic transfection. This study also demonstrated that miR-630 played an important role in the invasiveness and metastasis of gastric cancer cells. It is suggested that miR-630 might serve as a prognostic marker in clinical practice and that miR-630 might be useful as a new therapeutic method for the treatment of gastric cancer. Also, the beneficial role of miR-630 in the suppression of EMT should be further explored, as it could be considered as a novel drug target for gastric cancer treatment.