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Introduction

Gastric cancer (GC), one of the most common types of malignant tumor worldwide, is the second most common cause of cancer-related mortality globally (1). It is estimated that annually, there are ~1,000,000 new cases and >700,000 mortalities due to GC worldwide (2). As a result of higher Helicobacter pylori prevalence rates, >70% of patients with GC are in developing countries, particularly in China (3,4). Despite development in comprehensive treatment including surgery, radiotherapy and chemotherapy, the 5-year overall survival rate for GC remains poor (5). This poor survival rate is mainly due to recurrence and metastasis, even following subtotal gastrectomy (6). The tumorigenesis, development and metastasis of GC is multifactorial, and numerous genetic and epigenetic changes involving oncogenes, tumor suppressor genes and growth factors changes have been demonstrated to be involved in GC (7). However, the molecular mechanism underlying the tumorigenesis and development of GC remains unclear. Therefore, it is of great significance to investigate the molecular mechanisms underlying the initiation and progression of GC to explore new therapeutic treatments for GC.

MicroRNAs (miRNAs) are a group of non-protein-coding, highly conserved, single strand RNA molecules, which are 21–25 nucleotides in length (8). miRNAs primarily regulate target gene expression at the transcriptional or posttranscriptional level by binding to the 3′ untranslated region (3′UTR) of target genes (8,9). Previous studies have reported that miRNAs play important functions in various physiological and pathological processes, including cell proliferation, cell cycle, apoptosis, differentiation and metastasis; thereby affecting normal cell growth and development and leading to a variety of disorders including malignancies (10–12). In addition, the abnormal expression of miRNAs has been identified in various types of human malignant tumors, and their expression was significantly correlated with the carcinogenesis, progression and metastasis of these cancer types (13,14). Abnormally expressed miRNAs in human cancer are able to function as tumor suppressors or oncogenes depending on their target mRNAs (15,16). Therefore, it is important to further examine the functions of miRNAs in GC, in order to develop novel and efficient therapeutic strategies for GC.

The present study aimed to evaluate the expression, functions and mechanisms of microRNA-154 (miR-154) in GC. Firstly, the expression of miR-154 was measured in GC tissues and cell lines using reverse transcription-quantitative PCR (RT-qPCR). GC cells were then transfected with miR-154 mimics or negative control (NC) to evaluate the effects of miR-154 on the biological behavior of GC cells. Following transfection, bioinformatics analysis, Dual Luciferase reporter assay, RT-qPCR and western blot analysis were adopted to explore the molecular mechanisms underlying miR-154-inhibited growth and metastasis of GC cells.

Materials and methods

Tissue specimens

The present study was approved by the Research Ethics Committee of The First Hospital of Lanzhou University (Gansu, China). Full written informed consent was obtained from all patients with GC prior to the collection of tissue specimens. A total of 36 paired GC tissues and matched non-neoplastic gastric tissues (normal) were obtained from patients with GC who had undergone radical gastrectomy at The First Hospital of Lanzhou University. None of the patients with GC had been treated with any radiotherapy or chemotherapy prior to surgery.

Cell culture and transfection

The four human GC SGC-7901, AGS, MKN-1 and BGC-823 cell lines and the normal gastric epithelium GES-1 cell line were all ordered from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

miR-154 mimics, NC miRNA mimics, metadherin (MTDH) small interfering RNA (siRNA) and siRNA control were all purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Cells were transfected with mimics or siRNA using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

RT-qPCR

Total RNA was isolated from tissues and cells using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For mature miR-154 expression, the Taqman microRNA reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to synthesize cDNA, followed by RT-qPCR with a Taqman microRNA assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). RNU6B was used as an internal control for miR-154 expression analysis. The thermocycling conditions were as follows: 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 1 min, and a final elongation step at 72°C for 10 min. To quantify MTDH mRNA expression, total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). SYBR Green PCR Master mix (Applied Biosystems, Thermo Fisher Scientific, Inc.) was adopted to measure MTDH mRNA expression levels. The thermocycling conditions were as follows: 95°C for 10 min; 40 cycles at 95°C for 15 sec and 60°C for 1 min. The data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RT-qPCR was performed in triplicate on an AB7300 thermocycler (Applied Biosystems, Thermo Fisher Scientific, Inc.). The primer sequences were as follows: miR-154, forward, 5′-TGCGCTAGGTTATCCGTGTTG-3′ and reverse, 5′-CTCAAGTGTCGTGGAGTCGGCAA-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; MTDH forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH forward, 5′-CGTCTTCACCACCATGGAGA-3′ and reverse primer, 5′-CGCCCATCACGCCACAGTTT-3′. Relative expression level was calculated using the 2−ΔΔCT method (17).

Cell proliferation assay

The effects of miR-154 on cell proliferation were assessed with Cell Counting kit 8 assay (CCK8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). GC cells were seeded into 96-well plates at a density of 3,000 cells per well. Following transfection with mimics or siRNA, cells were incubated at 37°C in a humidified incubator for 24, 48, 72 and 96 h. Subsequently, 10 µl of CCK8 assay solution was added to each well of the 96 well plates and incubated at 37°C for an additional 2 h. The absorbance of each well at 450 nm was detected using a microplate reader (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). All experiments were performed in triplicate.

Transwell migration and Matrigel invasion assay

The effects of miR-154 on cell migration and invasion were evaluated using Transwell chambers with an 8 µm pore size (BD Biosciences, Bedford, MA, USA). For the Transwell migration assay, cells were collected, and 5×104 cells in 200 µl serum-free medium were seeded into the upper Transwell chambers 24 h subsequent to transfection with mimics or siRNA. The lower Transwell chambers were loaded with 500 µl medium supplemented with 20% FBS as a chemoattractant. For the Matrigel invasion assay, the Transwell chambers were pre-coated with 2% Matrigel (BD Biosciences, San Jose, CA, USA). Following incubation at 37°C for 24 h (migration assay) or 36 h (invasion assay), the cells on the upper surface of the Transwell chambers were removed carefully by cotton wool. The migrated and invaded cells were fixed and stained with 0.5% crystal violet. Subsequent to washing with PBS three times, the chambers were visualized with an IX71 inverted microscope (Olympus Corporation, Tokyo, Japan).

Bioinformatic analysis

Target genes of miR-154 were searched using TargetScan (http://www.targetscan.org).

Western blot analysis

In the present study, MTDH (1:1,000; catalog no. sc-517220) and β-actin (1:1,000; catalog no. sc-130301) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). At 72 h subsequent to transfection, transfected cells (mimics/siRNA) were lysed using a radioimmunoprecipitation assay buffer in the presence of a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The concentration of total cellular protein was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg) were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (PVDF, EMD Millipore, Billerica, MA, USA). Subsequent to blocking with 5% non-fat milk in TBS, the membranes were probed with primary antibodies overnight at 4°C, followed by incubation with goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000; catalog no. sc-2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The bands were detected with enhanced chemiluminescence solution (ECL; Pierce; Thermo Fisher Scientific, Inc.). The protein intensities were quantified using AlphaEase FC software. (version 4.1.0; Alpha Innotech, San Leandro, USA). This experiment was repeated three times.

Dual Luciferase reporter assay

For the luciferase reporter assay, the luciferase reporter vectors PGL3-MTDH-3′UTR wild type (Wt) and PGL3-MTDH-3′UTR mutant (Mut) were purchased from Shanghai GenePharma Co., Ltd. Cells were transfected with miR-154 mimics or NC, and PGL3-MTDH-3′UTR Wt or PGL3-MTDH-3′UTR Mut using Lipofectamine 2000, according to the manufacturer's protocol. The luciferase activities were measured using Dual Luciferase Reporter Assay system (Promega Corporation, Manheim, Germany) 48 h following transfection. Experiments were performed in triplicate and replicated 3 times.

Statistical analysis

Data are presented as the mean ± standard deviation, and compared using StataCorp LP 10.0 (College Station, TX, USA). Two-tailed P<0.05 was considered to indicate a statistically significant difference.

Results

miR-154 expression is downregulated in GC tissues and cell lines

To determine whether miR-154 was involved in the tumorigenesis and development of GC, its expression was measured using RT-qPCR in GC tissues and matched non-neoplastic gastric tissues. The results demonstrated that miR-154 was significantly downregulated in GC tissues in comparison with matched non-neoplastic gastric tissues (Fig. 1A). miR-154 expression levels were also detected in the four GC cell lines and the normal gastric epithelium cell line. The results revealed that all GC cell lines expressed lower levels of miR-154 compared with the expression levels in the normal gastric epithelium GES-1 cell line (Fig. 1B). Together, these results indicate that miR-154 is downregulated in GC.

Figure 1. Relative expression of miR-154 in GC. (A) Relative expression of miR-154 in GC tissues and matched non-neoplastic gastric tissues. (B) miR-154 was downregulated in the four GC cell lines (SGC-7901, AGS, MKN-1 and BGC-823) compared with that in the normal gastric epithelium GES-1 cell line. *P<0.05 compared with their respective controls. miR-154, microRNA-154; GC, gastric cancer.

Overexpression of miR-154 inhibits proliferation, migration and invasion of GC cells

To investigate whether miR-154 affects the proliferation of GC cells, miR-154 mimics or NC was introduced into GC cells. SGC-7901 and MKN-1 were selected for the present functional study due to their lower miR-154 expression levels. Following transfection, RT-qPCR was performed to measure relative miR-154 expression. As presented in Fig. 2A, miR-154 was significantly upregulated in SGC-7901 and MKN-1 cell lines. Cell proliferation was assessed using a CCK8 assay. As presented in Fig. 2B, overexpression of miR-154 inhibited the proliferation of SGC-7901 at 72 and 96 h following transfection. The overexpression of miR-154 also inhibited proliferation of MKN-1 cells at 96 h following transfection.

Figure 2. Over-expression of miR-154 inhibited the tumor behavior of GC cells. (A) Reverse transcription-quantitative polymerase chain reaction was performed to measure relative miR-154 expression in GC cells following transfection with miR-154 mimics or NC. (B) Cell proliferation assays were performed to evaluate the proliferation of SGC-7901 and MKN-1 cells following transfection with miR-154 mimic or NC. (C) Cell migration and invasion assays were performed in SGC-7901 and MKN-1 cells following transfection with miR-154 mimic or NC (magnification, × 200). *P<0.05 compared with their respective controls. miR-154, microRNA-154; GC, gastric cancer; NC, negative control.

Transwell migration and Matrigel invasion assays were performed to explore whether miR-154 affected the migration and invasion capacity of GC cells. As expected, the cell migration and invasion capacity in SGC-7901 and MKN-1 cells was significantly reduced following transfection with miR-154 mimics in comparison with NC (Fig. 2C). These findings indicate that miR-154 may function as a tumor suppressor in GC.

miR-154 directly targeted MTDH by interaction with the binding site in the 3′UTR

TargetScan was used to predicate potential target genes with complementary sites of miR-154 in their 3′UTR. The results revealed that MTDH contained a miR-154 seed match at 3′UTR of MTDH (Fig. 3A). To evaluate this possibility, luciferase reporter assays were performed. As presented in Fig. 3B, the relative luciferase activities of the PGL3-MTDH-3′UTR Wt were significantly decreased when miR-154 mimics were co-transfected. However, the luciferase activities of PGL3-MTDH-3′UTR Mut were unaffected by co-transfection with miR-154 mimics. Furthermore, RT-qPCR and western blot analysis were performed to explore whether miR-154 affects MTDH expression at transcriptional and translational levels. The results indicated that the levels of MTDH mRNA and protein expression in miR-154 transfected SGC-7901 and MKN-1 cells were significantly inhibited compared with those in NC-transfected cells (Fig. 3C and D). These results suggest that miR-154 directly targets MTDH in GC by interacting with the binding site in the 3′UTR of the MTDH gene.

Figure 3. miR-154 directly targeted MTDH. (A) The putative miR-154 binding sites in the 3′UTR of MTDH was shown. Mutation was generated on the MTDH 3′UTR sequence in the complementary site for the seed region of miR-154. (B) The relative luciferase activities of the PGL3-MTDH-3′UTR Wt were significantly decreased when miR-154 mimics was co-transfected. However, the luciferase activities of PGL3-MTDH-3′UTR Mut were unaffected by co-transfection with miR-154 mimics. (C) The expression of MTDH mRNA was determined by reverse transcription-quantitative polymerase chain reaction in GC cells following transfection with miR-154 mimics or NC. (D) The expression levels of MTDH protein were measured by western blot analysis in GC cells following transfection with miR-154 mimics or NC. β-actin was used as a control. *P<0.05 compared with their respective controls. miR-154, microRNA-154; MTDH, metadherin, 3′UTR, 3β untranslated region; Wt, wild type; Mut, mutant; GC, gastric cancer; NC, negative control.

MTDH siRNA inhibited proliferation, migration and invasion of GC cells

To explore the roles of MTDH in GC, GC SGC-7901 and MKN-1 cells were transfected with MTDH siRNA or siRNA as a control. Western blot analysis revealed that MTDH siRNA decreased MTDH expression in SGC-7901 and MKN-1 cells compared with that in siRNA control transfected cells (Fig. 4A). Cell proliferation assays, Transwell migration and Matrigel invasion assays were performed to investigate the effects of MTDH on cellular proliferation, migration and invasion of GC cells. As presented in Fig. 4B and C, MTDH siRNA significantly inhibited the proliferation (following incubation for 72 and 96 h) and motility of GC cells. These results indicate that the inhibition of MTDH performed similar functions to miR-154 overexpression in GC cells; therefore, MTDH may be a functional target of miR-154 in GC.

Figure 4. Knockdown of MTDH inhibited proliferation, migration and invasion of GC cells. (A) Expression levels of the MTDH protein were measured by western blot analysis following transfection with the MTDH siRNA or siRNA control. β-actin was used as a control. (B) MTDH siRNA inhibited the proliferation of GC cells. (C) MTDH siRNA inhibited the migration and invasion capacity of GC cells. *P<0.05 compared with their respective controls. MTDH, metadherin; GC, gastric cancer; siRNA, small interfering RNA; ctrl, control.

Discussion

GC is one of the most common types of malignant tumor and occurs as a result of genetic alterations and multiple environmental factors (18). The main therapy for GC has improved in recent decades. However, the prognosis of patients with advanced GC remains poor. Therefore, it is of great significance to understand the molecular mechanism underlying GC carcinogenesis and development. In addition, an increasing number of studies have indicated that abnormal expression of miRNAs plays an important function in the initiation and progression of GC, and that miRNAs may be investigated as a potential novel therapeutic target for the treatment of GC (19,20). The present study revealed that miR-154 was significantly downregulated in GC tissues and cell lines. Overexpression of miR-154 in GC cells resulted in suppression of cellular proliferation, migration and invasion. Furthermore, MTDH was validated as a potential functional target gene of miR-154 in GC. These findings suggest that miR-154 functions as a tumor suppressor in GC, and may have the potential to be investigated as an anticancer drug for GC.

miR-154 has been revealed to be downregulated in non-small cell lung (NSCLC) (21), colorectal (22,23) and prostate cancer (24). In NSCLC, the expression levels of miR-154 were significantly decreased in the tumor tissues compared with those in the matched non-tumorous lung tissues. Low miR-154 expression was significantly correlated with metastasis, larger tumor size and advanced tumor node metastasis (TNM) stage of patients with NSCLC (21). Kai et al (22) revealed that miR-154 levels in colorectal cancer tissues were significantly lower than those in non-cancerous tissues. Decreased expression levels of miR-154 were markedly associated with large tumor size, positive lymph node metastasis and advanced clinical stage. Univariate analysis also demonstrated that patients with colorectal cancer with low miR-154 expression levels had a poorer prognosis in this previous study. In addition, multivariate analysis confirmed that low miR-154 expression was an independent predictor of poor survival rate.

miR-154 has been demonstrated to be a tumor suppressor. In NSCLC, cells overexpression of miR-154 inhibits cell growth, colony formation, migration and invasion, and enhances cellular apoptosis and G0/G1 cell cycle arrest (21). Upregulation of miR-154 also suppresses the growth of NSCLC cell xenografts in vivo (21). Zhu et al (24,25) reported that enforced expression of miR-154 decreases the proliferation, colony formation, migration and invasion of prostate cancer cells via blockade of cyclin D2 and high mobility group AT-hook 2. Xin et al (23) indicated that miR-154 suppresses growth, colony formation and motility by directly targeting toll-like receptor 2 (TLR2). These findings indicate that upregulating miR-154 or providing analogous pharmaceutical compounds exogenously may be effective therapeutic strategies for these types of cancer.

Previous studies have demonstrated that miRNAs negatively regulate target gene expression by binding to the 3′UTR of target genes (26–28). In the present study, MTDH was identified as a novel target gene of miR-154 in GC. Firstly, MTDH was predicted as a target gene of miR-154 by using TargetScan. Secondly, Dual Luciferase reporter assays demonstrated that miR-154 significantly decreased the luciferase activity in GC cells transfected with MTDH-3′UTR Wt compared with MTDH-3′UTR Mut. Thirdly, over-expression of miR-154 suppressed MTDH mRNA and protein expression of GC cells. Finally, the functions of MTDH siRNA were similar to those induced by miR-154 in GC cells, which indicated that MTDH may be a functional target of miR-154 in GC. The identification of miR-154 target genes is essential for elucidating the functions of miR-154 in the carcinogenesis and progression of GC, and may provide promising therapeutic targets for patients with GC.

MTDH, located at chromosome 8q22, is a multifunctional oncogene that has been reported to be overexpressed in a variety of human cancers including glioma (29), hepatocellular carcinoma (30), colorectal (31) and breast cancer (32). Subsequent investigations have revealed that MTDH contributes to multiple biological processes in the course of cancer carcinogenesis and progression, including cellular growth, apoptosis, metastasis, invasion, chemoresistance and angiogenesis (33,34). The expression of MTDH mRNA and protein levels were also upregulated in GC tissues (35,36). In addition, high expression levels of MTDH were significantly associated with differentiation status, TNM stage, invasive depth and lymph node metastasis in GC (36). These studies all indicate that MTDH may be a novel and promising target for therapeutic intervention in GC. The present study demonstrated that MTDH was downregulated in GC cells following transfection with miR-154 mimics. Additionally, knockdown of MTDH inhibited growth and metastasis of GC cells. These results suggest that miR-154 may be investigated as a targeted therapy against MTDH and to block the growth and metastasis of GC.

In conclusion, the present study identified that miR-154 was downregulated in GC tissues and cells. Overexpression of miR-154 effectively inhibited the proliferation, migration and invasion of GC cells. Additionally, MTDH was demonstrated as a direct functional target gene of miR-154 in GC. The present study provides new insights into the tumorigenesis and development of GC. It also suggests that the miR-154/MTDH axis may act as a therapeutic target for patients with GC.

References