Gastric cancer is the fourth most common malignancy and the third leading cause of cancer-associated deaths worldwide. It has previously been demonstrated that microRNAs (miRNAs) are actively involved in the pathogenesis of gastric cancer. Therefore, miRNAs have been proposed as promising therapeutic targets in gastric cancer patients. MiR-744 is aberrantly expressed in different types of human cancer. However, the expression pattern and biological roles of miR-744 in gastric cancer remain unknown. The present study demonstrated that miR-744 expression was low in gastric cancer tissues and cell lines. Low expression levels of miR-744 was significantly correlated with lymph node metastasis, invasive depth and TNM staging in gastric cancer patients. The restoration of miR-744 expression inhibited cell proliferation and invasion
Gastric cancer is the fourth most common malignancy and the third leading cause of cancer-related deaths worldwide (
MicroRNAs (miRNAs) are 18–22 nt-long endogenous, noncoding and short RNAs which regulate gene expression through complete or partial base pairing with the 3′-untranslated regions (3′-UTRs) of their target genes, resulting in mRNA degradation or translation inhibition (
miR-744 is aberrantly expressed in different types of human cancer (
This study was approved by the Institutional Ethics Committee of the Cancer Hospital of Harbin Medical University. Written informed consent was also obtained from each patient. Fifty-four pairs of gastric cancer tissues and normal gastric tissues were collected from patients with gastric cancer who underwent surgery at the Cancer Hospital of Harbin Medical University between June 2014 and January 2016. None of these patients included in this research received chemotherapy or radiotherapy prior to surgical resection. Upon resection, tissue samples were immediately snap-frozen in liquid nitrogen and then stored at −80°C.
Five human gastric cancer cell lines, including AGS, MKN-45, SGC-7901, BGC-823, and MGC-803, were purchased from Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). One human gastric epithelial cell line GES-1 were obtained from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified incubator with 5% CO2 atmosphere at 37°C.
miR-744 mimics and negative control miRNA mimics (miR-NC) were bought form GenePharma Co., Ltd. (Shanghai, China). The pCMV6-BDNF plasmid and blank vector (pCMV6) were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). For transfection studies, cells were seeded into 6-well plates at a density of 5×105 cells/well. After incubation overnight, cells were transfected with miRNAs or vectors using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Then, the plates were incubated in a humidified atmosphere with 5% CO2 at 37°C.
Total RNA from tissues or cells was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. For detection of miR-744, the first-strand cDNA was synthesized using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed on an Applied Biosystems 7500 Sequence Detection system (Thermo Fisher Scientific) using TaqMan MicroRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific), with U6 as an internal control. For detection of brain-derived neurotrophic factor (BDNF) mRNA, total RNA was reverse-transcribed with a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) and qPCR was conducted with SYBR Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd.), with GAPDH as an internal control. The sequences of the primers were as follows: miR-744 forward, 5′-AATGCGGGGCTAGGGCTA-3′, and reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 forward, 5′-CGCTTCGGCAGCACATATAC-3′ and reverse, 5′-TTCACGAATTTGCGTGTCAT-3′; BDNF forward, 5′-AGCCTCCTCTTCTCTTTCTGCTGGA-3′ and reverse, 5′-TCCCGCCCGACATGTCCACT-3′; GAPDH forward, 5′-GACTCATGACCACAGTCCATGC-3′ and reverse, 5′-AGAGGCAGGGATGATGTTCTG-3′. Each sample was performed in triplicate and calculated using the 2−ΔΔCt method (
Cell proliferation was analyzed with CCK-8 assay (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Briefly, cells were plated into 96-well plates at a density of 3×103 cells per well. Cells were transfected with miRNAs or vectors. After incubation of 0, 24, 48, and 72 h, 10 µl CCK-8 regent was added into each well and incubated at 37°C for additional 2 h. The optical density (OD) at 450 nm was determined with a multifunction microplate reader (BioTek, Winooski, VT, USA). Each experiment was performed in triplicate and repeated three times.
Cell invasion assay was performed using a Transwell chamber (8.0-µm pores) coated with Matrigel (both from BD Biosciences, San Jose, CA, USA). After transfection 48 h, transfected cells were collected, and a number of 5×104 transfected cells were plated on the upper Transwell chambers. DMEM medium containing 10% FBS was added into the lower chamber as a chemoattractant. Subsequent to 24 h incubation, the cells remaining in the upper chamber were removed with a cotton swab. The invasive cells were fixed with 100% methanol and stained with 0.5% crystal violet. The invasive cells were imaged and counted using an inverted microscope (Olympus Corporation, Tokyo, Japan). Each experiment was repeated at least three times.
Total protein was extracted from cells or tissue samples using radioimmunoprecipitation (RIPA) lysis buffer supplemented with protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). We detect the protein concentration by using a BCA protein assay kit (Beyotime, Nanjing, China). Subsequently, equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After that, the membranes were blocked in 5% nonfat milk in Tris-based saline Tween-20 (TBST) for 1 h at room temperature, and further incubated overnight at 4°C with mouse anti-human BDNF monoclonal antibody (sc-546; 1:1,000 dilution) or mouse anti-human GAPDH monoclonal antibody (sc-47724; 1:1,000 dilution) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being washed three times for 10 min, the membranes were further incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000 dilution; sc-2005; Santa Cruz Biotechnology) for 1 h at room temperature. Finally, the protein bands were shown with the application of ECL Immunoblot Detection system (Pierce Biotechnology, Inc., Rockford, IL, USA). GAPDH was used as a loading control, and protein expression was measured using Quantity One software (Bio-Rad, Hercules, CA, USA).
The potential targets of miR-744 were analyzed using TargetScan (
For luciferase reporter assays, pMIR-BDNF-3′-UTR wild type (Wt) vector and pMIR-BDNF-3′-UTR mutant (Mut) vector were obtained from GenePharma Co., Ltd. Cells were seeded into 24-well plates and cotransfected with pMIR-BDNF-3′-UTR Wt or pMIR-BDNF-3′-UTR Mut, and miR-744 mimics or miR-NC using Lipofectamine 2000 reagent. Following incubation at 37°C for 48 h, the firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega, Madison, WI, USA), according to the manufacturer's protocol. Renilla luciferase was chosen as the normalization. Three independent experiments were performed.
Data were presented as mean ± standard deviation, and analyzed using (SPSS) version 18.0 (SPSS Inc., Chicago, IL, USA). The differences between two groups were analyzed using Students t-test, or assessed by one-way ANOVA when there were more than two groups. A two-tailed value of P less than 0.05 was considered statistically significant.
To determine whether miR-744 expression is associated with gastric cancer, we measured the miR-744 expression of five human gastric cancer cell lines (AGS, MKN-45, SGC-7901, BGC-823 and MGC-803) and that of a human gastric epithelial cell line (GES-1). Results showed that miR-744 was significantly downregulated in the gastric cancer cell lines compared with that in GES-1 (
To clarify the potential clinical significance of miR-744 in gastric cancer, all patients were divided into two groups according to the median miR-744 value: Low-miR-744 group (n=28) and high-miR-744 group (n=26). As shown in
To investigate the biological relevance of miR-744 downregulation on gastric cancer progression, we transfected the gastric cancer cell lines SGC-7901 and BGC-823 with either miR-744 mimics or miR-NC. Transfection efficiency was confirmed in both cell lines using RT-qPCR (
To elucidate the underlying mechanism by which miR-744 affects the biological functions of gastric cancer cells, the potential targets of miR-744 were determined using bioinformatic prediction. Among these predicted targets, NKD1 (
We detected BDNF expression in gastric cancer and normal gastric tissues to further explore the association between miR-744 and BDNF in gastric cancer. RT-qPCR and Western blot analyses indicated that BDNF expression was upregulated in gastric cancer tissues compared with normal gastric tissues at both mRNA (
After identifying BDNF as a direct target of miR-744 in gastric cancer, we next focused on whether BDNF could mediate the biological roles of miR-744 in gastric cancer. SGC-7901 and BGC-823 cells were transfected with miR-NC, miR-744 mimics or miR-744 mimics combined with pCMV6-BDNF. Western blot analysis confirmed that the ectopic expression of miR-744 suppressed BDNF protein expression, whereas co-transfection with pCMV6-BDNF could recover the BDNF expression in SGC-7901 and BGC-823 cells (
An increasing number of evidence demonstrated that miRNAs are aberrantly expressed in various types of human cancer (
The deregulation of miR-744 has been reported in different types of human cancer. For example, miR-744 is downregulated in both hepatocellular carcinoma tissues and cell lines (
Previous studies showed that miR-744 serves as a tumor suppressor in the development of human cancers. Lin
BDNF, which is located on the short arm of chromosome 11 (11p13), is an important neurotrophin in the brain (
In conclusion, miR-744, which is downregulated in gastric cancer tissues and cell lines, significantly correlates with lymph node metastasis, invasive depth and TNM staging in gastric cancer patients. Furthermore, miR-744 overexpression suppresses the proliferation and invasion of gastric cancer cells by directly targeting BDNF. Further research exploring the anti-cancer role of miR-744 in gastric cancer may contribute to the development of new therapeutic strategies for patients with this disease.
Expression of miR-744 in gastric cancer cell lines and tissue samples. (A) Expression of miR-744 was examined through RT-qPCR in gastric cancer cell lines (AGS, MKN-45, SGC-7901, BGC-823 and MGC-803) and a human gastric epithelial cell line (GES-1). *P<0.05 vs. GES-1. (B) Expression of miR-744 was determined via RT-qPCR in fifty-four pairs of gastric cancer and normal gastric tissues. *P<0.05 vs. normal gastric tissues.
Effect of miR744 on the proliferation and invasion of gastric cancer cells. (A and B) Transfection of miR-744 mimics into SGC-7901 and BGC-823 cells markedly increased miR-744 expression as indicated by the results of RT-qPCR analysis. *P<0.05 vs. miR-NC. (C) CCK-8 assays were utilised to evaluate the effect of miR-744 overexpression on the proliferation of SGC-7901 and BGC-823 cells. *P<0.05 vs. miR-NC. (D) Transwell cell invasion assay was performed to measure the invasiveness of SGC-7901 and BGC-823 cells transfected with miR-744 mimics or miR-NC. *P<0.05 vs. miR-NC.
BDNF is a direct target of miR-744 in gastric cancer. (A) Putative binding sites for miR-744 in the 3′-UTR of BDNF were determined using bioinformatic prediction. Mutated sites within the binding sites are shown. (B) Luciferase activities was detected in SGC-7901 and BGC-823 cells co-transfected with reporter plasmids carrying the wild-type (Wt) or mutant (Mut) BDNF 3′-UTR and with miR-744 mimics or miR-NC. *P<0.05 vs. miR-NC. (C) RT-qPCR was used to measure BDNF mRNA expression in SGC-7901 and BGC-823 cells transfected with miR-744 mimics or miR-NC. *P<0.05 vs. miR-NC. (D) Western blot was adopted to detect BDNF protein expression in SGC-7901 and BGC-823 cells transfected with miR-744 mimics or miR-NC. *P<0.05 vs. miR-NC. BDNF, brain-derived neurotrophic factor; CCK-8, Cell Counting Kit-8; 3′-UTR, 3′-untranslated region.
BDNF expression is upregulated in gastric cancer tissues and inversely correlated with miR-744 level. (A) Relative mRNA expression of BDNF was determined through RT-qPCR in gastric cancer and normal gastric tissues. *P<0.05 vs. normal gastric tissues. (B) Western blot analysis of BDNF protein expression in gastric cancer and normal gastric tissues. (C) Spearman's correlation analysis was conducted to explore the correlation between miR-744 and BDNF mRNA expression in gastric cancer tissues. r=−0.6833, P<0.0001. BDNF, brain-derived neurotrophic factor.
BDNF overexpression partially rescued the miR-744-inhibited proliferation and invasion of gastric cancer cells. (A) Western blot analysis of BDNF protein expression in SGC-7901 and BGC-823 cells co-transfected with miR-NC or miR-744 mimics alone or miR-744 mimics together with pCMV6-BDNF. *P<0.05 vs. miR-NC and miR-744 mimics together with pCMV6-BDNF. (B and C) BDNF upregulation reverted the suppressive effects of miR-744 overexpression on the proliferation and invasion of SGC-7901 and BGC-823 cells. *P<0.05 vs. miR-NC and miR-744 mimics together with pCMV6-BDNF. BDNF, brain-derived neurotrophic factor.
Relationship between microRNA-744 expression and clinicopathological factors of patients with gastric cancer.
microRNA-744 expression | ||||
---|---|---|---|---|
Clinicopathologic factors | No. of cases | Low | High | P-value |
Age (years) | 0.377 | |||
<60 | 22 | 13 | 9 | |
≥60 | 32 | 15 | 17 | |
Gender | 0.607 | |||
Male | 31 | 17 | 14 | |
Female | 23 | 11 | 12 | |
Tumor size (cm) | 0.599 | |||
<4 | 25 | 12 | 13 | |
≥4 | 29 | 16 | 13 | |
Differentiation | 0.761 | |||
Well and moderate | 30 | 15 | 15 | |
Poor and signet | 24 | 13 | 11 | |
Lymph node metastasis | 0.030 |
|||
No | 25 | 9 | 16 | |
Yes | 29 | 19 | 10 | |
Invasive depth | 0.005 |
|||
T1+T2 | 31 | 11 | 20 | |
T3+T4 | 23 | 17 | 6 | |
TNM staging | 0.006 |
|||
I–II | 21 | 6 | 15 | |
III–IV | 33 | 22 | 11 |
P<0.05.