Abstract
Gastric cancer (GC) is the fifth most common type of malignant tumor worldwide and the most common cause of cancer-associated mortality in China. Recent studies revealed that microRNAs (miRNAs) function in the pathogenesis of GC, and that miR-155-5p expression is downregulated in GC tissues. However, the function of miR-155-5p has not been fully identified. In the present study, it was demonstrated that overexpression of miR-155-5p inhibited GC-cell proliferation and promoted apoptosis, while downregulation of miR-155-5p promoted GC-cell proliferation and decreased the cisplatin sensitivity of GC cells. Mitogen-activated protein kinase kinase kinase 10 was demonstrated to be a potential target gene of miR-155-5p. In conclusion, an antitumor role of miR-155-5p in gastric cancer was suggested.
Keywords: microRNA-155-5p, proliferation, apoptosis, gastric cancer
Introduction
Gastric cancer (GC) is the fifth most common type of malignant tumor worldwide and the third most common cause of cancer-associated mortality (1,2). Approximately 50% of cases occur in Eastern Asia (mainly in China) (3). Therefore, GC is a major public health problem, particularly in China. In the majority of cases, GC patients are diagnosed at advanced stages, at which effective therapeutic strategies are limited and the prognosis is relatively poor (4–6). Invasion and metastasis are hallmarks of advanced GC progression; therefore, investigation of the molecular pathogenesis of GC is critical to improve the survival of GC patients.
miRNAs are evolutionary conserved small non-coding RNAs and are involved in the regulation of gene expression and protein translation (7). miRNAs function in the pathogenesis of GC (8–14).
The role of miR-155-5p in various types of cancer has been revealed in recent studies. In colorectal carcinoma (CRC), miR-155-5p expression is upregulated and has been indicated to promote the proliferation, invasion and metastasis of CRC cells (15). In osteosarcoma, increased miR-155-5p and reduced miR-148a-3p expression were demonstrated to contribute to the suppression of tumor cell death (16). In GC, miR-155-5p was demonstrated to be downregulated in GC tissues (17), and miR-155-5p inhibition promoted the transformation of bone marrow mesenchymal stem cells into GC tissue-derived MSC-like cells via nuclear factor-κB p65 activation (18). However, the role of miR-155-5p in GC has not been fully elucidated.
In the present study, the effect of miR-155-5p on GC-cell proliferation and apoptosis was investigated. The data achieved may provide a novel therapeutic target for further investigation.
Materials and methods
Patients and tissues
A total of 14 GC tissues and corresponding adjacent normal tissues were collected from 14 patients with GC who underwent surgery at the Department of Gastroenterology, Center Hospital of Nanchong City. College (Nanchong, China) from January 2013 to December 2013. The histopathological diagnoses of all patients were confirmed by a senior pathologist at West China Hospital (Chengdu, China). The information of the 14 GC patients (average age: 60.2 years, age range: 36–63 years) are listed in Table I. The sex ratio was 50:50. The present study was approved by the ethics committee of Northern Sichuan Medical College (Nanchong, Sichuan), and all patients provided written informed consent.
Table I.
Patient | Age (years) | Sex | Stage | Differentiation |
---|---|---|---|---|
1 | 45 | Female | III | Low |
2 | 63 | Female | II | Moderate |
3 | 56 | Female | II | Low |
4 | 45 | Female | III | Moderate |
5 | 59 | Male | III | Moderate |
6 | 50 | Male | IV | Moderate |
7 | 56 | Male | III | Moderate |
8 | 77 | Female | IV | Low |
9 | 66 | Male | II | Moderate |
10 | 73 | Female | II | Moderate |
11 | 48 | Male | III | Low |
12 | 67 | Male | III | Low |
13 | 53 | Female | IV | Moderate |
14 | 50 | Male | III | Moderate |
Cell lines and reagents
The GC cell lines, AGS and SGC-7901, were purchased from the Chinese Academy of Sciences (Shanghai, China). The human gastric epithelial mucosa cell line, GES-1, was gifted by the Department of Gastrology, West China Hospital, Sichuan University (Chengdu, China). Cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) (19). Cisplatin was purchased from Hanson Pharma (Lianyungang, China; http://www.hansoh.cn/). Cisplatin was added to the cultures at a final concentration of 25 µM, as previously described (20).
miR-155-5p mimics and oligonucleotide transfection
The miR-155-5p mimics, miR-155-5 oligonucleotides, miR-155-5p antisense oligonucleotides (ASO) and negative controls were purchased from Dharmacon, Inc. (Chicago, IL, USA). Cells were seeded at 2×105 per well in 6-well plates, and transfected with mimics (50 nM), oligonucleotides (50 nM) or controls (50 nM) using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.), according to the standard protocol. The sequence of these molecules were listed as following: miR-155-5p mimics, 5′-UUAAUGCUAAUCGUCAUAGGGGU-3′; miR-155-5p-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; miR-155-5p ASO, 5′-ACCCCUAUCACGAUUAGCAUUAA-3′; miR-155-5p ASO-NC, 5′-CAGUACUUUUGUGUAGUACAA-3′. A total of 24 h following transfection, the miR-155-5p levels were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
Western blot analysis
The lysates were prepared with lysis buffer (Abcam, Cambridge, UK) containing protease inhibitors and then centrifuged (12,000 × g for 5 min in 4°C). The protein levels were determined by BCA protein quantification kit (cat. no. ab102536; Abcam). A total of 20 µg protein was separated by SDS-PAGE (10%) and transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% skimmed milk for 15 min at room temperature. Following washing with PBS for 15 min, the membranes were incubated with MA3PK10 (cat. no. orb127031; 1:1,000; BioPike, LCC, Shanghai, China), or b-actin (1:4,000; cat. no. ab8227; Abcam) primary antibodies overnight at 4°C. The membranes were then washed by PBS three times. Next the membranes were incubated with a rabbit anti-mouse secondary antibody conjugated with horseradish peroxidase (1:5,000; cat. no. ab6728; Abcam) for 2 h at room temperature. The proteins were visualized using an enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.).
Cell proliferation assay
Cell proliferation was assessed by MTT assay. Cells were seeded into 96-well plates at a density of 5×105/well. MTT was added to the medium at a final concentration of 0.1 mg/ml. A total of 100 µl dimethyl sulfoxide was used to dissolve the formazan crystals in each well. The OD was measured using a microplate reader at a wavelength of 570 nm.
RT-qPCR
GC tissues and cell lines were incubated with TRIzol® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Total RNA was isolated from the tissues and cell lines using a mirVana miRNA Isolation kit (Thermo Fisher Scientific, Inc.). Reverse transcription was performed using an All-in-one™ miRNA First-Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). The primers were synthesized by the Sangon Biotech Company (Shanghai, China, http://www.sangon.com/). PCR assay was performed as previously described, with miR-155-5p expression normalized to U6 snRNA expression (14,21–24). The primers were: miR-155-5p, forward, 5′-UAAUACCGUCUUAAAACCGU-3′, and reverse, 5′-UUCUGGGAACGUGAAACCT-3′; and U6 snRNA, forward, 5′-CGCTTCGGCAGCACATATACTAAAATTGGAAC-3′, and reverse, 5′-GCTTCACGAATTTGCGTGTCATCCTTGC-3′. All the reaction mixtures were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 40 sec.
Cell apoptosis analysis
Cell apoptosis were analyzed by Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Staining Detection kit (cat. no. ab14085; Abcam) according to the manufacturer's protocol. In detail, 1×105 cells were suspended in 1X Annexin V Binding Buffer, then 5 µl Annexin V-FITC and 5 µl Propidium Iodide was added for incubation at room temperature for 5 min in the dark. Samples were analyzed using a flow assisted cell sorting analyzer instrument (BD LSR II, FACSDiva software v.1.1.0; BD Biosciences) using the 488 nm excitation line and emission was detected at 530 nm (green, FITC) and 575–610 nm (orange, PI) (22).
Dual luciferase reporter assays
Cells were seeded at 2×105 cells/well and were serum-starved for 6 h pre-transfection. A mutated version of the mitogen-activated protein kinase kinase kinase 10 (MAP3K10) 3′untranslated region (3′UTR) was generated using a Site-Directed Mutagenesis kit (Promega Corporation), as previously described (25). The intact 3′UTR of MAP3K10 and the mutated version were cloned into a luciferase reporter plasmid (500 ng; cat. no. K801-200; NeoBioscience, Shanghai, China; http://www.nbs-bio.com/). The plasmid, and a pGL3-control (100 ng; Thermo Fisher Scientific, Inc.) were co-transfected using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. A total of 24 h later, cells were harvested and the luciferase activities were analyzed using a dual-luciferase reporter assay system (Promega Corporation, Madison, WI, USA). The luciferase activity was normalized to Renilla luciferase activity.
Prediction of the possible targets of miR-155-5p
Targetscan software (http://www.targetscan.org/) (26–31) was used to predict possible targets of miR-155-5p.
Statistical analysis
Statistical analysis was performed using SPSS 10.0 software (SPSS, Inc., Chicago, IL, USA). The difference between two groups was analyzed using a two-tailed Student's t-test. Analysis of variance was used to analyze differences among ≥3 groups, with post hoc contrasts performed using Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.
Results
miR-155-5p expression is low in GC tissues
To investigate the function of miR-155-5p in the pathogenesis of GC, the miR-155-5p expression levels in 14 GC tissues were investigated by RT-qPCR. It was demonstrated that all GC tissues exhibited low levels of miR-155-5p compared with their matched adjacent normal tissues (Fig. 1A). As expected, the mean expression of miR-155-5p in GC tumor tissues was low compared with that in normal control tissues (Fig. 1B). Thus, we hypothesized that miR-155-5p may serve an antitumor role in GC.
Overexpression of miR-155-5p inhibits GC cell proliferation and promotes apoptosis
Subsequently, miR-155-5p expression levels were analyzed in the GC cell lines, AGS-1 and SGC-7901, using the gastric epithelial mucosa cell line, GES-1, as a negative control. Using RT-qPCR, it was demonstrated that AGS-1 and SGC-7901 cells expressed increased levels of miR-155-5p compared with GES-1 cells (Fig. 2A). Overexpression of miR-155-5p in AGS-1 and SGC-7901 was confirmed by RT-qPCR (Fig. 2B). Following transfection with miR-155-5p mimics, proliferation was analyzed by MTT assay. This revealed that miR-155-5p overexpression inhibited AGS-1 and SGC-7910 cell proliferation (Fig. 2C). A total of 48 h post-transfection, apoptosis was analyzed by Annexin V-FITC and PI staining. This indicated that miR-155-5p overexpression increased the apoptotic rates in AGS-1 and SGC-7910 cells (Fig. 2D).
Downregulation of miR-155-5p promotes GC cell growth and decreases the sensitivity of GC cells to cisplatin
It was demonstrated that miR-155-5p ASO transfection downregulated miR-155-5p expression levels in AGS-1 and SGC-7901 cells (Fig. 3A). miR-155-5p ASO transfection also promoted the proliferation of AGS-1 and SGC-7901 cells (Fig. 3B). miR-155-5p ASO transfection also decreased the apoptotic rates of AGS-1 and SGC-7901 cells following treatment with cisplatin. Untransfected cells treated with cisplatin exhibited an increased apoptotic rate compared with untreated untransfected cells. Thus, miR-155-5p reduced the effect of cisplatin-induced apoptosis (Fig. 3C).
MAP3K10 is targeted by miR-155-5p
A previous study demonstrated that MAP3K10 was overexpressed in pancreatic ductal adenocarcinoma (PDAC), and that it promoted proliferation and decreased therapeutic impact in pancreatic cancer cells (32). Bioinformatics analysis suggested that MAP3K10 is a targeted gene of miR-155-5p (Fig. 4A). The luciferase activity of the reporter carrying the mutated 3′UTR of MAP3K10 was not significantly different in miR-NC and miR-155-5p-transfected cells, whereas miR-155-5p was demonstrated to reduce the luciferase activity of the wild type 3′UTR reporter (Fig. 4B). To investigate whether miR-155-5p reduced MAP3K10 protein levels, western blotting was performed 48 h following miR-155-5p mimics transfection. It was demonstrated that miR-155-5p mimics significantly decreased the protein expression level of MAP3K10 proteins in AGS-1 cells, compared with negative control (Fig. 4C).
Discussion
In a previous study, patients with GC exhibiting low expression of miR-124-3pm miR-146a-5p, miR-335-5p and miR-155-5p were associated with increased lymph node metastasis, lymphatic invasion, venous invasion, high Bormann stage, lymphatic invasion and poor differentiation compared with those exhibiting high expression (17). The present may contribute to the elucidation of the underlying molecular mechanism of the clinical significance of miR-155-5p. Low expression of miR-155-5p was demonstrated to promote cellular proliferation and decrease the cisplatin-sensitivity of GC cells. MAP3K10 was also indicated to be a target gene of miR-155-5p. MAP3K10 is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, it activates the C-Jun N-terminal kinase (JNK) signaling pathway and the p38 mitogen-activated-protein kinase (MAPK) pathway, and regulates apoptosis in numerous neurodegenerative diseases (32–34). It was demonstrated in the present study that MAP3K10 may be targeted by miR-155-5p in GC cell lines.
MAP3K10 has been demonstrated to promote the proliferation of pancreatic cancer cells and decrease the sensitivity to gemcitabine by upregulating the expression of GLI family zinc finger (Gli)-1 and Gli-2 (32). Whether Gli-1 and Gli-2 promote or inhibit GC tumor growth requires further investigation. In conclusion, the present study suggests that miR-155-5p serves an antitumor role in GC.
Acknowledgements
The authors would like to thank Ms. Miao Chen (Clinical Laboratory Department, The Affiliated Hospital of North Sichuan Medical College, Nanchong, China) for technical her assistance.
Funding
No funding was received.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
SL, TZ and XZ collected patient data and performed cell experiments. ZD and FC performed RT-qPCR, western blot analysis and other molecular experiment. JL and QL contributed to study design and manuscript writing.
Ethics approval and consent to participate
The present study was approved by the ethics committee of Northern Sichuan Medical College (Nanchong, China), and all patients provided written informed consent.
Consent for publication
All patients gave informed consent for the use of their tissues and publication of the data and images.
Competing interests
The authors declare that they have no competing interests.
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Data Availability Statement
All data generated or analyzed during this study are included in this published article.