Gastric cancer (GC) is one of the most common malignancies with a high worldwide incidence rate. The association between microRNAs (miRs) and malignancy has been widely studied in recent years. The aim of the present study was to assess the clinical value of miR-4636 in patients with GC and its effect on the proliferation, migration and invasion of GC cells. Reverse transcription-quantitative PCR was used to detect the expression of miR-4636. Receiver operating characteristics curve, Kaplan-Meier survival curve and Cox regression analyses were used to evaluate the diagnostic and prognostic value of miR-4636. Transwell migration and MTT assays were used to assess the regulatory effects of miR-4636 expression on the biological function of GC. The results demonstrated that the expression of miR-4636 was significantly downregulated in GC serum and tissue samples, as well as in GC cell lines. The aberrant miR-4636 expression was closely associated with lymph node metastasis and TNM stage, and had considerable diagnostic and prognostic significance in patients with GC. Cellular experiments revealed that the overexpression of miR-4636 inhibited GC cell proliferation, migration and invasion, while the knockdown of miR-4636 led to opposite effects on the biological function of GC. In summary, decreased miR-4636 expression may serve as a biomarker for the diagnosis and prognosis of GC. Furthermore, miR-4636 overexpression significantly inhibited GC cell proliferation, migration and invasion, indicating the potential of miR-4636 as a therapeutic target for GC treatment.
Gastric cancer (GC) is the third most common malignant tumor and a leading cause of cancer-related mortality worldwide according to statistics from 2008 (
MicroRNAs (miRNAs or miRs) are small non-coding RNAs approximately 18–25 nucleotides in length with important regulatory effects on gene expression at the post-transcriptional level (
To further improve the treatment of GC, the present study analyzed the expression levels of miR-4636 in patients with GC and in GC cell lines to evaluate the diagnostic and prognostic significance of miR-4636 and its biological function in GC progression. The results may provide a potential novel biomarker and therapeutic target for the diagnosis, prognosis and treatment of GC.
A total of 112 patients (65 males and 47 females) who underwent surgery at Zibo Central Hospital (Zibo, China) between June 2009 and May 2013 were recruited. The mean age of the patients was 64.87±11.31 years (range, 45–85 years). The inclusion criteria were as follows: i) All the patients were pathologically diagnosed with GC; ii) none of the patients had received any therapy prior to surgery; and iii) had complete clinicopathological data. The exclusion criteria were as follows: i) Patients younger than 18 years or older than 80 years; ii) pregnant or lactating patients; and iii) patients that had autoimmune diseases or other malignancies. Furthermore, 60 healthy individuals with an average age of 65.23±10.96 years (range, 46–84 years) were recruited during the same time period. The healthy volunteers included 35 males and 25 females, and had no history of malignancy. Venous blood was obtained from all the participants and serum was isolated by centrifugation at 1,500 × g for 15 min at 4°C, that was then stored at −80°C until further use. Additionally, GC tissue samples and adjacent normal tissue samples, which were located 3 cm from the edge of the tumors, were collected from the patients and frozen with liquid nitrogen at −80°C for future use. All the patients received a 5-year follow-up survey and their survival information was recorded. Prior to sampling, all the patients and healthy volunteers provided written informed consent for clinical sample use and analysis, and the experimental methods were approved by the Ethics Committee of Zibo Central Hospital (approval no. ZCHh-090618).
The GC cell lines AGS, HGC27, HS746T and MKN45 and the gastric epithelial cell line GES-1 were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium (BioTek Corporation) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2.
GC cell lines AGS and MGN45 were subjected to cell transfection. Sequences for 50 nM miR-4636 mimics (5′-AACUCGUGUUCAAAGCCUUUAG-3′), 100 nM miR-4636 inhibitors (5′-CUAAAGGCUUUGAACACGAGUU-3′) or their respective negative controls (50 nM mimic NC, 5′-UUCUCCGAACGUGUCACGU-3′ and 100 nM inhibitor NC, 5′-CAGUACUUUUGUGUAGUACAA-3′) were transfected into AGS and MGN45 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C according to the manufacturer's protocol. After 48 h of cell transfection, the cells were used for subsequent experiments. The cells transfected with only transfection reagents were used as a mock group.
Total RNA was extracted from GC tissues and cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized from RNA using a PrimeScript RT reagent kit (Takara Bio, Inc.), according to the manufacturer's protocol. The expression levels of miR-4636 were detected by qPCR, which was conducted using a SYBR-Green I Master Mix kit (Invitrogen; Thermo Fisher Scientific, Inc.) on a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C, 10 min for initial denaturation, 40 cycles of 95°C, 30 sec for denaturation, 58°C, 20 sec for annealing, 72°C, 30 sec for elongation, and 72°C, 10 min for final extension. U6 was used as the internal control and the final expression values of miR-4636 were quantified using the 2−ΔΔCq method (
At 48-h post-transfection, AGS and MKN45 cells (3×103 cells/well) were seeded into 96-well plates and the cell proliferation was analyzed using a MTT assay. The cell plates were retained in an incubator at 37°C for 3 days. A total of 10 µl MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA) was added to each well every 24 h followed by incubation for a further 4 h. Following this, 150 µl DMSO was added in each well to dissolve the violet formazan crystals. Following incubation, the absorbance of the cultures was determined at a wavelength of 570 nm using a microplate reader.
The invasion and migration abilities of AGS and MKN45 cells were analyzed using Transwell chambers with 8 µm pore membranes (Corning, Inc.) precoated with Matrigel (for invasion assays) or without Matrigel (for migration assays). GC cells (3×105 cells/well) were seeded into the upper chamber with serum-free RPMI-1640 medium. The lower chambers contained medium supplemented with 10% FBS. Following 24 h of incubation at 37°C, the cells in the lower chambers were stained using 0.1% crystal violet for 10 min at room temperature, and were calculated under an inverted light microscope (Olympus Corporation).
The statistical analyses were performed using SPSS (version no. 21.0; IBM Corp.) and GraphPad Prism (version no. 7.0; GraphPad Software, Inc.) software. Differences between groups were analyzed using Student's
The serum expression levels of miR-4636 in patients with GC were significantly lower compared with healthy controls (P<0.001;
As presented in
A ROC curve based on serum miR-4636 levels was constructed, which demonstrated that the area under the curve (AUC) was 0.963, indicating the diagnostic accuracy of serum miR-4636 in patients with GC (
By evaluating the 5-year follow-up data, the KM method was used to construct the overall survival and disease-free survival curves of patients with GC. Patients with GC and low miR-4636 expression had shorter survival times compared with patients with a high miR-4636 expression (log-rank, P<0.001;
The functional role of miR-4636 in GC progression was further investigated. GC cell lines AGS and MKN45 were selected for the cell experiments due to their significantly lower expression of miR-4636 expression compared with the normal cell line GES-1. The expression of mi-4636 was upregulated by miR-4636 mimics and downregulated by miR-4636 inhibitors compared with the mock group in the AGS and MKN45 cell lines (all, P<0.001;
The Transwell assay results demonstrated that the overexpression of miR-4636 inhibited GC cell migration, while the miR-4636 knockdown promoted cell migration in the AGS and MKN45 cell lines compared with the mock group (P<0.01 or P<0.001;
GC is a heterogeneous malignant disease (
miR-4636 has been reported to be deregulated in certain human cancers. For instance, Yin
Given the results by Yin
Numerous downregulated miRNAs in human malignancies have been reported to be involved in the regulation of tumor progression (
In conclusion, the present study demonstrated that miR-4636 expression was decreased in the serum and tissues of patients with GC and that miR-4636 dysregulation may serve as a biomarker for the diagnosis and prognosis of GC. Additionally, the results revealed a significant inhibitory effect of miR-4636 on GC cell proliferation, migration and invasion, indicating that may miR-4636 be a potential therapeutic target for the treatment of GC. Methods to increase miR-4636 expression may help in the development of novel therapeutic approaches for GC therapy.
Not applicable.
No funding was received.
All data generated or analyzed during this study are included in this published article.
JT and CG conducted the clinical study, analyzed clinical data and wrote the manuscript. YH and CZ performed the cell experiments. All authors read and approved the final manuscript.
Prior to sampling, all the patients and healthy volunteers provided written informed consent for clinical sample use and analysis, and the experimental methods were approved by the Ethics Committee of Zibo Central Hospital, Zibo, China (approval no. ZCHh-090618).
Written informed consent for publication was obtained.
The authors declare that they have no competing interests.
Expression of miR-4636 in patients with GC and GC cell lines. (A) Expression of miR-4636 in serum samples collected from healthy individuals and patients with GC. ***P<0.001 vs. healthy controls. (B) Expression of miR-4636 in GC tissues and adjacent normal tissues. ***P<0.001 vs. normal tissues. (C) Expression of miR-4636 in the GC cell lines. *P<0.05, **P<0.01 and ***P<0.001 vs. GES-1 cells. miR, microRNA; GC, gastric cancer.
Clinical significance of miR-4636 in the diagnosis and prognosis of GC. (A) A receiver operating characteristics curve based on serum miR-4636 levels of patients with GC (AUC, 0.963). (B) The Kaplan-Meier overall survival curves for patients with GC and different expression patterns of miR-4636 (log-rank, P<0.001). (C) The disease-free survival curves for patients with GC with high or low miR-4636 expression (log-rank, P=0.006). miR, microRNA; GC, gastric cancer; AUC, area under the curve.
Dysregulation of miR-4636 regulates cell proliferation of AGS and MKN45 cells. The expression of miR-4636 in (A) AGS and (B) MKN45 cells was upregulated by miR-4636 mimics and downregulated by miR-4636 inhibitors. Proliferation in (C) AGS and (D) MKN45 cells was inhibited by the overexpression of miR-4636 and promoted by the knockdown of miR-4636. *P<0.05 and ***P<0.001 vs. the mock group. miR, microRNA; NC, negative control.
Effects of miR-4636 on cell migration and invasion in AGS and MKN45 cells. (A) Cell migration was inhibited by the upregulated expression of miR-4636 and enhanced by decreased miR-4636 expression in both cell lines. (B) Cell invasion in AGS and MKN45 cells was significantly suppressed by the upregulation of miR-4636 and promoted by the downregulation of miR-4636. Magnification, ×200. **P<0.01 and ***P<0.001 vs. the mock group. miR, microRNA; NC, negative control.
Association between miR-4636 and the clinical characteristics of patients with gastric cancer.
miR-4636 in serum | miR-4636 in tissues | ||||||
---|---|---|---|---|---|---|---|
Characteristic | Total (n=112) | Low (n=60) | High (n=52) | P-value | Low (n=62) | High (n=50) | P-value |
Age (years) | 0.821 | 0.734 | |||||
<60 | 40 | 22 | 18 | 23 | 17 | ||
≥60 | 72 | 38 | 34 | 39 | 33 | ||
Sex | 0.945 | 0.572 | |||||
Female | 47 | 25 | 22 | 24 | 22 | ||
Male | 65 | 35 | 30 | 38 | 28 | ||
Tumor size (cm) | 0.143 | 0.068 | |||||
<5 | 52 | 24 | 28 | 24 | 28 | ||
≥5 | 60 | 36 | 24 | 38 | 22 | ||
Differentiation | 0.054 | 0.052 | |||||
Well/moderate | 58 | 26 | 32 | 27 | 31 | ||
Poor | 54 | 34 | 20 | 35 | 19 | ||
Lymph node metastasis | 0.026 | 0.010 |
|||||
Negative | 52 | 22 | 30 | 22 | 30 | ||
Positive | 60 | 38 | 22 | 40 | 20 | ||
TNM stage | 0.017 | 0.019 |
|||||
I–II | 49 | 20 | 29 | 21 | 28 | ||
III–IV | 63 | 40 | 23 | 42 | 22 |
P<0.05. miR, microRNA.
Cox regression analysis in patients with gastric cancer.
Parameter | HR value | 95% CI | P-value |
---|---|---|---|
miR-4636 | 3.522 | 2.016–6.154 | <0.001 |
Age | 1.681 | 0.997–2.829 | 0.053 |
Sex | 1.397 | 0.880–2.217 | 0.156 |
Tumor size | 1.377 | 0.855–2.217 | 0.189 |
Differentiation | 1.149 | 0.717–1.842 | 0.564 |
Lymph node metastasis | 1.527 | 0.927–2.516 | 0.097 |
TNM stage | 1.800 | 1.093–2.966 | 0.021 |
miR, microRNA.