MicroRNAs (miR) serve important roles in the development and progression of tumors by targeting different genes. miR-520a-3p reported in lung and breast cancers as a tumor suppressor gene. However, the expression and functional significance of miR-520a-3p is not completely understood in gastric cancer (GC). In the present study, it was demonstrated that the expression levels of miR-520a-3p were significantly downregulated in GC tissues and cells using RT-qPCR. In addition, downregulated expression of miR-520a-3p was associated with the clinical stage of the tumor and invasion in patients with GC. Furthermore, overexpression of miR-520a-3p significantly inhibited cell proliferation, invasion and migration in SGC-7901 and MGC-803 GC cell lines using proliferation, wound healing and cell invasion assays. Spindle and kinetochore associated 2 (SKA2) was upregulated in GC cells using western blot analysis and a target gene of miR-520a-3p; miR-520a-3p mimics significantly reduced SKA2 expression. In addition, upregulation of SKA2 protein expression SKA2 reversed the miR-520a-3p-mediated inhibition of SGC-7901 cell proliferation, migration and invasion. In conclusion, miR-520a-3p functioned as a tumor suppressor gene by targeting SKA2 in GC cell lines, and may serve as a novel prognostic and potential therapeutic marker.
Gastric cancer (GC) is a common cancer worldwide and remains the third leading cause of cancer-related death, following lung and liver cancer, accounting for 1.3 million cases of GC and 819,000 GC-associated cases of mortality in 2015 (
MicroRNAs (miRNAs/miRs) can function as oncogenes or tumor suppressor genes, and regulate gene expression through translational repression or mRNA degradation by binding to the 3′-untranslated region (3′UTR) of their target mRNAs (
Previous studies have demonstrated that the expression levels of miR-520a-3p are downregulated in non-small cell lung cancer cells. Decreased levels of miR-520a-3p expression were associated with poorer overall survival (
The present study examined the expression of miR-520a-3p in GC tissues and cells and demonstrated the functions of miR-520a-3p overexpression in GC cell proliferation, invasion and migration. Together, these data demonstrate that miR-520a-3p may be a novel therapeutic target for the treatment of GC.
GC specimens and adjacent tissues were received from patients who were diagnosed with GC and underwent surgery at Ningbo No. 2 Hospital (Zhejiang, China) between August 2016 and May 2017. A total of 80 female patients, aged between 41–75 (55.3±11.4) years, and had not received local or systemic therapy prior to surgery at Ningbo No. 2 Hospital. The present study was approved by The Research Ethics Committee of Ningbo No. 2 Hospital, and all subjects provided the information written consent. The samples were graded according to the 8th edition of the AJCC staging classification system (
All GC cell lines (SGC-7901, BGC-823, and MGC-803) and a normal gastric epithelial cell line (GES-1) used in the present study were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China) and maintained in DMEM (HyClone; GE Healthcare Life Sciences) medium (SGC-7901, BGC-823) or RPMI 1640 (HyClone; GE Healthcare Life Sciences) medium (MGC-803, GES-1) containing 10% FBS (Shanghai Ex CellBiology, Inc.) at 37°C, in a humidified incubator with 5% CO2 (Memmert GmbH).
Cells (SGC-7901 and MGC803) were transfected with miR-520a-3p mimics (100 pmol; forward, 5′-AAAGUGCUUCCCUUUGGACUGU-3′ and reverse, 5′-AGUCCAAAGGGAAGCACUUUUU-3′) or miR-520a-3p negative control (NC; 100 pmol; forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′; both from Shanghai GenePharma Co. Ltd.), or spindle and kinetochore associated 2 (SKA2) cDNA plasmid (pLenti-SKA2-Puro) or NC (pLenti-C-Myc-DDK-P2A-Puro) (4 µg; both from OriGene Technologies, Inc.) using Lipofectamine™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Cells were incubated with the transfection reagent for 24 h, following which RNA and protein were extracted. No treatment or transfection was performed in the MOCK group.
The SKA2 cDNA plasmid (without the 3′UTR) and miR-520a-3p mimics were co-transfected into SGC-7901 cells, which subsequently underwent western blot analyses, cell proliferation assays, wound healing assay and cell invasion assays.
Total cell protein was extracted by 1XSDS (Sigma-Aldrich; Merck KGaA) and quantified by a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Cellular proteins (40–50 µg) were loaded onto a 12% polyacrylamide gel and resolved by SDS-PAGE. After separation, the proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore) for immunoblotting. Membranes were blocked with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used were Ki-67 (1:1,500; Abcam; cat no. ab15580), matrix metalloproteinase (MMP)-2 (1:1,500; Abcam; cat no. ab7033), MMP-9 (1:1,500; Abcam; cat no. ab137651), SKA2 (1:1,500; Abcam; cat no. ab91551) and GAPDH (1:2,000; Cell Signaling Technology, Inc.; cat. no. 5174). The membranes were subsequently incubated for 1 h at room temperature with goat anti-rabbit IgG-horseradish peroxidase (HRP) or goat anti-mouse IgG-HRP (1:5,000; Wuhan Boster Biological Technology, Ltd.; cat. nos. BA1054 and BA1050, respectively). The protein signals on the membrane were detected using an ECL reagent (Absin Biotechnology Co., Ltd.) and visualized using a chemiluminescence imaging system (LI-COR Biosciences).
The expression levels of miR-520a-3p and SKA2 were determined using RT-qPCR. Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The RNA was reverse transcribed to cDNA using a TaqMan reverse transcription kit (Thermo Fisher Scientific, Inc.) with the following RT protocol; 15 min at 37°C and 5 sec at 85°C. cDNA was amplified using SYBR-Green PCR Master mix (Roche Diagnostics) on a LightCycler 480 system. The thermocycling conditions were as follows: 95°C for 10 min; and followed by 45 cycles, 95°C for 10 sec and 60°C for 60 sec. Fold changes were calculated by relative quantification (2−∆∆Cq) using endogenous U6 and GAPDH as references for miR-520a-3p and SKA2 expression, respectively (
miR-520a-3p mimics, SKA2 cDNA and negative control-transfected GC cells (SGC-7901 and MGC803) were seeded in 96-well plates (1×104 cells/well). A total of 20 µl Cell Titer 96® AQueous One Solution (Promega Corporation) was added to each well to determine cell viability at 4, 24, 48, 72 and 96 h after seeding. Cells were incubated at 37°C, in a humidified incubator with 5% CO2 for 3 h. Subsequently the absorbance of each well was measured at 490 nm on a spectrophotometer (Beckman Coulter, Inc.).
Cells were cultured in 6-well plates (1×105 cells/well) for 24 h. Once the cellular density had reached ~100%, a vertical line was scraped in the culture using a 1-ml pipette tip. The cells were rinsed with PBS and incubated with fresh serum-free medium at 37°C. The widths of the gap at 0 and 24 h were compared to assess the distance of migration using fluorescence microscopy.
Cell invasion assays were performed using a Matrigel invasion chamber (24-well plates; 8-µm pore size; Corning Inc.) according to the manufacturer's protocol. A total of 5×104 cells were seeded in the upper chambers of the wells in 100 µl FBS-free medium, and the lower chambers contained DMEM supplemented with 20% FBS. The cells were incubated for 24 h at 37°C, after which the cells on the filter surface were stained with 0.1% crystal violet for 30 min at room temperature and images were captured with an inverted fluorescence microscope (Nikon Corporation), representative images were captured at ×100 magnification. The absorbance was measured at 590 nm.
Potential miR-520a-3p targets were predicted and analyzed using the following two publicly available databases: TargetScanv7.2 (
The predicted miR-520a-3p binding sites on the 3′UTR of wild-type (WT) SKA2 (MirTarget-SKA2-3U-WT), together with a corresponding mutant (mut) miR-520a-3p binding sites on the 3′UTR of SAK2 (MirTarget-SKA2-3U-Mut), were synthesized and transfected into the pGL3 vector (Promega Corporation). SGC-7901 cells were seeded in a 24-well plates (5×104 cells/well), the WT or Mut 3′UTR vectors, and miR-520a-3p mimics or NC were co-transfected using Lipofectamine™ 2000. The relative luciferase activity was measured after 48 h using a Dual-Luciferase Reporter assay system according to the manufacturer's protocols (Promega Corporation). Luciferase activity was normalized to
All experiments were repeated three times and data are presented as the mean ± standard deviation. One-way ANOVA and Fisher's least significant difference tests were used to calculate P-values between multiple groups. Data in
miR-520a-3p expression levels in 80 GC samples and adjacent tissue samples were measured by RT-qPCR, and the levels were normalized to U6. The results demonstrated that miR-520a-3p expression levels were significantly decreased in GC tissues compared with the adjacent normal tissues (P<0.01;
To investigate the clinical significance of miR-520a-3p in patients with GC, miR-520a-3p expression in a cohort of 80 specimens was analyzed (
To investigate the functional effects of miR-520a-3p expression in GC, SGC-7901 and MGC-803 cells were transfected with MOCK, miR-NC or miR-520a-3p mimics. Treatment with miR-520a-3p mimics significantly increased the expression levels of miR-520a-3p (P<0.001), compared with the MOCK group (
To examine how miR-520a-3p affects the migration and invasion of GC cells, the effects on cell migration and invasion were investigated
Interactions between cell-surface proteins and the extracellular matrix (ECM) serve a critical role in tumor migration and invasion (
To investigate the molecular mechanism underlying the effects of miR-520a-3p on GC cellular functions, SKA2 mRNA was identified as a potential miR-520a-3p target using bioinformatics (
To determine whether SKA2 overexpression reversed the inhibitory effects of miR-520a-3p mimics on SGC-7901 cells, SKA2 cDNA without the 3′UTR and miR-520a-3p mimics were co-transfected into SGC-7901 cells (P<0.01;
miR-520a-3p serves an tumor suppressive role in certain types of cancer (
The results demonstrated that miR-520a-3p was downregulated both in human GC tissues and GC cell lines compared with adjacent tissues and a normal gastric cell line. In addition, the expression of miR-520a-3p was associated with clinical stage and tumor invasion in patients with GC. Subsequent experimentation revealed that the upregulation of miR-520a-3p inhibited the proliferation, migration and invasion of GC cells (SGC-7901 and MGC-803), potentially by directly inhibiting SKA2. Furthermore, miR-520a-3p significantly inhibited the level of SKA2, and overexpression of SKA2 rescued the miR-520a-3p-mediated inhibition of SGC-7901 cell proliferation, migration and invasion. Therefore, the present study further confirmed the several biological functions attributed to miR-520a-3p, including the reduction in GC cell viability, and inhibition of cell migration and invasion following transfection with miR-520a-3p mimics, as well as the molecular mechanism underlying the miR-520a-3p-mediated inhibitory effect (
As a potential target gene of miR-520a-3p predicted by miRanda and TargetScan software, the present study analyzed the relationship between SKA2 and miR-520a-3p in SGC-7901 cells. The results demonstrated that miR-520a-3p mimics significantly attenuated the mRNA and protein expression of SKA2. Furthermore, downregulation of SKA2 by miR-520a-3p mimics was reversed by SKA2 cDNA plasmid. These results suggested that miR-520a-3p directly repressed the expression of SKA2 in GC by binding to its 3′UTR.
A previous study reported that miR-520d-3p inhibited GC cell growth and metastatic activity by targeting EphA2 (
In summary, miR-520a-3p was downregulated in GC tissues and cell lines. miR-520a-3p also serves a role as a tumor suppressor gene in GC cell lines, decreasing cell proliferation, invasion and migration by targeting SKA2. The novel interaction between miR-520a-3p and SKA2 may contribute to the development of improved targeted therapies for patients with GC. However, due to the complex signaling pathways regulated by microRNAs and the limited sample size, further investigation into the function and regulatory mechanisms of miR-520a-3p
Not applicable.
No funding was received.
All data generated or analyzed during this study are included in this published article.
HS and FR collected and analyzed the data, and wrote and revised the manuscript. XF conceived and designed the experiments. HJ and YC conducted the experiments. All authors read and approved the final manuscript.
The present study was approved by The Research Ethics Committee of Ningbo No. 2 Hospital (Zhejiang, China), and all patients provided written informed consent.
Not applicable.
The authors declare that they have no competing interests.
miR-520a-3p expression is decreased in GC tissues and cell lines. (A) miR-520a-3p expression was detected via reverse transcription-quantitative PCR analysis in 80 GC tissues compared with adjacent non-tumor tissues. **P<0.01 vs. adjacent non-tumor tissues (B) miR-520a-3p expression was analyzed in three GC cell lines (SGC-7901, BGC-823, and MGC-803) compared with in a normal gastric epithelial cell line (GES-1). The levels of miR-520a-3p expression were normalized to U6. *P<0.05, **P<0.01 vs. GES-1. miR, microRNA; GC, gastric cancer.
Overexpression of miR-520a-3p inhibits the proliferation of GC cells. (A) Following transfection with miR-520a-3p mimics, the mRNA expression levels of miR-520a-3p were significantly increased in SGC-7901 and MGC-803 cells compared with the respective MOCK and NC groups. ***P<0.001 vs. MOCK and NC. (B) GC cell viability in SGC-7901 and MGC-803 cells was examined by cell proliferation assay following the overexpression of miR-520a-3p. Transfection of the mimics significantly reduced proliferation. *P<0.05, **P<0.01 vs. MOCK and NC. (C) Western blot of the Ki-67 expression levels normalized to GAPDH. ***P<0.001 vs. MOCK and NC. miR, microRNA; GC, gastric cancer; MOCK, mock transfected cells; NC, negative control; OD, optical density.
Overexpression of miR-520a-3p inhibits the migratory and invasive capabilities of GC cells. (A) Migratory distance and (B) number of invasive cells in the miR-520a-3p mimics group were significantly decreased compared with the MOCK and NC groups in the wound healing and Transwell invasion assays. *P<0.05 vs. MOCK and NC. (C) Protein expression levels of MMP-2 and MMP-9 were significantly lower in the miR-520a-3p mimics group compared with the MOCK and NC groups. ***P<0.001 vs. MOCK and NC. Magnification, ×100. Scale bar, 100 µm. miR, microRNA; GC, gastric cancer; MOCK, mock transfected cells; NC, negative control; MMP, matrix metalloproteinase.
SKA2 is a direct target gene of miR-520a-3p in GC cells. (A) Sequence alignment of miR-520a-3p with 3′UTR of SKA2 predicted by TargetScan and the Mut 3′UTR miR-520a-3p binding sequence. (B) Luciferase activity of SGC-7901 cells was examined via a luciferase reporter assay. **P<0.01 vs. mutant. miR-520a-3p decreased SKA2 expression at both the mRNA (C) and protein (D) levels compared to MOCK in SGC-7901 cells. **P<0.01, ***P<0.001 vs. MOCK group. (E) SKA2 expression was analyzed in three GC cell lines (SGC-7901, BGC-823 and MGC-803) and a normal gastric epithelial cell line (GES-1). ***P<0.001 vs. GES-1. miR, microRNA; GC, gastric cancer; MOCK, mock transfected cells; NC, negative control; WT, wild-type; SKA2, spindle kinetochore-associated protein 2; UTR, untranslated region.
SKA2 regulates miR-520a-3p-mediated inhibition of SGC-7901 cells proliferation, migration and invasion. (A) After cells were transfected with a SKA2 cDNA plasmid, the expression of SKA2 was notably increased in SGC-790 cells. Following co-transfection of miR-520a-3p mimics and SKA2 cDNA into SGC-7901 cells, (B) expression of SKA2 and (C) cell viability were examined by western blotting and cell proliferation assay, respectively. (D) Migration distance and (E) number of invasive cells in GC cells following treatment with miR-520a-3p mimics and SKA2 cDNA were detected by wound healing and Transwell assays, respectively. **P<0.01, ***P<0.001 vs. the MOCK group. ×100 magnification. Scale bar, 100 µm. SKA2, spindle kinetochore-associated protein 2; miR, microRNA; GC, gastric cancer; MOCK, mock transfected cells; NC, negative control; OD, optical density.
Association between miR-520a-3p expression and clinicopathological factors in 80 primary gastric cancer tissues.
miR-520a-3p expression level | ||||
---|---|---|---|---|
Characteristic | Patients, n | Low, n (%) | High, n (%) | P-value |
Age, years | 0.990 | |||
≤50 | 38 | 28 (73.7) | 10 (26.3) | |
>50 | 42 | 31 (73.8) | 11 (26.2) | |
Tumor diameter, cm | 0.067 | |||
≤3 | 24 | 21 (87.5) | 3 (12.5) | |
>3 | 56 | 38 (67.9) | 18 (32.1) | |
Tumor TNM staging | 0.016 |
|||
I+II | 43 | 27 (62.8) | 16 (37.2) | |
III+IV | 37 | 32 (86.5) | 5 (13.5) | |
Tumor histological overall NHS grade | 0.305 | |||
1 | 7 | 4 (57.1) | 3 (42.9) | |
2 | 41 | 33 (80.5) | 8 (19.5) | |
3 | 32 | 22 (68.8) | 10 (31.3) | |
Invasion | 0.001 |
|||
T1+T2 | 27 | 14 (51.9) | 13 (48.1) | |
T3+T4 | 53 | 45 (84.9) | 8 (15.1) | |
Pathological type | 0.412 | |||
Adenocarcinoma | 69 | 52 (75.4) | 17 (24.6) | |
Non-adenocarcinoma | 11 | 7 (63.6) | 4 (36.4) |
P<0.05
P=0.001. miR, microRNA; TNM, Tumor-Node-Metastasis; NHS, National Health Service.