Background
Gastric cancer (GC) is the second most frequent cause of cancer deaths in the world, and remains the type of cancer with the highest incidence in northeast Asia [
1]. Despite a variety of strategies to improve the survival rate of patients with GC, the survival time of patients with advanced GC is still short [
2‐
4], and effective therapies are limited [
5,
6]. Therefore, identification of new early diagnostic biomarkers and development of new anticancer targeted therapies is imperative.
MicroRNAs (miRNAs) are a class of small, non-coding RNA species, approximately 19–25 nucleotides in length. They can bind to the 3′-untranslated region (UTR) of the target mRNA and promote or inhibit the expression of target genes at the post-transcriptional level [
7,
8]. Moreover, they are also able to bind to the 5′UTR region and ORF (open reading frame) [
9]. Studies have shown that many miRNAs function as tumor suppressors or oncogenes in cancer cells, and participate in various biological processes such as proliferation, differentiation, apoptosis, and metastasis [
10,
11]. Dysregulation of numerous miRNAs, such as mir-122, mir-26a, and mir-200c, has already been identified in GC [
12‐
14], which suggests that miRNAs could serve as a class of new molecular biomarkers for GC [
15,
16].
Mir-1236-3p, an intronic miRNA, is located in the Chr6p21.33 and embedded within the intron of the NELFE gene. Accumulating evidence predicts that miR-1236-3p may act as a tumor suppressor gene, and downregulation of miR-1236-3p has been demonstrated in some cancers. In hepatocellular carcinoma, miR-1236-3p down-regulates alpha-fetoprotein (AFP), thus causing PTEN accumulation, which inhibits the PI3K/Akt pathway [
17]. In high-grade serous ovarian carcinoma, miR-1236-3p represses cell migration and invasion abilities by targeting ZEB1 [
18]. In renal cell carcinoma, miR-1236-3p directly targeted the p21 promoter, and increased miR-1236 expression inhibited cell proliferation, and decreased CDK4/6 and cyclin D1 expression [
19]. Moreover, miR-1236-3p down-regulation was also reported in bladder cancer, lung cancer, and breast cancer [
20,
21], however, its biological function in GC is remains unclear.
Through preliminary screening we found that miR-1236-3p is significantly down-regulated in GC, and may serve as a potential diagnostic biomarker and therapeutic target in GC. The purpose of this study is to investigate miR-1236-3p expression in GC and cell lines. The inhibition of miR-1236-3p expression promoted cell proliferation, migration, invasion, cell epithelial–mesenchymal transition (EMT) processes, and the PI3K/Akt signaling pathway. Furthermore, we showed that miR-1236-3p could directly target at MTA2 and regulate its expression. Thus, we demonstrated that miR-1236-3p functioned as a tumor suppressor in GC at least partly through inhibition of the EMT process and PI3K/Akt signaling pathway by targeting MTA2.
Materials and methods
Clinical samples
Eighty-three paired specimens of cancerous tissues and adjacent non-tumorous tissues were obtained from patients with GC who underwent surgery at the Cancer Research Institute of China Medical University (Shenyang, China) between 2013 and 2016. All specimens had been diagnosed with GC using histopathological confirmation. None of the patients underwent chemotherapy before surgery. Tissue samples were snap-frozen in liquid nitrogen after surgical removal. Informed written consents were obtained from all patients. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of China Medical University. A summary of the clinicopathological data from the study is given in Table
1.
Table 1
Correlation between miR-1236-3p expression and clinicopathological features in 83 GC patients
Age | | | | 0.644 |
< 60 | 24 | 15 | 9 | |
≥ 60 | 59 | 40 | 19 | |
Gender | | | | 0.306 |
Male | 62 | 43 | 19 | |
Female | 21 | 12 | 9 | |
Tumor invasion | | | | 0.520 |
T1–T2 | 23 | 14 | 9 | |
T3–T4 | 60 | 41 | 19 | |
Lymph node metastasis | | | |
0.018
|
Yes | 53 | 40 | 13 | |
No | 30 | 15 | 15 | |
Differentiation | | | |
0.015
|
Well/moderate | 35 | 18 | 17 | |
Poor | 48 | 37 | 11 | |
TNM stage | | | |
0.003
|
I–II | 32 | 15 | 17 | |
III–IV | 51 | 40 | 11 | |
Cell lines culture
The three human GC cell lines MKN-45, SGC-7901, MGC-803, and a normal human gastric cell line GES-1 were obtained from Typical China Academy Culture Collection Commission Cell Library (Shanghai, China). All cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, BRL, UK) and were maintained at 37 °C in 5% CO2.
RNA isolation and quantitative real-time PCR of miR-1236-3p
Total RNA was extracted using TaKaRa MiniBEST Universal RNA Extraction Kit (Takara, Dalian, China). Reverse transcription was performed with a Hairpin-it™ miRNA RT-PCR Quantitation Kit (GenePharma, Shanghai, China) at 25 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. U6 RNA was used as an internal control. Real-time PCR was performed in an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions with the following steps: 3 min at 95 °C; followed by 40 cycles of 12 s at 95 °C, and 40 s at 62 °C. The relative gene expression was calculated using the 2−ΔΔCt method. The PCR primers for mir-1236-3p were 5′-CCAATCAGCCTCTTCCCCTT-3′ (Forward) and 5′-TATGGTTGTTCACGACTCCTTCAC-3′ (Reverse). The primers for U6 were 5′-ATTGGAACGATACAGAGAAGATT-3′ (Forward) and 5′-GGAACGCTTCACGAATTTG-3′ (Reverse).
Quantitative real-time PCR of MTA2
MTA2 cDNA was reverse-transcribed according to the manufacturer’s instructions (Takara, Dalian, China). β-actin was used as the reference gene. Quantitative real-time PCR was carried out with SYBR Green (Solarbio, China) in a total volume of 20 μL using an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer, Daejeon, Korea). The reactions were incubated at 94 °C for 10 min; followed by 40 cycles of 94 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. Melting curves were generated to confirm the specificity of the amplification. Experiments were repeated in triplicate. Relative gene expression was calculated using the 2−ΔΔCt method. The primers for MTA2 were 5′-ATCATTACCAGCCACCCA-3′ (Forward) and 5′-CGATTATCAGATTCTCCCTC-3′ (Reverse). The primers for β-actin were 5′-CTTAGTTGCGTTACACCCTTTCTTG-3′ (Forward) and 5′-CTGTCACCTTCACCGTTCCAGTTT-3′ (Reverse).
Cell transfection
The lentivirus-hsa-miR-1236-3p mimic, the lentivirus-hsa-miR-1236-3p inhibitor, and a miRNA negative control (NC) were purchased from Genechem (Shanghai, China). The miR-1236-3p mimic and the negative control were transfected into SGC-7901 cells, and the miR-1236-3p inhibitor and the negative control were transfected into MKN-45 cells.
For lentivirus infection, cells were incubated with lentivirus and 5 μg/mL polybrene for 24 h. After infection, cells were measured using a fluorescent inverted microscope. The efficiency of lentivirus infection was evaluated by real-time PCR.
Cell proliferation assays
Transfected cells were grown on 96-well plastic dishes in normal culture medium. Then, Cell Counting Kit-8 (CCK-8) working solution (Keygen, Jiangsu, China) was added into the medium according to the manufacturer’s protocol. Cells were incubated with 10 μL/well of CCK-8 solution during the last 4 h of the culture. Then, a microplate reader was used to detect the absorbance of the wells at 0, 24, 48, and 72 h. Each experiment was performed three times.
Wound-healing assays
For wound-healing assays, transfected cells were plated in six-well plates. When the cell confluence reached 90–100%, a linear scratch was created using a 200 µL pipette tip. The wounded monolayer was washed with phosphate buffered solution (PBS). An inverted microscope was utilized to visualize wound healing at 0, 24, 48, and 72 h. Wound-healing assays were conducted in triplicate.
Cell migration and invasion assays
For migration assays, transfected cells were resuspended in 200 μL of serum-free RPMI 1640 medium and seeded onto transwell chambers (Corning, NY, USA) with 8-μm pore membranes (5 × 103 cells/well). The chambers were then incubated in RPMI 1640 with 30% FBS at 37 °C in 5% CO2. After 48 h, the cells adhering to the chamber’s lower surface were fixed, whereas cells remaining on the upper surface were removed. After staining in a dye solution containing 0.5% crystal violet, the cells from three randomly selected high-power fields were counted under a microscope (Olympus, Tokyo, Japan). Migration assays were conducted in triplicate. For the invasion assay, the upper surface of the insert membrane was first coated with matrigel (BD Biosciences, San Jose, CA, USA). Transfected cells were cultured into the upper chamber with serum-free medium, and the lower chamber was filled with medium containing 30% FBS. After 48 h incubation, the cells adhering to the lower surface were fixed with methanol, stained with crystal violet and counted under a microscope. The cells were counted under a microscope in three randomly selected fields. Each sample was performed three times.
miR-1236-3p target gene prediction
We predicted potential direct common target genes of miR-1236-3p using miRNA databases including TargetScan [
22], miRanda [
23], and miRDB [
24]. At the intersection of three databases, the MTA2 gene had a lower mirSVR score and was selected as a prediction target gene.
Protein extraction and western blotting
Cells were washed with PBS and radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) was utilized to extract total protein in accordance with the manufacturer’s instruction. After detecting the protein concentration, 40 μg of protein was loaded onto the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinyl fluoride membrane. The membranes were blocked with 5% non-fat milk in TBST for 1.5 h at room temperature and incubated overnight at 4 °C with the respective primary antibodies. The primary antibodies used were as follows: β-actin (1:1000, Absin Bioscience Inc, USA), MTA2 (1:500, Novus, USA), anti-E-cadherin (1:1000, CST, USA), anti-vimentin (1:1000, CST, USA), anti-N-cadherin (1:1000, CST, USA), anti-p-AKT (Ser 473) (1:1000, CST, USA), and anti-AKT (1:1000, CST, USA). After washing, membranes were incubated with the goat anti-rabbit secondary antibody (Beyotime, Shanghai, China) at room temperature for 2 h. Finally, blots were detected using an enhanced chemiluminescence system (Millipore Corporation, Temecula, CA, USA).
Luciferase reporter assays
For the luciferase reporter assays, miR-1236-3p mimics, scramble, and the wild-type or mutant 3′-UTR of MTA2 were co-transfected into H-293T cells for 48 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Renilla luciferase was used for normalization. Each sample was performed three times.
Statistical analyses
All experiments were performed at least three times. Prism 6.07 software (GraphPad, Inc., La Jolla, CA, USA) and SPSS 19.0 (SPSS Inc., Chicago, IL, USA) was used to conduct the statistical analysis. The data are presented as the mean ± SD, and Student’s t test was performed for comparisons between two groups. P < 0.05 was considered statistically significant.
Discussion
Previous studies have indicated that miRNAs can function as tumor suppressors or promoters in human cancers. For example, miR-203 could suppress the proliferation and metastasis of hepatocellular carcinoma by targeting ADAM9 [
28]. miR-592 functioned as a tumor suppressor in lung cancer by targeting SOX9 [
29]. miR-92a promoted cell proliferation and invasion by targeting FBXW7 [
30]. miR-519d could promote melanoma progression by downregulating EphA4 [
31], among other mechanisms. Recent studies revealed that miR-1236-3p played a critical role in human cancers. It functioned as a tumor suppressor in hepatocellular carcinoma, ovarian carcinoma, renal cell carcinoma, bladder cancer, lung cancer, and breast cancer [
17‐
21]. In this study, we examined the roles of miR-1236-3p in GC. miR-1236-3p down-regulation was observed in GC tissues specimens and cell lines. Moreover, miR-1236-3p down-regulation was clearly relative to advanced clinical stage. To further explore the function of miR-1236-3p in GC, we performed a series of functional analyses. These experiments demonstrated that up-regulation of miR-1236-3p significantly inhibited the cell proliferation and reduced the number of migrated and invaded cells, and down-regulation of miR-1236-3p promoted cell proliferation and increased the number of migrated and invaded cells. These results suggested a tumor suppressor role for miR-1236-3p in GC progression. To explore the potential mechanism of miR-1236-3p in GC, the miR-1236-3p candidate target genes were predicted with TargetScan, miRanda, and miRDB, and MTA2 was selected as a potential target gene for miR-1236-3p. MTA2 is a member of the metastasis-associated gene family that has been reported to be closely associated with tumor progression [
25]. MTA2 participated in the initiation and progression of a variety of tumors [
32‐
34], and MTA2 over-expression promoted the proliferation and invasion of GC cells [
26,
35]. MTA2 over-expression in ERa-positive breast cancer cells resulted in an enhanced anchorage-independent growth [
36]. This evidence provided support to the hypothesis that MTA2 could act as an oncogene in many cancers. In our study, luciferase assays were performed and MTA2 was identified as a novel target of miR-1236-3p. In addition, we found that MTA2 mRNA and protein levels were negatively related to miR-1236-3p in GC cells, indicating that miR-1236-3p can negatively regulate MTA2 expression in GC. EMT is an early stage of tumor invasion and metastasis. In this process, cell morphology changes from cobblestone to spindle, cell surface adhesion molecules decrease, cell adhesion ability decreases, invasion and migration capacity increases, and metastasis of cancer cells is promoted. In this study, we found that after overexpression of miR-1236-3p, the cell morphology showed an epithelialization trend. After inhibition of miR-1236-3p, the cell morphology showed a tendency of mesenchymal cells. And we demonstrated that miR-1236-3p inhibited EMT process in GC cells by up-regulating E-cadherin expression, and inhibited the expression levels of vimentin and N-cadherin, but down-regulated miR-1236-3p produced the opposite effects. We then investigated whether mir-1236-3p affected signaling pathways in GC cells. Several signaling pathways including the Wnt/β-catenin and PI3K/Akt pathways have been reported to be aberrantly activated in the progression of GC [
37,
38]. Our study revealed that miR-1236-3p over-expression decreases the phosphorylation of AKT and inhibition of miR-1236-3p increases the phosphorylation of AKT. Thus, these results suggested that miR-1236-3p inhibited the cell invasion by partly suppressing the PI3K/Akt signaling pathway. In summary, our results showed that miR-1236-3p functioned as a tumor suppressor to inhibit cell invasion in GC by targeting MTA2 and inhibiting the EMT process. The miR-1236-3p-MTA2 axis provides insight into the mechanisms underlying tumor metastasis, and may serve as a promising therapeutic target for GC treatment.
Authors’ contributions
DQD and JXA designed the experiments; JXA and MHM performed the experiments; SS and NMZ collected the data; JXA analyzed the data; MHM and CDZ provided the human samples; JXA wrote the manuscript. All authors read and approved the final manuscript.