Gastric cancer (GC) is one of the most common malignant tumors in the world and microRNAs (miRNAs) play an important role in GC. In this study, we found miR-497 played an important role and served as a novel biomarker in GC. Quantitative real-time PCR (qRT-PCR) was used to measure the miR-497 expression in GC cell lines and 86 paired GC samples and we also analyzed its correlation with GC clinicopathological parameters. A series of cellular function experiments were applied to validate the effects of miR-497 on GC. In addition, methylation-specific PCR (MSP) was applied to detect the gene methylation status. Finally, the correlation between miR-497 and the target gene was analyzed by western blotting assay. miR-497 was reduced obviously in GC cells and tissues and significantly associated with the pathologic stage. Low expression of miR-497 significantly inhibited the proliferation, invasion and migration of GC cell lines and accelerated apoptosis. Moreover, we found that the aberrant expression of miR-497 may be ascribed to DNA methylation.
Gastric cancer (GC) is a highly malignant digestive tract tumor, and most GC patients have a poor prognosis due to lack of an early diagnostic indicator (
miRNAs are short endogenous RNAs and they have no function of protein coding. Numerous studies have shown that dysregulated expression of miRNA participates in GC malignant phenotype. Wu
miR-497 was first reported in breast cancer and is located on chromosome 17p13.1 (
In this investigation, we validated that miR-497 was reduced significantly in GC and repressed GC cell proliferation, invasion and metastasis. Further research indicated that gene promoter hypermethylation might contribute to dysregulation of miR-497. We also revealed that miR-497 mediated GC progression by retrograde targeting RAF1.
Human GC cell lines SGC-7901, MGC-803, MKN-45, BGC-823 and a normal gastric mucomembrane cell line GES-1 were obtained from Chinese Academy of Sciences, Shanghai Institutes for Cell Resource Center. Cells were cultivated in RPMI-1640 medium (Corning, Manassas, VA, USA) containing 10% FBS (Corning). All cells were maintained in 5% CO2 at 37°C. Eighty-six cases of GC tissues were collected from 2013 to 2015 at Cancer Research Institute of China Medical University, tissues were histologically confirmed and stored at −80°C after surgical removal until further analysis. The study was approved by the Ethics Committee of China Medical University.
For cell transfection, cells were grown in 6-well culture plates for 24 h, then, 50 µM miR-497 mimics or mimics control (mimics-NC) (GenePharma, Shanghai, China) was added into the culture medium using Lipofectamine 3000 reagent (Invitrogen, Calsbad, CA, USA) following the manufacturer's instructions. For 5-Aza-dC (Sigma, St. Louis, MO, USA) and trichostatin A (TSA) (Sigma) treatment assay, SGC-7901 and MGC-803 cells were first incubated in 6-well culture dishes for 24 h. Then, for 5-Aza-dC treatment group, 0, 0.5, 1 or 1.5 µmol/l 5-Aza-dC were mixed with the cell culture medium for 72 h. For TSA treatment group, cells were treated with 0.3 µmol/l TSA for 24 h. For combination of drugs intervention group, 1 µmol/l 5-Aza-dC was first mixed with the medium for 48 h and then 0.3 µmol/l 5-Aza-dC was added for another 24 h.
RNA Extraction kit (Takara, Dalian, China) was used to purify total RNA from GC cells and tissues. Reverse transcription and qRT-PCR were performed using SYBR Green qRT-PCR kit (GenePharma). U6 SnRNA was identified as an internal reference. The U6 primers used were 5′-ATTGGAACGATACAGAGAAGATT-3′ and 5′-GGAACGCTTCACGAATTTG-3′, the miR-497 primers used were 5′-GACGACCTCAGCAGCACACT-3′ and 5′-CAGAGCAGGGTCCGAGGTA-3′. We conducted the PCR assay according to our previous report (
A GenElute™ Mammalian Genomic DNA Miniprep kit (Sigma) was used to separate the genetic DNA from the paired GC tissues. Then, purified DNA was conducted with a bisulfite treatment using the EZ DNA Methylation-Gold kit™ (Zymo Research, Irvine, CA, USA), this assay was carried out according to the manufacturer's instructions. MethPrimer 1.0 was used to design MSP primer for methylation-specific PCR (MSP). The methylated primers used were 5′-TTTGTTTTTGTGTTAGAGAGGGTTC-3′ (forward) and 5′-TTTGTTTTTGTGTTAGAGAGGGTTC-3′ (reverse), the unmethylated primers used were 5′-TGTTTTTGTGTTAGAGAGGGTTTG-3′ (forward) and 5′-ACTAACAATACAACTACATCCCATA-3′ (reverse). We conducted the PCR assay according to Jia
Cell Counting Kit-8 (Dojindo, Kumamato, Japan) was used to detect the proliferation rates of GC cells. First, 3,000 SGC-7901 and MGC-803 cells were incubated into 96-well plates for 24 h. Then, 10 µl CCK-8 was added into each well, proliferation rates of GC cells were surveyed at 0, 24, 48 and 72 h after transfection. This experiment was repeated for three times. Annexin V-PE/7AAD Apoptosis Detection kit (KeyGen, Jiangsu, China) was applied to perform the cell apoptosis assay according to the manufacturer's instructions and this experiment was repeated in triplicate.
Transfected cell lines were grown in 6-well plates for 24 h and linear wounds were scratched on the confluent cells using a 200-µl pipette tip. The serum-free medium was replaced by cell culture and we used an inverted microscope to visualize migrated cells and wound healings. For cell invasion assay, Matrigel-coated membrane matrix (Dojindo) was smeared onto the upper chamber for 24 h, then, 1×105 transfected cells were grown in the upper chambers with serum-free medium and the lower chambers was added with medium containing 10% FBS. Twenty-four hours later, non-invasive cells were removed using a cotton tip and the invasive cells were stained using crystal violet (Tiangen, Beijing, China). Images were taken using an inverted microscope. Each sample was repeated in triplicate.
Proteins were extracted from GC cells and clinical samples using RIPA buffer (Beyotime, Shanghai, China) and BCA Protein assay kit (Takara) was used to measure the protein concentration. Then, 30 µg proteins were added into 10% SDS-PAGE (Takara) and polyvinyl fluoride membranes (Beyotime) were conducted as a transmembrane. Membranes were blocked for 2 h with BSA (Sigma) and kept at 4°C overnight with anti-RAF1 antibody and we set an internal control using β-actin. The dilution ratio was 1:1,000. Thereafter, the membrances were incubated with secondary antibody (Sigma) at room temperature for 2 h. At last, images were determined using an enhanced chemiluminescence system. For histology, first, buffered formalin was used to fix tissues and then we embedded tissues in paraffin. Second, paraffin-embedded blocks were cut into 3 µm slices and stained with hematoxylin and eosin (H&E).
miR-497 mimics or mimics control (mimics-NC), reporter construct or control vector (GenePharma) were transfected in GC cells for 24 h. Dual Luciferase assay kit (KeyGen) was used to detect luciferase activity. The ratio of luciferase intensities was calculated and normalized to controls. Each sample was carried out in triplicate.
We used SPSS 19.0 to conduct statistical analyses and Student's t-test, an ANOVA test and χ2 test was utilized to analyze P-values. P<0.05 was considered to indicate a statistically significant difference (two-sided).
We first examined the expression level of miR-497 in four GC cell lines (SGC-7901, MGC-803, MKN-45, BGC-823) and four paired GC tissues (
To illuminate the relevance between miR-497 expression and GC clinicopathological parameters, we separated the patients into two groups according to the mean level of miR-497. Our data showed low miR-497 expression was apt to have an advanced GC TNM stage (P=0.014). However, no obvious relationship was found between miR-497 expression and tumor size or location (
In order to analyze the role of miR-497 in GC tumorigenesis and progression, miR-497 mimics were transfected in SGC-7901 and MGC-803 cells using Lipo 3000. To measure the transfection efficiency, qRT-PCR was applied to determine miR-497 expression level after cell transfection, the data exhibited that miR-497 was upregulated 154.33±11.50 times and 96.74±6.82 times in SGC-7901 and MGC-803 cells (
To explore whether miR-497 could regulate the metastasis ability of GC, a wound healing assay was performed to measure the GC cell migration ability. As showed in
In order to investigate the regulatory mechanism of low expression of miR-497 in GC, human genome database was used to find CpG islands around miR-497 (
miRNAs play their biologic role through downregulating their target genes.
Previous studies suggested that miRNAs functioned as oncogenes or tumor suppressors in various carcinomas and miRNAs played important roles in GC carcinogenesis and progression (
Yan
Accumulating studies illuminated that miRNAs were closely related to tumorigenesis, however, the exact mechanism involved were unknown. To demonstrated the effect of miR-497 on GC carcinogenesis and progression, we conducted cellular function tests including cell proliferation assay, cell invasion assay and cell apoptosis assay. The result proved that restoration of miR-497 obviously inhibited GC cell proliferation, migration, and invasion, the result also indicated that overexpression of miR-497 significantly induced GC cell apoptosis. Apoptosis can be seen as a stage-dependent process from its induction to early, intermediate and late-stage apoptotic events. Early apoptosis is represented by changes to, and ultimate loss of, the mitochondrial membrane potential. Our study indicated that low expression of miR-497 in GC was a frequent event and might play crucial roles in GC tumorigenesis. Recently, numerous studies proved that downregulation of miRNAs was associated with epigenetic modifications, including DNA methylation and histone modification (
Moreover, our study found RAF1 harboring miR-497 binding site and RAF1 was considered to be a potential target of miR-497. Particularly, RAF1 gene was located in 3p25.2 chromosome and its aberrant expression was reported to be associated with tumor malignant biological properties (
In conclusion, our results indicated that miR-497 had a tumoral suppression function through targeting RAF1 in GC and decreased expression of miR-497 might due to DNA hypermethylation. GC patients who possessed low miR-497 level were apt to have an advanced GC clinical stage. Restoration of miR-497 obviously inhibited GC cell proliferation, migration and invasion. The result proved that miR-497 could act as a novel biomarker and therapeutic target in gastric cancer.
This study was supported in part by a grant from the National Natural Science Foundation of China (no. 30572162), the Liaoning Province Science and Technology Plan Project (no. 2013225021) and the Natural Science Foundation of Liaoning Province (no. 201602817).
miR-497 is downregulated in GC cells and tissues. (A) Four paired GC samples stained with hematoxylin and eosin (H&E). C, cancer tissues; N, normal tissues. (B) The relative expression of miR-497 in GC cell lines, and a normal gastric cell line and four paired GC samples. (C) Expression of miR-497 in 86 paired GC samples was measured by qRT-PCR. (D) The expression level of miR-497 is related to advanced clinical stage in GC (**P<0.01).
Ectopic expression of miR-497 regulates SGC-7901 and MGC-803 proliferation and apoptosis. (A) The transfection efficiency of miR-497 in SGC-7901 and MGC-803 cells. (B) The proliferation ability of GC cells transfected with miR-497 mimics or mimics-NC was detected by CCK-8 at different time-points. (C) miR-497 significantly accelerated GC cell apoptosis. *P<0.05; **P<0.01.
Effect of miR-497 on GC cell migration and invasion. (A) Overexpression of miR-497 enhances the migration ability of GC cells. (B) miR-497 obviously suppressed GC cells invasion. *P<0.05; **P<0.01.
Low expression of miR-497 in GC is related to DNA methylation. (A) Arrow is CpG island location. (B) Methylation status in GC cell lines and a normal gastric cell line, methylation status in GC cell lines treated with 5-Aza-CdR or not. (C) 5-Aza-CdR obviously enhanced the expression level of miR-497. Ctrl, black control group; Aza, 5-Aza-CdR treatment group; TSA, TSA treatment group. Aza+TSA, combination of drugs intervention group. (D) Methylation status in 8 paired GC tissues. M, methylated; U, unmethylated. C, cancer tissues; N, normal tissues. The hypermethylation samples are marked. *P<0.05; **P<0.01.
miR-497 directly targets RAF1. (A) The predicted miR-497 binding site on the RNF1 mRNA 3′-UTR. (B) Luciferase activity of wild-type (WT-UTR) or mutant (MUT-UTR). (C) Western blot analysis of RAF1 expression in miR-497-transfected SGC-7901 and MGC-803 GC cells. u, untreated; m, mimics; NC, mimics control. (D) RAF1 protein expression level in 6 paired GC tissues. T, cancer tissues; N, normal tissues. *P<0.05; **P<0.01.
Clinicopathological characteristics of patients with gastric cancer (GC).
miR-497 expression | ||||
---|---|---|---|---|
Parameter | Total samples (n) n=86 | Low expression (%) | High expression ( |
P-value |
Age (years) | 48 | 38 | 0.681 | |
≥60 | 23 | 12 (52.2) | 11 (47.8) | |
<60 | 63 | 26 (57.1) | 27 (42.9) | |
Sex | 0.310 | |||
Male | 66 | 39 (59.1) | 27 (40.9) | |
Female | 20 | 9 (45.0) | 11 (55.0) | |
Location | 0.557 | |||
Proximal | 12 | 5 (41.7) | 7 (58.3) | |
Body | 20 | 12 (60.0) | 8 (40.0) | |
Distal | 54 | 31 (57.4) | 23 (42.6) | |
Tumor size | 0.730 | |||
T1-T2 | 38 | 22 (57.9) | 16 (42.1) | |
T3-T4 | 48 | 26 (54.2) | 22 (45.8) | |
Borrmann type | 0.459 | |||
Borrmann 1+2 | 11 | 5 (45.5) | 6 (54.5) | |
Borrmann 3+4 | 75 | 43 (57.3) | 32 (42.7) | |
Grade | 0.298 | |||
Well and moderately differentiated | 31 | 15 (48.4) | 16 (51.6) | |
Poorly differentiated | 55 | 33 (60.0) | 22 (40.0) | |
TNM stage | 0.014 | |||
I+II | 35 | 14 (66.7) | 21 (33.3) | |
III+IV | 51 | 34 (55.8) | 17 (44.2) |