Introduction
Gliomas are the most common and lethal primary brain tumors in adults. Patients with newly diagnosed glioblastoma multiforme (GBM), the most malignant histological subtype of glioma, have a median survival period of approximately one year [
1-
3]. Despite comprehensive therapies including surgical resection, radiation, and chemotherapy, the final prognosis of glioma remains extremely bad [
4]. The significant property of a glioma usually develops in the craniocerebral depths and always infiltrates into adjacent normal brain tissue, so complete resection is difficult finished and very dangerous for patients with gliomas [
5]. Therefore, novel biological molecular therapies that inhibit tumor cell growth and invasion are urgently needed.
MicroRNAs are endogenous small noncoding RNAs with lengths of approximately 18–25 nucleotides. They regulate gene expression at the transcriptional and post-transcriptional level by completely or incompletely binding to the 3′-UTR (untranslated regions) of their target gene messenger RNA (mRNA) and by repressing the translation or promoting the degradation of the target gene to exert biological functions [
6-
8]. There is mounting evidence confirming that microRNAs play pivotal roles in tumorigenic processes including cellular differentiation, proliferation, angiogenesis, cell death, apoptosis, and invasion [
9]. MicroRNAs will hopefully be applied in clinical cancer diagnosis, therapy, and prognosis [
10]. miR-184 is particularly enriched in the brain and testes in humans and is located in region 25.1 on the q-arm of chromosome 15. Its corresponding transcript is comparatively small (84 bp) and is not encoded near other clustered miRNAs [
11,
12]. Recently, researchers have shown that miR-184 functions as a potential oncogene in human hepatocellular carcinoma by suppressing Sox7 expression [
13]. The overexpression of miR-184 might play an onco-miRNA role in the anti-apoptotic and proliferation processes. The plasma expression levels of miR-184 were also associated with the presence of primary tumors and might be used as a novel cancer marker in tongue squamous cell carcinoma [
14]. However, the high expression of miR-184 reportedly causes a decrease in cell numbers and increases apoptosis in neuroblastoma cell-lines [
15]. In concordance with the previous results, Tivnan
et al. have demonstrated that miR-184 mediated inhibition tumor growth and prolonged the survival time in an orthotopic murine model of neuroblastomas [
16]. However, miR-184 functions both as oncogene and tumor suppressor in the development and progression of numerous cancers. For glioma, miR-184 exhibited a progression-associated down-regulation miRNA; Overexpression of miR-184 in A172 and T98G glioma cells significantly decreased cell viability and proliferation [
17]. Besides, a study by Emdad et al. confirmed that miR-184 is down-regulated in human malignant glioma cells and tumor tissue as compared with their non-neoplastic counterpart [
18]. The functions and exact mechanisms of miR-184 in glioma are poorly uderstood.
TNFα-induced protein 2 (TNFAIP2) maps to chromosome 14q32, encodes a 654 amino acid protein, belongs to the Sec6 family, is differentially expressed in capillary tube-like formation
in vitro, and can be induced to express itself with a TNFα treatment [
19]. TNFAIP2 was highly expressed in marrow from patients with acute myelogenous leukemia subtypes M0-M2 but was repressed in marrow cells from M3 patients. It was also a target gene of PML-RARs (promyelocytic leukemia-retinoic acid receptor α) in APL (acute promyelocytic leukemia) [
20]. Recently, Chen
et al. found that TNFAIP2 is highly expressed in tumor cells compared to adjacent normal tissue, is closely related to invasion and metastasis, and may serve as an independent prognostic indicator for nasopharyngeal carcinoma (NPC) [
21]. A previous study showed that miR-184 directly targeted the 3′-UTR of TNFAIP2 using a dual-luciferase reporter assay in lung, head, and neck cancer cell-lines. The miR-184 binding site single nucleotide polymorphisms [SNP (rs8126 T > C)] in the 3′-UTR of TNFAIP2 modulated TNFAIP2 expression and contributed to susceptibility to squamous cell carcinoma of the head and neck (SCCHN) [
22].
The expression and function of TNFAIP2 have not been studied in gliomas; the relationship between TNFAIP2 and miR-184 also has not been studied. This study confirmed that miR-184 was lower in glioma cell-lines and in 49 glioma specimens than in normal brain tissues. The up-regulation of miR-184 inhibited glioma cell proliferation and invasion and induced apoptosis. It also increased the cell population in the G0/G1 phase and reduced the percentage of U87 and U251 cells in the S phase. The expression of TNFAIP2 was higher in 81 glioma samples compared to noncancerous brain tissues. The results of this experiment implied that miR-184 might be a suppressor gene and conformed that miR-184 could target TNFAIP2 in gliomas.
Materials and methods
Human tissue samples
Tissue samples were obtained from March 2009 and September 2011 from the Department of Neurosurgery of the First Affiliated Hospital of Soochow University. There were a total of 81 glioma samples (29 cases low-grade WHO grades I and II, 52 cases high-grade WHO grades III and IV) and 8 non-neoplastic brain tissues obtained from patients who suffered severe cerebral injury. Their injuries required a reduction in increased intracranial pressure by removing a partial amount of normal brain tissue. The mean age of the patients at the time of surgery was 48.9 years for 31 women and 49.76 years for 50 men. There were 2 cases of pilocytic astrocytoma (grade I), 22cases of diffuse astrocytoma and 5 cases of pleomorphic xanthoastrocytoma (grade II), 19 cases of anaplastic astrocytoma and 5 cases of oligoastrocytoma (grade III), 28 cases of primary brain glioblastoma (grade IV). All tissue specimens were immediately collected and stored in liquid nitrogen after resection. This study was authorized by the local ethics committee of our hospital. All patients were informed of their participation in the study before surgery and gave their consent.
Cells and cell culture
The glioma cell-lines U87, U373, U251, and A172 were purchased from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). U373 cells were retired from the ATCC catalogue, since similar characteristics were found between U373 and U251 cells. SHG44 and normal human astrocyte 1800 cells were conserved and provided by our Brain and Nerve Research Laboratory. All cell-lines were maintained in Dulbecco’s modified eagle’s medium (DMEM, Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 U of penicillin/ml, 100 mg of streptomycin/ml, and 10% fetal bovine serum (FBS, Gibco) at 37°C under a humidified atmosphere of 5% CO2.
Transfection
U87 and U251 cells were transfected with miR-184 mimic, miR-184 inhibitor, and their corresponding negative control (miR-NC and anti-NC) by Lipofectamine2000 (Invitrogen Inc., Carlsbad, CA, USA), which were designed and synthesized by Invitrogen. The sequences were as follows: miR-184 mimic, 5′-UGGACGGAGAACUGAUAAGGGUCCUUAUCAGUUCUCCGUCCAUU-3′; the negative control (miR-NC), 5′-UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT-3′; miR-184 inhibitor, 5′-ACCCUUAUCAGUUCUCCGUCCA-3′; and the negative control (anti-NC), 5′-CAGUACUUUUGUGUAGUACAA-3′. Mock group was untreated by anything. To achieve stable transfectant overexpression of miR-184 and negative control oligonucleotide, pLenti-miR-184-GFP and pLenti-NC-GFP (Genechem Co., Ltd., Shanghai, China) were applied to transfect U87 cells.
Quantitative RT-PCR
RNA was extracted using TRIzol reagent (Invitrogen Inc., Carlsbad, CA, USA), which was quatified by spectrophotometer . Only the RNA samples with 260/280 ratios of 1.8–2.0 were used for further investigation. The miR-184 expression level was measured using All-in-One™miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA) according to the instructions. Briefly, 100 ng RNA was used to synthesize cDNA in a 25 μl reaction system containing 5 μl 5× Reaction Buffer, 1 μl RTase Mix, and 1 μl 2.5 U/μl Poly A Polymerase. All reaction systems were incubated at 37°C for 60 min and 85°C for 5 min. The PCR conditions for miRNA quantification were as follows: 10 min at 95°C, then 40 cycles of 10 s at 95°C, 20 s at 62°C, and 30 s at 72°C. The relative level of TNFAIP2 mRNA was examined using SYBR green qRT-PCR (Applied LightCycler480). Subsequently, 2 μg of total RNA were reverse transcribed in a 20 μl reaction containing 10 units of M-MLV reverse transcriptase and 0.5 μg of oligo (dT) primer. A total of 2 μl of cDNA was used for qPCR. The expression level of GAPDH was used as an internal control for mRNAs , and the endogenous U6 snRNA as an internal control for miRNAs. The following primers were used: TNFAIP2 forward primer, 5′-CCTGCTCTCCCTACGC-3′, reverse primer, 5′-CGTCCAAGATGCTCCG-3′ [
19]; and GAPDH forward primer, 5′-AACGGATTTGGTCGTATTG-3′, reverse primer, 5′-GGAAGATGGTGATGGGATT-3′. The PCR conditions for relative quantification were as follows: initial denaturation at 95°C for 5 min, then 40 cycles consisting of 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. The relative expression of each gene was calculated and normalized using the 2
−ΔΔCt method. Each sample was tested in triplicate.
Cell proliferation, cell cycle, and cell apoptosis
Cell proliferation was measured by a cell counting kit-8 (CCK-8) (Beyotime, Shanghai, China). Cells (2 × 103 per well) were seeded in a 96-well plate and incubated for 24 h. Then, the cells were transfected with miR-184 mimic, miR-184 inhibitor, or the negative control miRNA (Negative)at a final concentration of 50 nmol/L. CCK-8 (10 μl) was added to each well at 24, 48, and 72 h after transfection, and plates were incubated for 4 h at 37°C. Absorbance was measured at a wavelength of 450 nm. For cell cycle analysis, transfected cells were fixed in 75% ethanol at 4°C and stained with propidium iodide (PI). The cell cycle distribution was analyzed by flow cytometer (CYTOMICS, FC, 500 Beckman-coulter, CA, USA). Cell apoptosis was also detected using the Annexin V PE Apoptosis Detection Kit PE (eBioscience, San Diego, CA, USA) by flow cytometer. Cells transfected with miR-184 mimic, inhibitor, or negative control miRNA were collected and resuspended in 400 μl of 1× binding buffer containing 5 μl 7-AAD (7-amino-actinomycin D) and 5 μl PI at room temperature in the dark for 10–15 min. All experiments were performed in triplicate.
Wounding healing and invasion assay
For the cell migration and cell invasion assay, all the cells (U87, U251) were used after transfection. For the cell motility assay, cells were seeded in six-well plates and cultured to 70-80% confluence. A 200 μl pipette tip was applied to generate a linear wound. The floating cells were rinsed with culture medium. Cells cultured for 48 h and were recorded under a microscope (Olympus, Tokyo, Japan) at 0, 6, 12, 24, and 48 h. For the cell invasion assay, Transwell chambers were covered with Matrigel (BD Bioscience, San Jose, CA, USA), and 1 × 105 cells suspended in 100 μl serum-free medium were added to the upper chambers. The lower chambers were filled with 750 μl DMEM with 10% fetal bovine serum. After 48 h of incubation, non-invading cells on the upper surface of the membrane were removed using a cotton swab. The invaded cells were fixed in 4% formaldehyde for 30 min and stained with 0.1% crystal violet for 5 min. Then, cells were imaged and counted under a microscope. The number of cells that penetrated the Matrigel was counted from 6 randomly selected fields.
Western blotting
Total protein was isolated by RIPA and quantified using a BCA (bicinchonininc acid) assay kit (Beyotime, Shanghai, China). Equal amounts of protein samples were separated by 12% SDS-PAGE and transferred into nitrocellulose membranes. After blocking, the membranes were incubated with primary antibodies overnight at 4°C. After washing, membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies at a dilution of 1:4000 (Prosci Inc., Poway, CA, USA) at room temperature for 2 h. Then, the membranes were detected and recorded using ECL Western blotting detection reagents. The primary antibodies used were rabbit anti-TNFAIP2 at a dilution of 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-GAPDH at a dilution of 1:2000 (Abcam, Tokyo, Japan).
Xenograft experiments
To select stably expressed cells, U87 cells were transfected with pLenti-miR-184-GFP or pLenti-NC-GFP. U87-miR-184 and U87-miR-NC cells were established and inoculated into nude mice (4 to 5 weeks old) in intracranial (1 × 105) and subcutaneous (1 × 106) fashions, respectively (n = 6 /group). Caliper measurements were performed to assess tumor growth. The size is represented by the width of subcutaneous tumor multiply the length of subcutaneous tumor (W*L). MRI (Magnetic Resonance Imaging) was also used to observe tumor growth. Each nude mouse was anesthetized by 200 μl 4% chloral hydrate, then scanned by Signa HDX model 3.0 T. MRI machine (GE., Detroit, USA). T2-weight images were acquired, and the following parameters were used: repetition time 2160.00 milliseconds, echo time 108.80 milliseconds, field of view 10 × 10 cm, and matrix 256 × 256. About 5 weeks post-implantation, the nude mice were euthanized and the tumors obtained. All mice experimental procedures were performed according to the First Affiliated Hospital of Soochow University policies.
Immunohistochemistry
Formalin-fixed paraffin-embedded U87 intracranial tumors were cut into 6-μm-thick sections on a microtome. Immunohistochemical staining was performed according to standard procedures. Antigen retrieval was conducted in 0.1 M citrate buffer (pH 6.0) at 95°C for 16 min and cooled at 25°C for 1 h. After blocking, slides were incubated with primary antibodies TNFAIP2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SOX2 (1:200, Boster Bioengineering Co., Wuhan, China) and ki-67 (1:200, Boster Bioengineering Co., Wuhan, China). Next, according to the manufacturer’s instructions, slides were treated with the Cell & Tissue Staining Kit HRP-DAB system (R&D Systems, Minneapolis, MN, USA). The immunohistochemical staining results were evaluated by two experienced pathologists.
Statistical analysis
Statistical analyses were performed using GraphPad PRISM4.0 software (GraphPad, La Jolla, CA, USA) and SPSS version 13.0 (SPSS, Chicago, IL, USA). Experimental data are all presented as the means ± standard deviation (SD). Statistical analyses were performed using a two-tailed Student’s t-test or ANOVA and P < 0.05 was considered statistically significant.
Discussion
MiRNAs directly modulate and inversely regulate different genes via inducing mRNA cleavage or translation repression and function as oncogenes or tumor suppressors depending on the target genes [
23,
24]. Accumulated evidence shows that miRNAs play a pivotal role in the development of the malignant phenotype of glioma cells, including cell survival, proliferation, angiogenesis, differentiation, and stem cell generation [
25]. The effects of miR-184 on malignant progression are debated because it can act as a tumor promoter or suppressor in some solid tumors [
14,
13,
26]. Previous experimental studies have demonstrated that miR-184 acted as a modulator in the malignant progression of gliomas [
17] and was expressed lower in glioma cells and tissues [
18]. The collective data provided sufficient indication that miR-184 might serve as a tumor suppressor gene in gliomas. Gliomas are characterized by high invasion, migration, and proliferation abilities [
27], so it is necessary to study the molecular mechanisms of miR-184 modulate gliomas. Even though previous studies have confirmed that miR-184 was down-regulated in gliomas and decreased with the increasing degree of malignancy, the number of cases used was too limited. The present study increased the glioma cases to further confirm the expression and function of miR-184 in gliomas.
The present study’s results were in accordance with those from previous studies. The miR-184 expression levels were determined in 49 glioma tissues and 5 glioma cell-lines by qRT-PCR analysis, which displayed a remarkable down-regulation of miR-184 in gliomas compared to 6 non-tumor brain tissues. Also, miR-184 down-regulation might be linked to glioma development. Wound healing and Matrigel invasion assays were performed to conclude that the overexpression of miR-184 markedly suppressed the invasiveness of U87 and U251 cells in comparison with the negative control group. miR-184 performed an important function in glioma invasion.
In vitro, the proliferation of glioma cells was found to be significantly inhibited by the forced expression of miR-184.
In vivo, the growth curve of tumor xenografts showed that high expression level of miR-184 obviously slowed tumor growth. Foley
et al. found that miR-184 ectopic overexpression in neuroblastoma cell-lines had pro-apoptotic and anti-proliferation functions through inhibiting AKT2, which was one downstream gene of the PI3K/AKT pathway [
28]. Whether miR-184 inhibits glioma survival by blocking the PI3K/AKT2 pathway needs further investigation. The present study identified that miR-184 induced the apoptosis of U87 and U251 cells, increased the cell population in the G0/G1 phase, and reduced the percentage of cells in the S phase. It is reasonable to hypothesize that miR-184 inhibited the proliferation of glioma cells by increasing the percentage of early apoptotic cells, and it was suggested that miR-184 might be a novel specific biomarker for gliomas. In additional, Yuan et al. reported that miR-184 was significantly upregulated in human glioma cells. Our results differ from those by presented in the study by Yuan et al. [
29]. It is reasonable to hypothesize that these differences are due to gliomas cell lines may have undergone mutations during culture.
Biological information software also predicted that miR-184 could directly target TNFAIP2 mRNA sequences at the region from 1870 to 1876. In gastric cancer and SCCHN, miR-184 bonded to the 3′-UTR of TNFAIP2, and the miR-184 binding site single nucleotide polymorphisms in TNFAIP2 contributed to tumor susceptibility [
22,
30]. TNFAIP2, which can be induced by treatment of TNF-α that with miR-184 can directly target TNFAIP2 in carcinoma (NPC) tissues, and is closely related to invasion and metastasis and poor survival in NPC patients [
21]. A previous study demonstrated that latent membrane protein 1 (LMP1), an Epstein-Barr virus oncoprotein, induced TNFAIP2 expression via NF-κB pathway and TNFAIP2 also contribute to LMP1-induced cell motility [
31]. Researchers have suggested that TNFAIP2 may play multiple roles in the development of cancer and may be particularly closely related to tumor metastasis. However, the biological function and molecular mechanism of TNFAIP2 in gliomas remain unclear. The present study is first to report that both mRNA and protein expression levels of TNFAIP2 are up-regulated in glioma cell-lines and tissues compared to non-neoplastic brain tissues. As in other reports, the results of the present study indicated that miR-184 could regulate TNFAIP2 in glioma cells. The up-regulated expression of miR-184 in U87 and U251 cells led to the obvious down-regulation of TNFAIP2 mRNA and protein expressions. Reducing the expression of miR-184 resulted in the significant up-regulation of TNFAIP2 mRNA and protein. The IHC results of the nude mice intracerebral transplantation tumors further demonstrated that U87 cells transfected with miR-184 had reduced TNFAIP2 compared to the miR-NC groups. The results confirmed that both TNFAIP2 mRNA and protein expressions were substantially down-regulated by the high expression of miR-184 in gliomas. In human gliomas, TNFAIP2 was one of the specific targets of miR-184. The study’s results showed that the expression level of TNFAIP2 was higher in human gliomas than in noncancerous brain tissues. Also, miR-184 targeted TNFAIP2
in vitro and
in vivo, but it was neither elaborated upon nor proven whether the high expression of TNFAIP2 led to the process of invasion and proliferation in gliomas or whether miR-184 suppressed the survival and invasion of gliomas by down-regulating the expression of TNFAIP2. The function and mechanism of TNFAIP2 in gliomas need further investigation.
The present study demonstrated that miR-184 was markedly down-regulated in human glioma cells and tissues, TNFAIP2 was up-regulated in human glioma cells and tissues, and TNFAIP2 expression was inversely correlated with miR-184 expression. Also, the overexpression of miR-184 led to the down-regulation of TNFAIP2, and miR-184 regulated the expression of TNFAIP2 by binding to the 3′-UTR of TNFAIP2 mRNA. miR-184 had a significant suppressive effect on glioma proliferation, migration, and invasion. All the experiments showed that miR-184 was a suppressor gene in the malignant procession and carcinogenesis of gliomas and may be used to develop a miRNA-based therapeutic strategy against glioma.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CC conceived, designed the experiments and wrote the paper. CC, XTL, ZWW, TS, XSX, YLH and GLC performed the experiments. HZW analyzed the data. ZWD and YXZ supervised the whole experimental work and revised the manuscript. All authors read and approved the manuscript.