Introduction
A glioma is the most common form of neural malignancy. High grade glioma, especially glioblastoma, is a leading cause of brain cancer fatality involving highly invasive and neoplastic growth. Despite therapeutic advances, many patients suffer from tumor recurrence due to chemo- and radio- therapy resistance[
1‐
3]. Increasing evidence suggests that the progression of a glioma is relative to the rate of both cell proliferation and apoptosis. Therefore, understanding the main regulatory mechanism of gliomas is key to the development of effective therapeutic approaches against this malignancy.
MicroRNAs (miRNAs) are small, endogenous, non-coding RNA molecules, which usually result in gene silencing by binding to complementary sequences in the three prime untranslated regions (3-UTRs) of target messenger RNA transcripts (mRNAs)[
4‐
9]. The deregulation of miRNAs has been observed in various types of human malignancies, including lymphoma, colorectal cancer, lung cancer, breast cancer, papillary thyroid carcinoma, hepatocellular carcinoma and glioblastoma[
10‐
13]. Accounting for approximately 1% of all the expressed human genes, miRNAs are predicted to regulate the expression of up to 1/3 of human protein-coding genes[
14‐
17]. A few studies suggest that the downregulation of miRNAs may play a critical role in cancer progression by affecting not only proliferation but also apoptosis[
18‐
20]. Primary brain tumors expressed higher levels of miR-92b than both primary tumors in other tissues and their metastases to the brain[
21]. In neuroblastoma, mir-92b was reported to modulate the expression of the inhibitory protein-coding Dickkopf-3 gene (
DKK3)[
22]. However, the underlying mechanism of mir-92b in gliomas has not been identified so far.
In the current study, we demonstrate that high levels of miR-92b expression in gliomas confer highly aggressive invasion and poorer overall survival. Knockdown of miR-92b decreased glioma cell prolifirelation, reduced apoptosis and up-regulated the expression of the target, DKK3, whereas ectopic expression of miR-92b exhibited the opposite effects. Furthermore, miR-92b could regulate the expression of downstream genes of the Wnt/beta-catenin signaling pathway, such as Bcl2, c-myc and p-c-Jun. These findings indicate that DKK3 is a critical target of miR-92b and that the microRNA could be critical therapeutic targets and survival predictors in glioma.
Materials and methods
The human glioma tissue samples and their corresponding nontumorous tissues were collected at the time of surgical resection at the Department of Pediatric Neurosurgery, Xinhua Hospital, Shanghai Jiao Tong University. Twenty frozen glioma specimens with clinical data were collected from January 2008 to June 2013, including 9 grade I-II tumors, 8 grade III tumors and 3 grade IV tumors. The glioma samples were deep-frozen using liquid nitrogen, stored at −80°C and were quantified by Real-time PCR. This study was approved by the Institutional Review Board of Xinhua hospital. Patients were followed by clinical and laboratory monitoring on a regular basis starting at definitive diagnosis. Disease-specific survival time was defined as the time from definitive diagnosis to disease-specific death.
Reagents
The antibodies aganist c-jun, phospho-c-jun, JNK, phospho-JNK, DKK3, beta-catenin, Bcl-2, β-actin, caspase-3, Bax, c-myc were purchased from Santa Cruz Biotechnology (California, USA). The dual luciferase reporter assay system, the PGL3-Promoter, the PGL3-Basic and PRL-TK vectors were purchased from Promega (Promega Corporation, Wisconsin, USA). The miRNA mimics and siRNA were purchased from Biomics Biotechnologies (Nantong, China). All other chemicals were from Sigma- Aldrich unless otherwise stated.
Cell cultures and transfection
The human glioma cell lines U251, U87, A172 and SHG44, and human astrocytes (HA) (Cell Bank of the Chinese Academy of Science, Shanghai, China), were maintained in RPMI 1640 medium (Gibco Industries, Inc. Carlsbad, CA) with 10% fetal bovine serum (Gibco Industries, Inc.) at 37°C in a humid atmosphere wih 5% CO2. Cell transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
MicroRNA microarrays
Total RNA was extracted from eight glioma tissues using the miRVana miRNA Isolation Kit (Ambion, Carlsbad, USA) according to the manufacturer’s instructions. The samples were subsequently submitted to Shanghai Biotechnology Corporation (Shanghai, China) for array hybridization on an Agilent Human miRNA array (v.12.0). Each microarray chip was hybridized with a single sample labeled with either Cy3 or Cy5. Background subtraction and normalization were performed. The raw data were deposited at Shanghai Biotechnology Corporation (Shanghai, China) and have not been reported publicly up till the present moment. We selected the miRNAs that exhibited a difference in expression levels of at least 2-fold (p<0.05) between the glioma tissue samples and their corresponding nontumorous tissues.
RNA extraction and quantification
Total RNA was extracted using Trizol (Invitrogen Corporation, California, USA) according to the manufacturer’s instructions. Reverse transcription was performed using One Step PrimeScript®miRNA cDNA Synthesis Kit (Takara Bio Inc, Dalian, China). Real-time PCR was performed using SYBR®Premix Ex TaqTM II (Takara Bio Inc, Dalian, China) with an iCycler®thermal cycler (Bio-Rad, Hercules, USA).U6 RNA was used as a miRNA internal control. The primers of miR-92b was as follows: 5′-TATTGCACTCGTCCCGGCCT-3′.
U251 and U87 cells were transfected with miR-92b mimics, a control oligonucleotide and a miR-92b inhibitor. After the transfection, the U251 and U87 cells were counted and seeded in 12-well plates (in triplicates) at a density of 50 and 60 cells per well, respectively. The culture medium was replaced every 3 days. The number of colonies was counted on the sixth day after seeding. The rate of colony formation was calculated with the following equation: colony formation rate (%) = (number of colonies number of seeded cells) × 100%.
MTT assays
The MTT assay was used to determine cell viability. All the cells were seeded into 96-well culture plates (2×103 cells/well) in regular growth medium. The cells transfected with miR-92b mimics. control and inhibitors were grown for 4 days. One plate was developed immediately after the medium change and other plates were developed every 24 hours for 4 days. Assays were initiated by adding 20 L of MTT substrate to each well and incubating the cells for an additional 3 hours. Finally, the medium was removed and 200 L DMSO was added to each well. The absorbance was measured at 492 nm using an Automated Microplate Reader (Multiskan Ex, Lab systems, FIN).
RT-PCR
Analysis was used to determine the relative expression levels of miRNAs. Total RNA was isolated using TRIZOLTM reagent (Promega Corporation, Wisconsin, USA) according to the supplier’s instruction. Reverse transcription was done using One Step PrimeScript® miRNA cDNA Synthesis Kit (TaKaRa Biotechnology Ltd, Shandong, CHN). Real-time PCR was performed using SYBR Green Supermix with an iCycler®thermal cycler (Bio-Rad Laboratories Inc, California, USA).Primers of all genes were in Supple-mentary. The data were collected and analyzed using the comparative Ct (threshold cycle) method using GADPH as the reference gene.
MicroRNA target prediction
The target genes of miR-92b were predicted by the following computer-aided algorithms: TargetScan Human Release 6.2 (http://www.targetscan.org).
Luciferase assay
The 3′UTR of human
DKK3, containing the putative target sites for miR-92b, was amplified by PCR. The wild-type and mutant inserts were transfected into the PGL3-promoter vector. Dual-Luciferase reporter assays were performed according to the manufacturer’s instructions (Promega, Madison, WI) as previously described[
23].
Flow cytometric analysis of apoptosis
Cells (6 *104/well) were plated in 6-well plates in antibiotic-free medium and transfected with control oligonucleotide (100 nM) or inhibitor (100 nM) using Lipofectamine 2000 (Invitrogen 4 Corporation, California, USA) according to the manufacturer’s recommendation. Luciferase and renilla signals were measured 48 h after transfection using the Annexin V-FITC apoptosis detection kit (Invitrogen Corporation, California, USA) as described by the manufacturer’s instructions. Three independent experiments were performed and the data are presented as the mean + SD.
Western blot analysis
Total cell lysates (50 ug) were fractionated by SDS/PAGE. The proteins were electroblotted onto nitrocellulose membranes and Western blot analyses were carried out according to standard procedures as previously described[
24]. β-actin was used as the loading control in the Western blots.
Statistical analysis
The statistical analyses were performed using the Statistical Package for the Social Sciences software using the two-tailed Student’s t-test. The significance was determined at the 95% confidence interval. All the data were expressed as the mean ± standard deviation (SD) from a representative experiment.
Discussion
MicroRNAs play a crucial role in the process of tumor formation. They impact the dynamic balance between oncogenes and tumor suppressor genes by degrading target genes, thereby contributing to cancer progression[
30]. Previous studies have shown that miR-92b is over-expressed in brain primary tumor, as compared to primary tumors from other tissues and their metastases to the brain[
21]. Based on topological and functional analyses, it was also reported that miR-92b could play important roles related to the Notch signaling pathway in Glioblastoma multiforme (GBM) tumors[
31]. However, there were no reports about the association of miR-92b and survival.
In our study, we focused on the regulatory mechanisms of the miR-92b in gliomas. Initially, the miRNA array results showed that miR-92b was upregulated in gliomas, which suggested that miR-92b could play an important role in the development of gliomas as an oncogene. Thus, we hypothesized that the downregulation of miR-92b could promote apoptosis, providing a potential strategy for glioma treatment. In vitro, our studies demonstrated that the miR-92b inhibitor significantly promoted apoptosis and impeded cell viability and colony formation. To determine how miR-92b was involved in the development of gliomas, we used TargetScan and predicted that DKK3 was a probable target of miR-92b in the 3!UTR of DKK3. We proved that the miR-92b overexpression resulted in the downregulation of DKK3 at the protein level, whereas the functional inhibition of miR-92b led to the inhibition of DKK3, strongly suggesting that DKK3 is regulated by miR-92b in gliomas. Meanwhile, a dual luciferase reporter assay identified DKK3 as a direct target of miR-92b.
DKK3 is a critical antagonist of the Wnt/beta-catenin signaling pathway[
32], which has been shown to be inhibited by miR-92b in neuroblastomas, but the mechanism in gliomas has not been elucidated fully[
22]. A previous study showed that the Wnt/beta-catenin signaling pathway was activated in gliomas[
33]. Thus, we speculated that miR-92b played its role by regulating the Wnt/beta-catenin signaling pathway. To elucidate the mechanism, we detected the protein level of beta-catenin and the downstream genes of the Wnt/beta-catenin signaling pathway, such as Bcl2, c-myc, c-Jun and p-c-Jun. The results showed that the overexpression of miR-92b inhibited
DKK3 and increased the expression of beta-catenin (Figure
4A), which suggested that miR-92b modulated beta-catenin via
DKK3. To verify whether miR-92b could modulate the Wnt/beta-catenin signaling pathway, we measured the expression of the downstream genes Bcl2, c-myc, c-Jun and p-c-Jun by Western blotting. The results showed that the miR-92b inhibitor could modulate the expression of these genes. The protein expression of Bcl-2, which is not only a downstream gene of the Wnt/beta-catenin signaling pathway but is also an anti-apoptotic gene, was inhibited by miR-92b. This demonstrated that miR-92b could modulate the genes downstream of the Wnt/beta-catenin signaling pathway. Furthermore, it could modulate apoptosis. To testify how miR-92b affected apoptosis, we analyzed the apoptotic genes Caspase-3 and Bax. The results demonstrated that miR-92b increased the expression of Caspase-3 and Bax, indicating that Caspase-3 was activated after treatment with the miR-92b inhibitor (Figure
4B).
Recent data showed miR-92b could regulate Wnt/beta-catenin signaling via Nemo-like kinase[
34]. However, the significance of miR-92b in prognostic determination have not been shown in glioma. In this study, our data suggest that a high miR-92b expression level might be a valuable marker for pathological diagnosis and prognosis prediction in high-grade glioma; high miR-92b expression levels were significantly associated with poor survival in high-grade glioma patients as determined by Kaplan-Meier analysis.
In summary, our data demonstrated that the miR-92b could regulated glioma cell proliferation, apoptosis by directly targeting DKK3. We also provide direct evidence that high levels of miR-92b expression are significantly associated with poorer overall survival. To conclude, our data suggest that miR-92b could be a intrinsic regulator of progression in glioma cells and could function as a potential target and predictor of survival in glioma.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QL identified and recruited patients and organised sample collection, performed quantitative RT–PCR. KS performed western bolt, cell proliferation and Luciferase assays. YZ and CM performed MTT assay and construct of vectors. JL and JM participated in the project design, coordination the experiments, and manuscript preparation. All authors read and approved the final manuscript.