miR‑489 promotes apoptosis and inhibits invasiveness of glioma cells by targeting PAK5/RAF1 signaling pathways
Retraction in: /10.3892/or.2023.8564
- Authors:
- Published online on: October 22, 2019 https://doi.org/10.3892/or.2019.7381
- Pages: 2390-2401
-
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Glioma is the most common and aggressive malignant primary tumor of the human central nervous system (1,2). Presently available therapies include surgical resection, radiation, chemotherapy and combination therapies. However, despite significant advances in treatment options, patients with glioma frequently display rapid progression and a high rate of recurrence after the initial resection (3). An improved understanding of the underlying molecular pathology and signaling pathways involved in the progression of glioma may uncover novel potential targets in order to design innovative therapies for preventing glioma recurrence and, thus, prolonging survival (4).
p21-activated kinase 5 (PAK5) is a member of the PAK family of Ser/Thr protein kinases. PAK5 is activated by Cdc42/RAC1 and a range of effectors, including hepatocyte growth factor and epidermal growth factor (5). PAK5 is predominantly expressed in the brain and plays a role in multiple signaling pathways, including cytoskeletal regulation, cell motility, cell survival, apoptosis and proliferation (6). PAK5 upregulation is frequently observed in patients with glioma, and has been demonstrated to contribute to glioma cell survival, anti-apoptosis, invasion and progression (7,8). Studies of PAK5 in glioma, particularly on the effects of inactivation of PAK5, are limited.
A wide range of microRNAs (miRNAs) have been demonstrated to be involved in the progression of different types of malignancies (9,10). The aim of the present study was to identify PAK5-targeting miRNAs, and to assess whether these miRNAs possess prognostic or therapeutic value in glioma. miR-489, a PAK5-targeting miRNA, has been shown to regulate glioblastoma progression through the LINC01446/miR-489-3p/TPT1 axis (11). As one of the effectors of the long non-coding RNA (lncRNA) ENST01108, miR-489 targets SIK1 and suppresses glioma progression (12). By targeting SPIN1-mediated regulation of the PI3K/AKT pathway, miR-489 induces apoptosis and arrests cell cycle progression (13). Recent studies have demonstrated that miR-489 acts as a tumor suppressor miRNA by targeting various oncogenic cascades in a number of different types of cancer, indicating that miR-489 targets different oncogenes in glioma as a tumor suppressor miRNA. The aim of the present study was to determine whether PAK5 is a target gene of miR-489, and elucidate the mechanism through which miR-489 suppresses glioma progression.
Materials and methods
Patients
A total of 40 glioma tissues and matched adjacent tissues were used in the present study. The diagnosis of glioma was performed by surgeons and pathologists. The exclusion criteria were as follows: Patients with recurrent glioma; patients who received immunotherapy, radiotherapy or chemotherapy prior to surgery; and other severe organ or autoimmune diseases. The protocol of the present study was approved by The Human Ethics Committee of The First Affiliated Hospital of China Medical University. All the experiments were performed in accordance with the Declaration of Helsinki and subsequent updates. Written informed consent was obtained from each patient.
miRNA microarray analysis
A total of 500 ng of RNA was subjected to Custom RT2 Profiler PCR Array (cat. no. 330171; Qiagen China Co., Ltd.). According to the list of gene names, symbols, UniGene and Genbank IDs, primers were synthesized by the manufacturer (GenePharma Co., Ltd.). Three biological replicates (experimental replicate) were used per group, and each was measured in duplicate (technical replicates). Comparisons for significance were performed using a Student's t-test.
Cell culture and transfection
The normal human astrocyte cell line CC-2565 was obtained from Lonza Group, Ltd. U87 MG cells, a glioblastoma cell line of unknown origin, were obtained from the American Type Culture Collection (cat. no. HTB-14). The human glioma cell line U251 (cat. no. KCB 200965YJ) was purchased from Kunming Institute of Zoology, Chinese Academy of Sciences. Normal human astrocytes (NHA) were cultured in Astrocyte Growth Medium (Lonza Group Ltd.) supplemented with 0.03% FBS (Thermo Fisher Scientific, Inc.). U87 and U251 cells were cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. Cells were maintained at 37°C in an incubator with a humidified atmosphere with 5% CO2 and 95% air. All the reagents were purchased from Gibco (Thermo Fisher Scientific, Inc.). miR-489 mimics, miR-489 inhibitor (miR-489 inh) and negative control RNA were purchased from GenePharma Co., Ltd. The oligonucleotide sequences were as follows: miR-489 mimic, 5′-GUGACAUCACAUAUACGGCAGC-3′; miR-489 inh, 5′-GCTGCCGUAUAUGUGAUGUCAC-3′; negative control (control), 5′-UUGUCCGAACGUGUCACGUTT-3′. Cells were transfected with Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. pDONR223 PAK5 (Addgene plasmid no. 60532) was a gift from Dr John Brognard (Professor of Signalling Networks in Cancer Group, Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester) (14). pDONR223 (Addgene plasmid no. 12536017) was obtained from Thermo Fisher Scientific, Inc.
Luciferase reporter assays
Wild-type (wt) or mutant (mut) 3′-untranslated region (3′-UTR) of the PAK5 promoter was amplified and cloned into a pGL474-vector, and the 3′-UTR of PAK5 and corresponding miRNA vectors were co-transfected into U87 and U251 cells, respectively. The primer sequences were as follows: Wt-PAK5 forward, 5′-CTTTGATGTCATGTAGCCATTG-3′ and reverse, 5′-CAAAGTTCCTAAAGAATCATTGGT-3′; and mut-PAK5 forward, 5′-CTTTGATGTCCTAATGCCATTG-3′ and reverse, 5′-CAAAGTTCCTAATTAGCATTGGT-3′. At 24 h after co-transfection, luciferase activity was measured using a Dual-Luciferase® Reporter Assay System (Promega Corporation).
Reverse transcription-quantitative (RT-q) PCR analysis
Total RNA was extracted using TRIzol® (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Reverse transcription of RNA to cDNA was performed using a reverse transcription system (Promega Corporation). PCR amplification of miR-489 and PAK5 mRNA was performed using an Mx3000P real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. U6 and GAPDH were used as the reference genes to normalize the expression of miR-489 and PAK5 mRNA, respectively. The primer sequences for the detection of mRNA expression were as follows: PAK5: Forward, 5′-GTCTCCTCTGACTTCAACAGC-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′; miR-489: Forward, 5′-GTGACATCACATATACGG-3′ and reverse, 5′-GAACATGTCTGCGTATCTC-3′; U6: Forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; c-Myc: Forward, 5′-CCTGGTGCTCCATGAGGAGAC-3′ and reverse, 5′-CAGACTCTGACCTTTTGCCAGG-3′; cyclin D1: Forward, 5′-ATGTTCGTGGCCTCTAAGATG-3′ and reverse, 5′-CAGGTTCCACTTGAGCTTGTTC-3′; Bcl-2: Forward, 5′-ATCGCCCTGTGGATGACTGAG-3′ and reverse, 5′-CCAGGAGAAATCAAACAGAGG-3′; Bax: Forward, 5′-TCAGGATGCGTCCACCAAGAA-3′ and reverse, 5′-TGTGTCCACGGCGGCAATCATC-3′; MMP2: Forward, 5′-AGCGAGTGGATGCCGCCTTTA-3′ and reverse, 5′-CATTCCAGGCATCTGCGATGAG-3′; GAPDH: Forward, 5′-GTCTCCTCTGACTTCAACAGC-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′.
Western blotting
Total proteins from 1×106 cells were extracted using lysis buffer (Thermo Fisher Scientific, Inc.) and protein concentration was determined using a bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.). A total of 40 µg protein was resolved on a 10% gel using SDS-PAGE (Thermo Fisher Scientific, Inc.). Subsequently, proteins were transferred to a PVDF membrane (EMD Millipore). The membranes were blocked in 5% skimmed milk in TBS-Tween for 1 h at room temperature, and probed with the following primary antibodies: Rabbit polyclonal antibodies PAK5 (cat. no. ab110069; Abcam), p-S338-RAF1 (cat. no. ab51042; Abcam), RAF1 (cat. no. ab137435; Abcam) and matrix metalloproteinase 2 (MMP2; cat. no. ab37150; Abcam); rabbit monoclonal antibodies c-Myc (cat. no. ab32072; Abcam), cyclin D1 (cat. no. ab134175; Abcam), Bcl-2 (cat. no. ab32124; Abcam) and Bax (cat. no. ab32503; Abcam) at 4°C overnight, after which time the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab222772 and ab222759; Abcam). GAPDH was used as the loading control (mouse monoclonal anti-GAPDH, KC-5G4, obtained from Zhejiang Kangchen Biotech Co., Ltd.). The dilution of primary antibodies was 1:1,000 and the dilution of secondary antibodies was 1:5,000. Signals were visualized using enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.).
MTT assay
Glioma cell lines were transfected with negative control (NC), miR-489 mimic or miR-489 inhibitor using Lipofectamine® 3000. After 24 h, 1×104 cells were plated in medium containing 10% FBS in 96-well plates. Cell proliferation was measured using a modified MTT assay. Briefly, MTT solution in PBS was added to each well. After a 4 h incubation at 37°C, the medium was replaced with DMSO. After a 15 min incubation at 37°C, the optical density at 490 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc.).
Cell Counting Kit-8 (CCK-8) assay
Glioma cells transfected with miR-489 mimic or inh were plated at a density of 2×103 cells/well into 96-well plates at 37°C in an incubator with 5% CO2. Cell proliferation was assessed every 24 h using a CCK-8 assay (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. For each sample at each time-point, six wells were analyzed and the experiment was repeated three times.
Migration and invasion assays
Migration and invasion assays were performed using modified Boyden chambers with Corning® Transwell® polycarbonate membrane cell culture inserts (Sigma-Aldrich; Merck KGaA). The invasion assay was performed using BioCoat Matrigel invasion chambers (BD Biosciences). A total of 1×105 cells in 100 µl serum-free DMEM supplemented with 0.1% BSA were placed in the upper part of each chamber, and the lower compartments were filled with 600 µl DMEM supplemented with 10% FBS. The cells were incubated for 18 h at 37°C, and the cells that had invaded or migrated through the membrane were stained with 0.5% crystal violet solution and counted.
Hoechst 33258 staining
Glioma cells were transfected with NC, miR-489 mimic, or miR-489 inh for 24 h. After incubation, the cells were fixed with 4% polyoxymethylene, washed twice with PBS, treated with 10 µg/ml Hoechst 33258 (Molecular Probes; Thermo Fisher Scientific, Inc.) for 5 min at room temperature, and subsequently washed with PBS three times, for 10 min per wash. Cells were observed using a fluorescence microscope (Leica Microsystems GmbH).
Statistical analysis
The results are presented as the mean ± standard deviation of at least three experimental repeats. Comparisons among different groups were performed using a two-way ANOVA. Dunnett's multiple comparisons test was performed to compare the mean of each group with the mean of the control group. Paired data were compared using a Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
Association between miR-489 and PAK5 in glioma
Recently, numerous miRNAs have been demonstrated to be aberrantly expressed in glioma tissues compared with adjacent tissues. In the present study, it was demonstrated that the difference in miR-489 expression between glioma and adjacent tissues was the largest amongst the various miRNAs measured (Table I). Using the miRDB database (9,15), miR-489 was identified as a candidate miRNA targeting PAK5. The relative RNA expression levels of miR-489 and PAK5 were determined in 40 samples from patients with glioma (Fig. 1). The results demonstrated that the expression of miR-489 was decreased in cancer tissues (0.583±0.080) compared with that in adjacent tissues (0.774±0.110), whereas the expression of PAK5 increased in cancer tissues (0.732±0.072) compared with that in adjacent tissues (0.572±0.070) (Fig. 1A and C). To determine whether there was an association between miR-489 expression and clinical outcome in patients with glioma, the 40 cases were divided into two groups according to miR-489 expression, the high miR-489 (n=14) and low miR-489 (n=26) groups. As shown in Fig. 1B, patients with higher miR-489 expression levels had longer survival. In addition, as shown in Fig. 1D, patients with lower PAK5 expression levels (n=20) had longer survival.
To further determine the association between miR-489 and PAK5 in glioma, a Spearman's rank correlation analysis was performed (Fig. 1E). miR-489 and PAK5 expression were found to be negatively correlated. These results suggest that miR-489 expression is negatively correlated with PAK5 expression and may be used to predict the clinical outcome in patients with glioma.
miR-489 decreases PAK5 expression by targeting the 3′-UTR of PAK5
miRNA target prediction data were analyzed using the miRDB database (http://mirdb.org/index.html), and it was predicted that miR-489 targeted the 3′-UTR of PAK5 (Fig. 2A). A dual luciferase reporter assay was performed in U87 and U251 cells to demonstrate the putative binding sites. Glioma cells were transfected with miR-489 mimic or miR-489 inh, combined with wt-PAK5 promoter or mut-PAK5 promoter.
As shown in Fig. 2B, the relative activity of the wt-PAK5 promoter was reduced in cells transfected with miR-489 mimic in U87 cells. Ectopic expression of miR-489 inh restored the relative activity of wt-PAK5 compared with the control. The relative activity of the mut-PAK5 promoter was not altered in cells transfected with either miR-489 mimic or miR-489 inh. Similar results were observed in the U251 cells (Fig. 2C).
To further investigate expression of miR-489 and PAK5 in glioma cells, RT-qPCR assays were performed. As shown in Fig. 2D, miR-489 expression was lower in U87 and U251 cells compared with NHA, whereas PAK5 expression was higher in U87 and U251 cells compared with NHA (Fig. 2E). Additionally, western blotting was performed to examine changes in PAK5 protein expression following transfection with miR-489 mimic or miR-489 inh. PAK5 expression increased following transfection of miR-489 inh, and decreased significantly following transfection with miR-489 mimic in both U87 and U251 cells (Fig. 2F). Similar changes were observed in the relative RNA expression levels (Fig. 2G and H). These results suggest that miR-489 targets the 3′-UTR of PAK5 and decreases the expression of PAK5 in glioma cells.
miR-489 suppresses cell viability through decreasing the expression of PAK5
Recent studies have demonstrated that miR-489 is a tumor suppressor targeting multiple oncogenes in a number of different types of cancer. miR-489 mimic or miR-489 inh was transfected into U87 and U251 cells to determine the effects of miR-489 on glioma cells in vitro. MTT assays were performed to determine the effects of miR-489 on cell viability. The results demonstrated that miR-489 mimic decreased cell viability, whereas miR-489 inh increased the viability of both U87 and U251 cells (Fig. 3A and B). The differences in cell viability after 36 and 48 h were significant. A CCK-8 assay was used to evaluate the effect of miR-489 on cell proliferation. As shown in Fig. 3C, proliferation was increased in cells transfected with miR-489 mimic, whereas it was decreased in cells transfected with miR-489 inh. Similar results were observed in both U251 and U87 cells (Fig. 3D).
As PAK5 phosphorylates RAF1, but not B-raf, at Ser338, thereby activating the RAF1/ERK/MAPK signaling pathway (16), the expression of phospho-Ser338 RAF1 (p-RAF1), RAF1, and the effectors c-Myc and cyclin D1 was determined in U87 and U251 cells using western blotting. As shown in Fig. 3E, following Ser338 phosphorylation, c-Myc and cyclin D1 decreased in U87 cells, whereas PAK5 expression was decreased following transfection with miR-489 mimic, although total RAF1 remained unchanged. By contrast, p-RAF1, c-Myc and cyclin D1 expression decreased following transfection with miR-489 inh. Similar results were observed in U251 cells (Fig. 3F). To confirm the changes in the protein expression levels, RT-qPCR assays were performed to evaluate changes in the relative RNA levels. As shown in Fig. 3G, transfection of miR-489 mimic decreased PAK5, c-Myc and cyclin D1 RNA expression levels significantly, but did not affect RAF1 levels in U87 cells. Transfection with miR-489 inh significantly increased PAK5, c-Myc and cyclin D1 levels (Fig. 3H). Similar results were observed in the U251 cells (data not shown). These results suggest that miR-489 decreases cell viability by inactivating PAK5/RAF1-mediated pathways.
miR-489 increases apoptosis by suppressing the RAF1/Bax pathways
As previously described, the pro-oncogene RAF1 and apoptosis-associated proteins Bax/Bcl-2 were involved in the regulation of apoptosis in a variety of different types of cancer (17,18). U87 and U251 cells were transfected with control, miR-489 mimic or miR-489 inh. Hoechst 33258 staining was performed to determine whether miR-489 increased the apoptosis of glioma cells. Increased condensation of chromatin was observed in glioma cells following transfection with the miR-489 mimic (Fig. 4A and C), whereas the opposite was observed in cells transfected with miR-489 inh (Fig. 4B and D). Furthermore, apoptosis was determined by flow cytometry 24 h after transfection, and the results demonstrated that apoptosis in cells transfected with miR-489 mimic was significantly increased, whereas apoptosis in cells transfected with miR-489 inh was significantly decreased compared with control.
Western blotting was performed to assess changes in expression of the pro-apoptotic protein Bax, and pro-survival protein Bcl-2. As shown in Fig. 4E and H, Bax expression decreased significantly, whereas Bcl-2 expression increased significantly following transfection with miR-489 mimic in both U87 and U251 cells. Transfection of miR-489 inh significantly upregulated Bax expression and downregulated Bcl-2 expression. Furthermore, RT-qPCR analysis demonstrated that transfection of miR-489 mimic increased Bax expression and decreased Bcl-2 expression (Fig. 4F), whereas the opposite results were observed in cells transfected with miR-489 inh in U87 cells (Fig. 4G). The effects of miR-489 on the expression of Bax and Bcl-2 in U251 cells were consistent with those in U87 cells (Fig. 4I and J). These results suggest that miR-489 induced apoptosis in glioma cells by inhibiting the PAK5/RAF1/Bax/Bcl-2 axis.
miR-489 suppresses cell motility by decreasing MMP2 expression
To investigate the effects of miR-489 on migration and invasion, Transwell assays with or without Matrigel® were performed. As shown in Fig. 5A and B, miR-489 mimic significantly decreased migration and invasion, whereas miR-489 inh significantly increased migration and invasion in both U87 and U251 cells. The cell counts of invading or migrating cells are presented in Table II. To further verify the results, western blotting and RT-qPCR analyses were performed in U87 and U251 cells. As shown in Fig. 5C-E, MMP2 expression increased significantly following transfection with miR-489 mimic, whereas the opposite was observed in cells transfected with miR-489 inh. Consistently with the results of RNA expression, the protein levels of MMP2 were decreased in cells transfected with miR-489 mimic, whereas they were significantly increased in cells transfected with miR-489 inh. Similar results were observed in U251 cells (Fig. 5F-H).
These results suggest that miR-489 decreased cell migration and invasion by attenuating the PAK5-MMP2 signaling pathway.
miR-489 suppresses glioma progression by targeting PAK5 and inhibiting PAK5/RAF1-mediated signaling
U251 cells were transfected with control, miR-489 mimic or a combination of miR-489 mimic and PAK5 for 24 h. MTT assays, flow cytometry and Transwell invasion assays were performed to assess cell viability, apoptosis and invasiveness, respectively. The results demonstrated that overexpression of PAK5 reversed the effects of miR-489 on cell growth (Fig. 6A), apoptosis (Fig. 6B) and invasion (Fig. 6C). Furthermore, western blot analysis demonstrated that the overexpression of PAK5 attenuated the miR-489-induced dephosphorylation of RAF1 (Fig. 6D). Additionally, RT-qPCR analysis confirmed that overexpression of miR-489 decreased the expression of c-Myc, cyclin D1, Bcl-2 and MMP2, whereas miR-489 enhanced Bax expression, which was accompanied by a reduction in PAK5 expression (Fig. 6E). Overexpression of PAK5 enhanced the expression of c-Myc, cyclin D1, Bcl-2 and MMP2, and reduced the expression of Bax. These results suggest that miR-489 targeted PAK5 and downregulated PAK5/RAF1-mediated signaling.
Discussion
Despite advances in our understanding of tumorigenesis and progression of glioma, presently available therapeutic approaches cannot cure glioma, and thus contribute little to survival time. At present, molecules and signaling pathways have been identified to be involved in the malignant properties of glioma, such as anti-apoptosis, invasion and chemo-resistance, the critical factors remain to be determined (19–21).
In the present study, miR-489 showed the largest fold change in expression in a miRNA microarray analysis. A number of studies have demonstrated that miR-489 is aberrantly expressed in several types of cancers, including breast cancer (22–26), colorectal cancer (27,28), glioma (11–13), hepatocellular carcinoma (29), melanoma (30), ovarian cancer (31,32) and prostate cancer (33). In particular, Soni et al (22) reported that miR-489 expression levels are associated with poor overall survival in patients with a mutant lysosomal protein transmembrane 4 beta in breast cancer (22). Gao et al (27,28) showed that patients with elevated miR-489 expression levels had reduced cancer free recurrence times.
In the present study, miR-489 expression levels in glioma tissues and matching adjacent normal tissues were determined. miR-489 expression was downregulated in glioma tissues compared with the matching normal tissues. Upregulated levels of miR-489 predicted longer overall survival of patients with glioma. Li et al (13) reported that patients with decreased levels of miR-489 expression had a markedly reduced overall survival (13).
Among 232 predicted targets of miR-489 in miRDB, PAK5, which is expressed predominantly in the central nervous system and regulates multiple cell behaviors, including cytoskeletal stabilization, cell migration, proliferation and cell survival was identified as the candidate (5,34,35). PAK5 expression was determined in the same paired tissues. Compared with matched tumor-adjacent tissues, PAK5 expression was upregulated in cancer tissues significantly, and patients with increased PAK5 expression levels exhibited less favorable clinical outcomes. Consistent with this result, previous studies showed that upregulated PAK5 expression was associated with significantly worse survival in patients with breast cancer (36), bladder cancer (37) and gastric cancer (38). The results of the present study provide evidence that increased PAK5 expression is associated with shorter overall survival in patients with glioma. miR-489 expression was negatively correlated with PAK5 expression. The correlation between miR-489 and PAK5 suggested that miR-489 targeted PAK5 and regulated PAK5-mediated signaling in glioma. There are numerous studies on PAK5 in different types of cancer, although the data of PAK5 in glioma is limited. Increased PAK5 expression in glioma tissues and cells promoted glioma progression by impairing cell cycle arrest and enhancing invasion (7,8). In addition, Zheng et al (39) showed that lncRNA colorectal neoplasia differentially expressed rescued apoptotic suppressor protein XIAP and PAK5 expression by inhibiting miR-186 expression, and thus promoted proliferation, migration, invasion and survival of glioma stem cells. In the present study, it was demonstrated that miR-489 targeted the PAK5 3′-UTR directly using a mut-PAK5 3′-UTR, resulting in suppression of PAK5 expression. Additionally, overexpression of miR-489 attenuated the PAK5/RAF1 axis, resulting in a decrease in cell survival. Overexpression of PAK5 reversed the miR-489 mediated effects on cell growth and invasion, suggesting that regulation of miR-489 on glioma cell growth and invasion is dependent on PAK5. Further experiments with glioma xenografts and integrated analysis of The Cancer Genome Atlas data are required to investigate this hypothesis.
In conclusion, the present study demonstrated that miR-489 was downregulated while PAK5 was upregulated in glioma tissues. miR-489 reduced cell viability and invasion while inducing apoptosis by targeting PAK5/RAF1-mediated pathways. The mechanism underlying the inhibition of malignant behavior was dependent on downregulation of PAK5, improving our understanding of PAK5-mediated signaling cascades in glioma. The present study highlights potentially novel therapeutic targets for treating patients with glioma.
Acknowledgements
Not applicable.
Funding
The Ministry of Education Personnel Returning from Overseas Project sponsored by the Scientific Research Foundation [left outside of the Teaching Department grant no. (2013)1792]; Liaoning Province Natural Science Foundation of China (grant no. 2015020460); Chinese Postdoctoral Science Foundation Funded Project on the Fifty-Ninth Batch of Surface (grant no. 2016M590240); Shenyang City Science and Technology Project (grant no. 17-230-9-13); the Scientific Research Foundation of the First Affiliated Hospital of China Medical University (grant no. FSFH201722).
Availability of data and materials
All the datasets generated and/or analyzed during the present study are included in this published article.
Authors' contributions
WW was responsible for the conception and design of the study, participated in all the experiments and drafted the manuscript; LZ was responsible for cell line culture, and performed the MTT and cell counting assays; WG participated in the western blot and apoptosis analyses; DZ participated in the Hoechst 33258 staining and western blotting; ZZ performed the RT-qPCR assays; YB critically revised the manuscript and provided final approval of the version to be submitted.
Ethics approval and consent to participate
The patients provided written consent for the use of clinical materials for research purposes, and approval was obtained from the First Affiliated Hospital of China Medical University. The patients' prior written consent was obtained according to institutional regulations.
Patient consent for publication
Patient consent for publication has been obtained according to institutional regulations.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
NHA |
normal human astrocytes |
PAK5 |
P21-activated kinase 5 |
3-UTR |
3′-untranslated region |
MMP2 |
matrix metalloproteinase 2 |
GAPDH |
glyceraldehyde 3-phosphate dehydrogenase |
References
Kumar V, Kumar V, McGuire T, Coulter DW, Sharp JG and Mahato RI: Challenges and recent advances in medulloblastoma therapy. Trends Pharmacol Sci. 38:1061–1084. 2017. View Article : Google Scholar : PubMed/NCBI | |
Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P and Ellison DW: The 2016 world health organization classification of tumors of the central nervous system: A summary. Acta Neuropathol. 131:803–820. 2016. View Article : Google Scholar : PubMed/NCBI | |
Lapointe S, Perry A and Butowski NA: Primary brain tumours in adults. Lancet. 392:432–446. 2018. View Article : Google Scholar : PubMed/NCBI | |
Lombardi MY and Assem M: Glioblastoma Genomics: A very complicated storyGlioblastoma. De Vleeschouwer S: Chapter 1. Codon Publications; Brisbane (AU): 2017, https://www.ncbi.nlm.nih.gov/books/NBK470004/doi: 10.15586/codon.glioblastoma.2017.ch1. | |
Ha BH, Morse EM, Turk BE and Boggon TJ: Signaling, regulation, and specificity of the type II p21-activated kinases. J Biol Chem. 290:12975–12983. 2015. View Article : Google Scholar : PubMed/NCBI | |
Wen YY, Zheng JN and Pei DS: An oncogenic kinase: Putting PAK5 forward. Expert Opin Ther Targets. 18:807–815. 2014. View Article : Google Scholar : PubMed/NCBI | |
Gu X, Wang C, Wang X, Ma G, Li Y, Cui L, Chen Y, Zhao B and Li K: Efficient inhibition of human glioma development by RNA interference-mediated silencing of PAK5. Int J Biol Sci. 11:230–237. 2015. View Article : Google Scholar : PubMed/NCBI | |
Han ZX, Wang XX, Zhang SN, Wu JX, Qian HY, Wen YY, Tian H, Pei DS and Zheng JN: Downregulation of PAK5 inhibits glioma cell migration and invasion potentially through the PAK5-Egr1-MMP2 signaling pathway. Brain Tumor Pathol. 31:234–241. 2014. View Article : Google Scholar : PubMed/NCBI | |
Wang X: Improving microRNA target prediction by modeling with unambiguously identified microRNA-target pairs from CLIP-ligation studies. Bioinformatics. 32:1316–1322. 2016. View Article : Google Scholar : PubMed/NCBI | |
Wen MM: Getting miRNA therapeutics into the target cells for neurodegenerative diseases: A mini-review. Front Mol Neurosci. 9:1292016. View Article : Google Scholar : PubMed/NCBI | |
Zhang L, Wang Q, Wang F, Zhang X, Zhang L, Tang Y and Wang S: LncRNA LINC01446 promotes glioblastoma progression by modulating miR-489-3p/TPT1 axis. Biochem Biophys Res Commun. 503:1484–1490. 2018. View Article : Google Scholar : PubMed/NCBI | |
Xu D, Liu R, Meng L, Zhang Y, Lu G and Ma P: Long non-coding RNA ENST01108 promotes carcinogenesis of glioma by acting as a molecular sponge to modulate miR-489. Biomed Pharmacother. 100:20–28. 2018. View Article : Google Scholar : PubMed/NCBI | |
Li Y, Ma X, Wang Y and Li G: MiR-489 inhibits proliferation, cell cycle progression and induces apoptosis of glioma cells via targeting SPIN1-mediated PI3K/AKT pathway. Biomed Pharmacother. 93:435–443. 2017. View Article : Google Scholar : PubMed/NCBI | |
Fawdar S, Trotter EW, Li Y, Stephenson NL, Hanke F, Marusiak AA, Edwards ZC, Ientile S Waszkowycz B, Miller CJ and Brognard J: Targeted genetic dependency screen facilitates identification of actionable mutations in FGFR4, MAP3K9, and PAK5 in lung cancer. Proc Natl Acad Sci USA. 110:12426–12431. 2013. View Article : Google Scholar : PubMed/NCBI | |
Wong N and Wang X: MiRDB: An online resource for microRNA target prediction and functional annotations. Nucleic Acids Res. 43:D146–D152. 2015. View Article : Google Scholar : PubMed/NCBI | |
Wu X, Carr HS, Dan I, Ruvolo PP and Frost JA: P21 activated kinase 5 activates Raf-1 and targets it to mitochondria. J Cell Biochem. 105:167–175. 2008. View Article : Google Scholar : PubMed/NCBI | |
Chai Z, Fan H, Li Y, Song L, Jin X, Yu J, Li Y, Ma C and Zhou R: MiR-1908 as a novel prognosis marker of glioma via promoting malignant phenotype and modulating SPRY4/RAF1 axis. Oncol Rep. 38:2717–2726. 2017. View Article : Google Scholar : PubMed/NCBI | |
Lee SH, Lee EH, Lee SH, Lee YM, Kim HD and Kim YZ: Epigenetic role of histone 3 lysine methyltransferase and demethylase in regulating apoptosis predicting the recurrence of atypical meningioma. J Korean Med Sci. 30:1157–1166. 2015. View Article : Google Scholar : PubMed/NCBI | |
Filbin MG and Suva ML: Gliomas genomics and epigenomics: Arriving at the start and knowing it for the first time. Annu Rev Pathol. 11:497–521. 2016. View Article : Google Scholar : PubMed/NCBI | |
Alifieris C and Trafalis DT: Glioblastoma multiforme: Pathogenesis and treatment. Pharmacol Ther. 152:63–82. 2015. View Article : Google Scholar : PubMed/NCBI | |
Karsy M, Guan J, Sivakumar W, Neil JA, Schmidt MH and Mahan MA: The genetic basis of intradural spinal tumors and its impact on clinical treatment. Neurosurg Focus. 39:E32015. View Article : Google Scholar : PubMed/NCBI | |
Soni M, Patel Y, Markoutsa E, Jie C, Liu S, Xu P and Chen H: Autophagy, cell viability, and chemoresistance are regulated by miR-489 in breast cancer. Mol Cancer Res. 16:1348–1360. 2018. View Article : Google Scholar : PubMed/NCBI | |
Jiang L, He D, Yang D, Chen Z, Pan Q, Mao A, Cai Y, Li X, Xing H, Shi M, et al: MiR-489 regulates chemoresistance in breast cancer via epithelial mesenchymal transition pathway. FEBS Lett. 588:2009–2015. 2014. View Article : Google Scholar : PubMed/NCBI | |
Chen X, Wang YW, Xing AY, Xiang S, Shi DB, Liu L, Li YX and Gao P: Suppression of SPIN1-mediated PI3K-Akt pathway by miR-489 increases chemosensitivity in breast cancer. J Pathol. 239:459–472. 2016. View Article : Google Scholar : PubMed/NCBI | |
Patel Y, Shah N, Lee JS, Markoutsa E, Jie C, Liu S, Botbyl R, Reisman D, Xu P and Chen H: A novel double-negative feedback loop between miR-489 and the HER2-SHP2-MAPK signaling axis regulates breast cancer cell proliferation and tumor growth. Oncotarget. 7:18295–18308. 2016. View Article : Google Scholar : PubMed/NCBI | |
Kuppa SS, Jia W, Liu S, Nguyen H, Smyth SS, Mills GB, Dobbin KK, Hardman WJ and Murph MM: Autotaxin exacerbates tumor progression by enhancing MEK1 and overriding the function of miR-489-3p. Cancer Lett. 432:84–92. 2018. View Article : Google Scholar : PubMed/NCBI | |
Gao S, Liu H, Hou S, Wu L, Yang Z, Shen J, Zhou L, Zheng SS and Jiang B: MiR-489 suppresses tumor growth and invasion by targeting HDAC7 in colorectal cancer. Clin Transl Oncol. 20:703–712. 2018. View Article : Google Scholar : PubMed/NCBI | |
Tao Y, Han T, Zhang T, Ma C and Sun C: LncRNA CHRF-induced miR-489 loss promotes metastasis of colorectal cancer via TWIST1/EMT signaling pathway. Oncotarget. 8:36410–36422. 2017. View Article : Google Scholar : PubMed/NCBI | |
Liu G, Wang H, Fu JD, Liu JY, Yan AG and Guan YY: A five-miRNA expression signature predicts survival in hepatocellular carcinoma. APMIS. 125:614–622. 2017. View Article : Google Scholar : PubMed/NCBI | |
Chen X, Dong H, Liu S, Yu L, Yan D, Yao X, Sun W, Han D and Gao G: Long noncoding RNA MHENCR promotes melanoma progression via regulating miR-425/489-mediated PI3K-Akt pathway. Am J Transl Res. 9:90–102. 2017.PubMed/NCBI | |
Dong P, Xiong Y, Yue J, Hanley SJB and Watari H: B7H3 As a promoter of metastasis and promising therapeutic target. Front Oncol. 8:2642018. View Article : Google Scholar : PubMed/NCBI | |
Wu H, Xiao Z, Zhang H, Wang K, Liu W and Hao Q: MiR-489 modulates cisplatin resistance in human ovarian cancer cells by targeting Akt3. Anticancer Drugs. 25:799–809. 2014. View Article : Google Scholar : PubMed/NCBI | |
Pashaei E, Pashaei E, Ahmady M, Ozen M and Aydin N: Meta-analysis of miRNA expression profiles for prostate cancer recurrence following radical prostatectomy. PLoS One. 12:e01795432017. View Article : Google Scholar : PubMed/NCBI | |
Kumar R, Sanawar R, Li X and Li F: Structure, biochemistry, and biology of PAK kinases. Gene. 605:20–31. 2017. View Article : Google Scholar : PubMed/NCBI | |
Rane CK and Minden A: P21 activated kinase signaling in cancer. Semin Cancer Biol. 54:40–49. 2018. View Article : Google Scholar : PubMed/NCBI | |
Zhang YC, Huo FC, Wei LL, Gong CC, Pan YJ, Mou J and Pei DS: PAK5-mediated phosphorylation and nuclear translocation of NF-kappaB-p65 promotes breast cancer cell proliferation in vitro and in vivo. J Exp Clin Cancer Res. 36:1462017. View Article : Google Scholar : PubMed/NCBI | |
Ismail AF, Oskay Halacli S, Babteen N, De Piano M, Martin TA, Jiang WG, Khan MS, Dasgupta P and Wells CM: PAK5 mediates cell: Cell adhesion integrity via interaction with E-cadherin in bladder cancer cells. Biochem J. 474:1333–1346. 2017. View Article : Google Scholar : PubMed/NCBI | |
Aburatani T, Inokuchi M, Takagi Y, Ishikawa T, Okuno K, Gokita K, Tomii C, Tanioka T, Murase H, Otsuki S, et al: High expression of P21-activated kinase 5 protein is associated with poor survival in gastric cancer. Oncol Lett. 14:404–410. 2017. View Article : Google Scholar : PubMed/NCBI | |
Zheng J, Li XD, Wang P, Liu XB, Xue YX, Hu Y, Li Z, Li ZQ, Wang ZH and Liu YH: CRNDE affects the malignant biological characteristics of human glioma stem cells by negatively regulating miR-186. Oncotarget. 6:25339–25355. 2015. View Article : Google Scholar : PubMed/NCBI |