miR-29a/b/c function as invasion suppressors for gliomas by targeting CDC42 and predict the prognosis of patients.

BACKGROUND: The lethality and poor outcome of high-grade gliomas result from the tumour relentless invasion. miR-29a/b/c downexpressions contribute to several human tumourigenesis. However, their relevance to prognosis and invasion in gliomas remains unclear.
METHODS: Relationships of miR-29a/b/c and CDC42 expressions to grade and survival-time in 147 human gliomas were analysed by in situ hybridisation and immunohistochemistry. Dual-luciferase reporter assay was used to identify CDC42 as a target of miR-29a/b/c. Underlining mechanisms by which miR-29a/b/c inhibited glioma cell migration and invasion were studied by in vitro and in vivo assays.
RESULTS: miR-29a/b/c expressions were inversely correlated with glioma grades, but positively correlated with patients' survival. Two distinct subgroups of grade I-IV glioma patients with different prognoses were identified according to miR-29a/b/c expressions. miR-29a/b/c overexpressions suppressed glioma cell migration and invasion through targeting CDC42 and subsequently decreasing phosphorylated PAK1/2/3, LIMK1/2 and cofilin, the pivotal downstream effectors of CDC42. Moreover, CDC42 expression was positively correlated with glioma grades, but inversely correlated with miR-29a/b/c expressions and patients' survival. In glioblastoma cell lines, CDC42-knockdown could mimic the anti-tumour effects of miR-29a/b/c.
CONCLUSIONS: miR-29a/b/c are important tumour suppressors and novel prognostic biomarkers of gliomas, and miR-29a/b/c and CDC42 are potential therapeutic candidates for malignant gliomas.

Gliomas are the most frequent primary brain tumours (Ricard ; Louis ). Malignant gliomas, especially glioblastoma, are characterised by relentless invasion and rapid growth, thus making radical resection almost impossible for them (Lefranc ). Despite the progress in radiotherapy and chemotherapy, the patients’ prognoses remain dismal (Stupp ). The recent studies have indicated that identification of prognostic biomarkers and therapeutic targets for gliomas is constantly optimising the molecular subclassification, diagnosis, prognostic evaluation and therapy of gliomas (Noushmehr ; Verhaak ; Tanaka ; Louis ).

Several miRNAs have been proved to be the important regulators of cancer biologic behavior and valuable prognostic biomarkers (Riddick and Fine, 2011; Li ; Liu ). miR-29 family has three closely related members, i.e., miR-29a, miR-29b and miR-29c (miR-29a/b/c; Lagos-Quintana ). Previous studies have shown that miR-29a/b/c are tumour suppressors and downexpression in colorectal cancer (Cummins ), lung cancer (Fabbri ; Cushing ), leukaemia (Garzon ), hepatocellular carcinoma (Xiong ) and pancreatic cancer (Kwon ). Moreover, the decrease of miR-29a/b/c expressions closely correlates with more aggressive phenotype and poorer prognosis (Xiong ; Zhao ), suggesting that they may serve as therapeutic candidates and prognostic biomarkers of these malignant tumours (Garzon ; Huang ). Furthermore, miR-29a/b/c may repress the cell proliferation and growth of glioblastoma via targeting SCAP and SREBP-1, and prolong the survival of glioblastoma-bearing mice (Ru ). However, the prognostic and therapeutic significances of miR-29a/b/c in gliomas remain elusive owing to the lack of large pools of clinical specimens for screening.

Cell division cycle 42 (CDC42), a small GTPase of Rho-subfamily, is overexpressed in various malignant tumours and accelerates the migration and invasion of tumour cells by inducing the rearrangement of actin cytoskeleton (Stengel and Zheng, 2011; Orgaz ). It has been known that miR-29a/b/c may promote the apoptosis of HeLa (cervical cancer) and SNU-638 (gastric cancer) cell lines, and osteoclastogenesis by targeting CDC42 (Park ; Franceschetti ). Our previous study has discovered that miR-29a/b/c can suppress the migration and invasion of glioma cells by targeting DNMT3A and 3B (Xu ). To our knowledge, it is not reported whether miR-29a/b/c inhibit the migration and invasion of glioma cells through directly targeting CDC42.

In this study, we confirmed for the first time that miR-29a/b/c downexpressions result in CDC42 overexpression in gliomas and miR-29a/b/c upregulation may restrain the migration and invasion of glioma cells through directly targeting CDC42, and identified miR-29a/b/c and CDC42 as predictors for the survival of glioma patients. Our findings also indicate that miR-29a/b/c and CDC42 are potential therapeutic candidates for malignant gliomas.

Materials and methods

Tissue samples and clinical data

The surgical specimens of 147 astrocytic gliomas and 20 nontumoural brain tissues (control) were collected from Tianjin Medical University General Hospital (TMUGH) with written consent. After surgical excision, specimens were fixed in 3.7% buffered formaldehyde solution immediately and embedded in paraffin afterwards (FFPE samples). Then, 5 μm continuous sections were prepared for HE staining, miR-29a/b/c in situ hybridisation and the immunohistochemical detection of CDC42. Pathological diagnoses were independently made by two neuropathologists according to the 2016 World Health Organization (WHO) classification of central nervous system tumours (Louis ). The WHO grades and patients’ clinical features were summarised in Supplementary Table S1. All the 147 glioma patients had complete information and were followed up after operation until 31 December 2013, with a follow-up time of 4.4 months to 15 years. This study was carried out in accordance with the principles of the Helsinki Declaration and approved by the Ethics Committee of TMUGH.

An independent cohort of 1002 human glioma specimens from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/) was used to validate the correlations between miR-29a/b/c expressions and DFS (756 cases) or OS (1002 cases) in glioma patients.

In situ hybridisation

In situ hybridisation (ISH) detection was performed as described previously (Liu ). The locked nucleic acid (LNA)-modified and digoxin-labelled oligonucleotide probes of miR-29a/b/c and scrambled control sequence (Scr; TaKaRa, China; Supplementary Table S2), Rhodamine (TRITC)-conjugated anti-digoxin antibody (Roche, Indianapolis, IN, USA) and 4′,6-diamidino-2-phenylindole (DAPI; Roche) were used for ISH. The hybridisation images were acquired under a DM6000B fluorescent microscope (Leica, Wetzlar, Germany) and the percentage ratio (Labelling index (%), LI) of positive cell number to total cell number in 6 randomly selected microscopic fields at × 400 was calculated with Image Pro Plus 5.0 software (Leica).

Cell culture, lentivirus and stable-infected cell line establishment

Human glioblastoma cell lines, U87MG and U251 cells, were used for present study. U87MG was obtained from the American Type Culture Collection and U251 was purchased from the China Academia Sinica Cell Repository (Shanghai). U87MG and U251 cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) in a humidified incubator with 5% CO2 atmosphere at 37 °C. The recombinant lentiviruses expressing miR-29a/b/c (miR-29s), the scrambled control sequence (Scr) and CDC42 were constructed and packaged by Genechem (Shanghai, China). U87MG stable sub-cell lines (U87MG-Scr, -miR-29s and -miR-29s+CDC42) were established by infecting the above lentiviruses, respectively.

Oligonucleotides, plasmids and cell transfection

The dsRNA oligonucleotides of miR-29a/b/c mimics, siRNA silencing CDC42 (si-CDC42) and scrambled control sequence (Scr; Ribobio, Guangzhou, China) were transfected into U87MG and U251 cells with X-tremeGENE siRNA Transfection Reagent (Roche). Their sequences have been listed in Supplementary Table S3. The mock control cells were treated only using the transfection reagent of the same volume. CDC42 expression plasmid (p-CDC42) carrying optimised full-length open reading frame of CDC42 was constructed by GeneCopoeia (Rockville, MD, USA) using pcDNA3.1 vector. p-CDC42 was prepared with EZgene Plasmid Miniprep Kit (Biomiga, San Diego, CA, USA) and transfected with X-tremeGENE HP DNA Transfection Reagent (Roche).

Quantitative RT–PCR

Quantitative RT–PCR (qRT–PCR) detection was performed as previously described (Liu ). miR-29a/b/c were quantified by Stem-loop Detection Kit (GenePharma, Shanghai, China) with U6 as the internal control and CDC42 mRNA level was detected by GoTaq qPCR Master Mix Kit (Promega, Fitchburg, WI, USA) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. Primers for CDC42 mRNA detection have been listed in Supplementary Table S4. The fold changes of miR-29a/b/c and CDC42 mRNA levels were calculated by 2−ΔΔCt method.

Transwell migration and invasion assays

For transwell assays, control and treated cells (2.5 × 104 per well) were seeded in the upper well with serum-free medium of the transwell chamber (Millipore, Billerica, MA, USA) coated with or without Matrigel (BD Bioscience, Mountain View, CA, USA), and allowed to migrate or invade towards the medium containing 10% FBS in lower compartment for 24 h. The cells reaching the filter lower surface of each chamber were fixed with methanol, stained with 0.1% crystal violet and counted in five randomly selected microscopic fields (× 200).

Target prediction and dual-luciferase reporter assay

The candidate target genes of miR-29a/b/c were predicted using Targetscan (http://www.targetscan.org/), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/) and PicTar (http://pictar.mdc-berlin.de/cgi-bin/PicTar_vertebrate.cgi). The cDNAs coding CDC42 3′-untranslated region (CDC42-3′-UTR-WT) and its mutants (CDC42-3′-UTR-MT1 and CDC42-3′-UTR-MT2) without predicted miR-29a/b/c target region 1 or 2 were acquired by RT–PCR or site-directed mutagenesis PCR (primers see Supplementary Table S5), and inserted downstream of firefly luciferase reporter gene in pEZX-MT01 vector (Genecopoeia). The recombinant reporter plasmids (p-WT, p-MT1 or p-MT2) of CDC42 3′-UTR were transfected in U87MG or U251 cells alone (Mock) or with miR-29a/b/c mimics and scrambled control (Scr) using X-tremeGENE HP DNA Transfection Reagent (Roche). Firefly and renilla luciferase activities were measured with Dual-luciferase Reporter Assay System (Promega). The results were presented as the firefly luciferase activities normalised against those of renilla.

Western blotting

Western blotting was carried out as previously described (Li ). The primary antibodies used in this study were as follows: rabbit anti-human CDC42 (Boste, Wuhan, China), rabbit anti-human p-PAK1/2/3 (Abcam, Cambridge, MA, USA), rabbit anti-human p-LIMK1/2 (CST, USA), rabbit anti-human p-Cofilin (Immunoway, Plano, TX, USA) and mouse anti-human β-actin (Boste).

Immunohistochemistry

Immunohistochemistry (IHC) staining was performed with rabbit anti-human CDC42 primary antibody (Boste) as described previously (Li ). IHC images were acquired and LI (Labelling index (%)) was calculated as described in ISH methods.

Tumour xenograft assay in nude mice

The animal experiments were conducted strictly in accordance with a protocol approved by the Institutional Animal Care and Use Committee of TMUGH. Four-week-old BALB/C athymic nude mice (the National Laboratory Animal Center, Beijing, China) were anaesthetised and intracranially injected with 5 × 105 U87MG cells (Mock) or the stable sub-cell lines (U87MG-Scr, -miR-29s or -miR-29s+CDC42). The brains were collected until 15 days after the transplantation and then sampled for HE staining.

Statistical analysis

All statistical analyses were performed using SPSS 21.0 software (IBM, Chicago, IL, USA). The data were presented as mean±standard deviation (s.d.). One-way ANOVA test, Pearson correlation analysis, Kaplan–Meier analysis and log-rank test were used to analyse corresponding data in this study. The median was used to determine the cutoff in each glioma cohort for survival analyses. Statistical significance was assigned at *P<0.05,** P<0.01 or ***P<0.001. All the experiments of cell lines were performed at least three times with triplicate samples.

Results

miR-29a/b/c correlate with grades and better prognosis in human gliomas

To identify the relationships between miR-29a/b/c expressions in gliomas and histopathological grades or patients’ prognoses, endogenous miR-29a/b/c levels in the FFPE specimens of 147 gliomas and 20 nontumoural control brain tissues from human were detected by ISH with LNA-modified probes. We found that miR-29a/b/c expressions in gliomas were lower than those in the control (P<0.001) and that their expressions were significantly decreased with glioma grade elevation and were the lowest in glioblastoma (P<0.001; Figures 1A and B, 2A and B, and 3A and B). Kaplan–Meier analyses revealed that glioma patients with higher levels of miR-29a/b/c had longer disease-free survival (DFS; P<0.0001) and overall survival (OS; P<0.0001; Figures 1C, 2C and 3C). Significantly, we discovered that patients with the same grade glioma could be divided into two subgroups with different outcomes based on miR-29a/b/c expressions, that is, the higher expressions of miR-29a/b/c were, the better prognosis of patients (DFS: P<0.0001; OS: P<0.0001; Figures 1D, 2D and 3D and Supplementary Figure S1A–S1C). The prognostic values of miR-29a/b/c in gliomas were further verified in glioma patients from TCGA database (DFS: P<0.01∼0.001; OS: P<0.01; Supplementary Figure S2A-S2C). These data indicate the inverse association of miR-29a/b/c expressions with glioma malignancy and reveal that they are potential prognostic biomarkers for glioma patients.

Figure 1

miR-29a expression correlates with glioma grades and patients’ prognoses. (A) Representative images of miR-29a ISH detection. Scale bar, 50 μm. (B) Comparisons among groups of miR-29a expression level (Labelling index, LI (%)) in the FFPE samples of 147 gliomas and 20 nontumoural control brain tissues. The miR-29a LI (%) of each sample was calculated with Leica Image Pro Plus 5.0 software according to percentage ratio of positive cell number to total cell number. The data in B are presented as the mean±s.d. ***P<0.001. (C and D) Kaplan–Meier analyses of the correlation between miR-29a and DFS (upper) or OS (under) of all grade glioma patients (C) and WHO grade IV glioblastoma patients (D). Patients were stratified into high and low expression subgroups using the median of miR-29a LIs.

Figure 2

miR-29b expression correlates with glioma grades and patients’ prognoses. (A) Representative images of miR-29b ISH detection. Scale bar, 50 μm. (B) Comparisons among groups of miR-29b expression level (Labelling index, LI (%)) in the above FFPE samples. The miR-29b LI (%) of each sample was calculated as described in Figure 1. The data in B are presented as the mean±s.d. ***P<0.001. (C and D) Kaplan-Meier analyses of the correlation between miR-29b and DFS (upper) or OS (under) of all grade glioma patients (C) and WHO grade IV glioblastoma patients (D). Patients were stratified into high- and low-expression subgroups using the median of miR-29b LIs.

Figure 3

miR-29c expression correlates with glioma grades and patients’ prognoses. (A) Representative images of miR-29c ISH detection. Scale bar, 50 μm. (B) Comparisons among groups of miR-29c expression level (Labelling index, LI (%)) in the above FFPE samples. The miR-29c LI (%) of each sample was calculated as described in Figure 1. The data in B are presented as the mean±s.d. ***P<0.001. (C and D) Kaplan–Meier analyses of the correlation between miR-29c and DFS (upper) or OS (under) of all grade glioma patients (C) and WHO grade IV glioblastoma patients (D). Patients were stratified into high and low expression subgroups using the median of miR-29c LIs.

miR-29a/b/c suppress the migration and invasion of human glioma cells

The relentless invasion of malignant gliomas is the major cause resulting in poor outcome and death of the patients. Prompted by the above findings, we examined the suppressive effects of miR-29a/b/c on glioma cell migration and invasion by transfecting their mimics. The transfection efficiency was verified by qRT–PCR (Supplementary Figure S3A and S3B). Transwell assays showed that all miR-29a/b/c could effectively suppress the migration and invasion of U87MG and U251 cells compared with Mock and Scr controls (P<0.001; Figure 4A–C), indicating that miR-29a/b/c are the effective inhibitors of cell migration and invasion of malignant gliomas.

Figure 4

miR-29a/b/c suppress glioma cell migration and invasion. (A) Transwell assays of the migration and invasion of U87MG and U251 cells untreated (Mock) and transfected with scrambled control sequence (Scr) or miR-29a/b/c mimics. (B and C) Quantitative analyses of the transmembrane migratory and invasive capabilities of the cells as indicated. The migratory and invasive cells were counted under the microscopic fields at × 200. All experiments were performed at least in triplicate and the data in B and C are presented as the mean±s.d. ***P<0.001.

CDC42 is a direct target of miR-29a/b/c in human glioma cells

Targetscan, miRTarBase and PicTar predictions revealed that the 3′-UTR of CDC42 mRNA contained two conserved miR-29a/b/c target regions (Figure 5A), which were confirmed by dual-luciferase reporter assays in U87MG and U251 cells (Figure 5B and C), indicating CDC42 as a target directly silenced by miR-29a/b/c in glioma cells. To further verify whether miR-29a/b/c directly knocked down CDC42, we monitored the changes of CDC42 mRNA and protein levels in U87MG and U251 cells transfected with miR-29a/b/c by qRT–PCR and Western blotting. As shown in Figure 5D and E, the mRNA and protein of CDC42 were significantly decreased in the cell lines transfected with miR-29a/b/c, as compared with Mock and Scr control ones (P<0.01∼0.001). The results reveal that miR-29a/b/c may directly bind with CDC42 3′-UTR and inhibit CDC42 protein expression through inducing its mRNA degradation in glioma cells.

Figure 5

miR-29a/b/c directly target CDC42 in glioma cells. (A) Two miR-29a/b/c target regions in CDC42 3′-UTR predicted by bioinformatics. (B) Wild (CDC42-3′-UTR-WT) and mutant (CDC42-3′-UTR-MT1 and CDC42-3′-UTR-MT2) CDC42 3′-UTRs carried in recombinant luciferase mRNAs transcribed by reporter plasmids (p-WT, p-MT1 and p-MT2). The target region 1 or 2 was deleted from CDC42-3′-UTR-MT1 or CDC42-3′-UTR-MT2. (C) Dual-luciferase reporter assays in U87MG and U251 cells transfected with p-WT, p-MT1 or p-MT2 alone (Mock), and cotransfected with p-WT, p-MT1 or p-MT2 and scrambled control sequence (Scr) or miR-29a/b/c mimics. (D and E) qRT–PCR and western blot analyses of CDC42 mRNA and protein in U87MG and U251 cells transfected with Scr or miR-29a/b/c mimics. Their relative expression levels were normalised against GAPDH mRNA or β-actin protein. The ratios of CDC42/GAPDH mRNAs and CDC42/β-actin proteins in untreated cells (Mock) were set to 1.0. All experiments were performed at least in triplicate and the data in C–E are presented as the mean±s.d. *P<0.05; **P<0.01; ***P<0.001.

CDC42 overexpression is associated with miR-29a/b/c downexpressions and poorer prognoses in human gliomas

We then detected CDC42 in the above FFPE specimens of gliomas and control brain tissues by IHC, and found that CDC42 expression was higher in gliomas than in brain tissues (P<0.001), and was significantly increased with glioma grade elevation and was the highest in glioblastoma (P<0.01–0.001; Figure 6A and B). Furthermore, CDC42 expression was negatively correlated with miR-29a/b/c expressions in gliomas (miR-29a: r=−0.870; miR-29b: r=−0.877; miR-29c: r=−0.883; P<0.0001; Figure 6C). Kaplan–Meier analyses demonstrated that the high level of CDC42 predicted a short-term DFS (P<0.0001) and OS (P<0.0001; Figure 6D) in glioma patients. Moreover, the patients with the same grade glioma could also be divided into two subgroups with different outcomes based on CDC42 expression, i.e., the higher expression of CDC42 was, the poorer prognosis of patients (DFS: P<0.0001; OS: P<0.0001; Figure 6E and Supplementary Figure S1D). These data identify the positive correlation of CDC42 expression with glioma grades and CDC42 as a potential prognostic biomarker for glioma patients, and indicate that miR-29a/b/c downexpressions are the important cause inducing CDC42 overexpression in gliomas.

Figure 6

CDC42 expression correlates with glioma grades, miR-29a/b/c expressions and patients’ prognoses. (A) Representative images of CDC42 IHC detection. Scale bar, 50 μm. (B) Comparisons among groups of CDC42 expression level (Labelling index, LI (%)) in the FFPE samples of 147 gliomas and 20 nontumoural control brain tissues. The CDC42 LI (%) of each sample was calculated as described in Figure 1. The data in B are presented as the mean±s.d. **P<0.01; ***P<0.001. (C) Pearson correlation analysis between CDC42 and miR-29a/b/c expressions in the above FFPE samples. (D and E) Kaplan–Meier analyses of the correlation between CDC42 and DFS (left) or OS (right) of all grade glioma patients (D) and WHO grade IV glioblastoma patients (E). Patients were stratified into high- and low-expression subgroups using the median of CDC42 LIs.

miR-29a/b/c suppress glioma cell migration and invasion by targeting CDC42

To determine whether miR-29a/b/c suppress glioma cell migration and invasion by silencing CDC42, U87MG and U251 cells were transfected with miR-29a/b/c mimic mixture (miR-29s) or CDC42 siRNA (si-CDC42) alone, and cotransfected with CDC42 expression plasmid plus miR-29s (miR-29s+CDC42) or si-CDC42 (si-CDC42+CDC42). Transwell assays showed that miR-29s also obviously restrained the migration and invasion of these cells, while CDC42 overexpression partially compromised the inhibitory effects of miR-29s on the migration and invasion (P<0.01∼0.001; Figure 7A). qRT–PCR and western blot analyses confirmed that si-CDC42 could specifically and effectively decrease the expressions of CDC42 mRNA and protein in U87MG and U251 cells as compared with Mock and Scr controls (P<0.001; Figure 7B and C). Moreover, si-CDC42 perfectly simulated the suppressive roles of miR-29s on glioma cell migration and invasion, while these effects of si-CDC42 were also partially reversed by CDC42 overexpression (P<0.001; Figure 7D). Consistent with the in vitro results, xenografted tumours formed by miR-29s-overexpressing U87MG cells showed less migration and invasion compared with those by control ones, while CDC42 overexpression might partially relieve the miR-29s-repressed migration and invasion (Supplementary Figure S4). All these findings indicate that miR-29a/b/c suppress glioma cell migration and invasion by directly targeting CDC42.

Figure 7

CDC42 is a target by which miR-29a/b/c restrain glioma cell migration and invasion. (A) Transwell representative images (left) and migratory and invasive cell numbers (right) of U87MG and U251 cells untreated (Mock) and transfected with scrambled control sequence (Scr), miR-29a/b/c mimic mixture (miR-29s) or miR-29s plus CDC42 expression plasmid (miR-29s+CDC42). (B and C) qRT–PCR and western blot analyses of CDC42 mRNA and protein in U87MG and U251 cells untreated (Mock) and transfected with Scr or CDC42 siRNA (si-CDC42). Their relative expression levels were normalised and quantified as described in Figure 5D and E. (D) Transwell representative images (left) and migratory and invasive cell numbers (right) of U87MG and U251 cells untreated (Mock) and transfected with Scr, si-CDC42 or si-CDC42 plus CDC42 expression plasmid (si-CDC42+CDC42). All experiments were performed at least in triplicate and the data in A–D are presented as the mean±s.d. **P<0.01; ***P<0.001.

miR-29a/b/c inhibit glioma invasion by interdicting CDC42-PAK pathway

To further determine the underlying mechanisms by which miR-29a/b/c suppress glioma cell migration and invasion, we focused on PAK1/2/3, LIMK1/2 and cofilin, the important downstream effectors of CDC42, to investigate whether they were responsible for the migration and invasion suppressed by miR-29a/b/c. Western blot analyses showed that phosphorylated PAK1/2/3 (p-PAK1/2/3), LIMK1/2 (p-LIMK1/2) and cofilin (p-cofilin) were significantly reduced in U87MG and U251 cells transfected with miR-29a/b/c mimics (P<0.01–0.001; Figure 8A). Moreover, the transfections of miR-29s and si-CDC42 not only inhibited CDC42 expression but also decreased p-PAK1/2/3, p-LIMK1/2 and p-cofilin, while CDC42 overexpression abrogated the inhibitory effects of miR-29a/b/c overexpressions and CDC42 knockdown on the phosphorylation of these proteins (P<0.05∼0.001; Figure 8B and C). The results reveal that miR-29a/b/c suppress the activation of CDC42-PAK pathway by directly targeting CDC42, and then decrease the phosphorylation activation of LIMK1/2 and the phosphorylation inactivation of cofilin, thereby inhibiting glioma cell migration and invasion (Figure 8D). It further verifies the dependability of the above results that CDC42 siRNA imitates the effects of miR-29a/b/c.

Figure 8

miR-29a/b/c and CDC42 siRNA suppress the activation of CDC42-PAK pathway. (A–C) Left: western blot analyses of CDC42, p-PAK1/2/3, p-LIMK1/2 and p-cofilin in U87MG and U251 cells untreated (Mock) and transfected with scrambled control sequence (Scr), miR-29a/b/c mimics, miR-29a/b/c mimic mixture (miR-29s) or CDC42 siRNA (si-CDC42) and CDC42 expression plasmid plus miR-29s (miR-29s+CDC42) or si-CDC42 (si-CDC42+CDC42). Right: Comparisons among groups of CDC42, p-PAK1/2/3, p-LIMK1/2 and p-cofilin levels in the cells as indicated. Their relative levels were normalised against β-actin protein. All experiments were performed at least in triplicate and the data in A–C are presented as the mean±s.d. *P<0.05; **P<0.01; ***P<0.001. (D) Schematic illustration of the molecular pathway by which miR-29a/b/c inhibit glioma cell migration and invasion.

Discussion

Previous reports have showed that miR-29a/b/c may function as tumour suppressors in different tumours (Cummins ; Fabbri ; Garzon ; Xiong ; Zhao ; Kwon ). However, the exact roles and clinical relevance of miR-29a/b/c in gliomas remain uncertain. In the present study, we identified miR-29a/b/c as tumour suppressors to inhibit the cell migration and invasion in astrocytic gliomas, representing the first comprehensive analysis of miR-29a/b/c in gliomas. Mechanistically, we verified CDC42 as a direct functional target of miR-29a/b/c in gliomas, which facilitated our understanding of the mechanisms underlying glioma malignant progression. Most importantly, we discovered that miR-29a/b/c and CDC42, not only were correlated with each other, but also predicted the survival of glioma patients, highlighting the potential values of miR-29a/b/c and CDC42 as novel prognostic biomarkers in human gliomas.

Our previous studies have found that several miRNAs and their target proteins have important values in the diagnosis, molecular subclassification and prognosis evaluation of gliomas (Li ; Shi ; Liu ). These discoveries provide new ideas for searching clinical biomarkers relating to the grades, specific subtypes or prognosis of gliomas. In this study, we demonstrated that miR-29a/b/c expressions were significantly decreased and CDC42 expression was observably increased with the grade elevation in 147 human glioma specimens of WHO grade I–IV, and that the glioma subgroups with higher levels of miR-29a/b/c and lower level of CDC42 had better prognoses, suggesting that they were the potential biomarkers in distinguishing glioma grades and the specific biomarkers for prognostic-based glioma subclassification. There was an inverse correlation between the expressions of miR-29a/b/c and CDC42, implying that miR-29a/b/c downexpressions resulted in CDC42 overexpression in gliomas. Furthermore, miRNAs stably exist in FFPE samples and can be easily detected by ISH (Li ; Liu ). Thus, miR-29a/b/c and CDC42 could be the novel and clinical feasible candidates for diagnosis and subclassification for gliomas.

Malignant gliomas, of which 60 to 70% are glioblastoma, are characterised by relentless invasive growth resulted from high-speed migration and invasion of tumour cells (Wen and Kesari, 2008; Li ; Liu ; Ostrom ). Our transwell and tumour transplantation assays showed that miR-29a/b/c could effectively suppress the migration and invasion of glioma cells in vitro and in vivo. These results indicated that miR-29a/b/c as tumour suppressors inhibited the migration and invasion of glioma cells, and suggested that CDC42 overexpression induced by miR-29a/b/c downexpressions was an important factor accelerating the migration and invasion of malignant glioma cells, highlighting the potential values of miR-29a/b/c and CDC42 in the therapy of malignant gliomas.

Small GTPase CDC42 is a molecular switch that cycles between inactive GDP-bound and active GTP-bound state (Dummler ; Stengel and Zheng, 2011; Orgaz ). CDC42-GTP-bound PAKs are activated by autophosphorylation and then catalyse phosphorylated activations of LIMKs (Edwards ; Dummler ; Radu ; Rane and Minden, 2014). Actin filaments (F-actin) drive cell directional migration through the cycle of ADP•F-actin depolymerisation and ATP•F-actin polymerisation (Bravo-Cordero ). Cofilin binds to and severs ADP•F-actin to generate free barbed end and ADP•G-actin-cofilin complex, while p-LIMKs phosphorylate and dissociate cofilin from ADP•G-actin which will be converted into ATP•G-actin used for ATP•F-actin polymerisation at the newly formed barbed ends, and then p-cofilin is dephosphorylated by SSH1 and recycled, thereby CDC42 overexpression plays a crucial role in the migration and invasion of malignant tumour cells (Edwards ; Bravo-Cordero ; Orgaz ).

We identified CDC42 as a direct functional target of miR-29a/b/c in glioma cells by bioinformatics prediction, luciferase reporter assay and the detection of CDC42 knockdown with miR-29a/b/c. Subsequently, we confirmed that miR-29a/b/c-induced knockdown of CDC42 could inactivate PAK1/2/3-LIMK1/2 pathway and reduce cofilin phosphorylation in glioma cells, and inhibited their migration and invasion. Moreover, siRNA knockdown of CDC42 perfectly imitated the above effects of miR-29a/b/c. All these findings were further validated by the rescue experiments. Combining with the inverse relevance of miR-29a/b/c and CDC42 expressions in the glioma specimens, our results indicated that CDC42 overexpression induced by miR-29a/b/c downexpressions promoted glioma cell migration and invasion by activating PAK1/2/3-LIMK1/2 pathway that phosphorylated cofilin, which enhanced our comprehension of the molecular mechanism of glioma malignant progression (Figure 8D) and suggested that miR-29a/b/c could be potentially applied in the therapy of malignant gliomas.

In summary, our study revealed that miR-29a/b/c inhibited glioma cell migration and invasion by directly targeting CDC42, and predicted better prognosis in human gliomas, especially in glioblastoma. Their downregulations were the important causes leading to glioma malignant progression. More importantly, miR-29a/b/c might be the novel biomarkers for molecular subclassification of malignant gliomas and the therapeutic candidates for these lethal diseases.