A novel functional polymorphism of GFAP decrease glioblastoma susceptibility through inhibiting the binding of miR-139

Glioblastoma (GBM) is the most commonly diagnosed solid tumor outside the central nervous system. However, genetic factors underlying GBM remain largely unclear. Previous studies indicated that Glial fibrillary acidic protein (GFAP) might play an important role in the aggressiveness of GBM and also contributed to its poor overall survival. The present study aims to test (1) the associations between GFAP single nucleotide polymorphisms (SNPs) and GBM cells chemoresistance and metastasis, and (2) the molecular mechanism accounting for their effects. Four tagging SNPs of GFAP were initially genotyped in 667 subjects and the significant SNP was further analyzed via online bioinformatical tools. SNP rs11558961 was found to be significantly associated with GBM susceptibility. It was predicted to influence microRNA(miR)-139 binding to 3'UTR of GFAP gene. In functional experiments, we found that cells transfected with rs11558961 G-allele constructs had lower baseline luciferase activities and were more responsive to miR-139 changes, compared to C-allele constructs. Moreover, rs11558961 C>G variant reduced the chemoresistance of GBM cells and migration capability. In conclusion, rs11558961 might influence the chemoresistance and progression of GBM cells via promoting the binding of miR-139, ultimately decrease the susceptibility of GBM. This investigation will shed light on the optimizing for clinical trial design and individualizing of therapeutic plans.


INTRODUCTION
AGING astrocytes [4]. Increasing evidences showed that GFAP level elevated in high-grade brain tumors, implicating that GFAP might be involved in the aggressiveness of brain tumors [5][6][7]. The polymorphism of GFAP gene was confirmed involving in neural dysfunction disease. C/C genotype at rs2070935 of the GFAP gene in Alexander disease was associated with an earlier onset and a more rapid progression of ambulatory disability compared with other genotypes [8]. According to candidate gene strategy, we chose 4 SNP located in GFAP gene and detected the association between SNPs and GBM risk.
miRNAs are small non-coding RNAs which participate in cell biological processes and always function as pivotal gene transcriptional regulators. They modulate gene expression via promoting mRNA degradation after binding to the 3'UTR [9]. With screening for functional SNPs associated with GBM risk, we found a significant SNP-rs11558961 locating in a miR-139 binding site. The variant affected GFAP expression with the presence of miR-139. Emerging evidences indicated that miR-139 was considered as a tumor suppressor in GBM, which played a role in repressing migration and malignantly proliferation of cancer cells [10,11]. This was in concordance with our findings, rs11558961 C>G variant resulted in a emerging miR-139 bind site and thus a reduced GBM risk. Taken together, to demonstrate the clear molecular mechanism between functional SNPs, miR-139, 3' UTR of GFAP mRNA, the malignant progression of cancer cells and the susceptibility for GBM is essential in the development of novel therapeutic strategies to suppress GBM progression.
In summary, we reported a functional SNP in GBM-rs11558961 in 3' UTR of GFAP. It significantly decreased GBM susceptibility and affected the binding of miR-139 to GFAP mRNA. This might be the potential mechanism of rs11558961 influencing the GBM susceptibility.

Association of SNPs with GBM susceptibility
rs11558961, rs1042329, rs8067254, and rs17027 were consistent with HWE in healthy controls, with the P

Association of SNP rs11558961 Genotypes with miR-139 binding and GFAP expression
Bioinformatical analyses suggested that rs11558961 was located at miR-139 binding site, the 3'UTR of GFAP. rs11558961 C>G variant affected secondary structure and minimum free energy of the 3'UTR mRNA (Fig. 1A, B). To evaluate the different affinity of C and G allele to miR-139, three luciferase reporter constructs were created ( Fig. 2A). The G allele was predicted to bind with miR-139 but C allele not. The binding site in mutant sequence was completely destroyed (Fig. 2B). In GBM cells transfected with rs11558961-G allele construct, miR-139 mimic attenuated the luciferase activity by 30.63% while compared with control group; however, miR-139 mimic only reduced the luciferase activity by 7.58% in rs11558961-C allele group (Fig. 2C). Antagomir-miR-139 increased the luciferase activity by 42.31% in G allele group than that by 9.86% in C allele group (Fig.  2D). Mutant construct lost the miR-139 binding site did not present any obvious change. Based on the above evidences, miR-139 mimic could interfere GFAP expression in both rs11558961-C and G allele groups.
Antagomir-miR-139 increased GFAP expression in C and G allele groups. Under the three conditions (control, miR-139 mimic, antagomir-miR-139), cells carrying G allele construct showed lower luciferase activity than those with C allele. Destruction of the miR-139 binding site prevented the effects of miR-139, antagomir-miR-139 on GFAP expression.
We further compared GFAP expression among GBM cells with different genotypes. The Western Blot data revealed that the expression level of GFAP were highest in CC group, and then in CG or GG group ( Fig. 3A-B).

rs11558961 C>G variant reduced chemoresistance and increased imatinib-induced apoptosis
To evaluate the biological effect of rs11558961 on malignant phenotype of GBM cells in vivo, we measured the chemoresistance in GBM cells from patients with CC, CG and GG genotypes. As shown in Fig. 4, the che- AGING moresistance was notably different among patients with rs11558961 CC, CG and GG genotypes. With the treatment of imatinib (10μg/ml, 50μg/ml), the average apoptotic cell ratio was lowest in CC group, followed by CG and then highest in GG group (10μg/ml: 36.21±6.03, 53.19±10.32 and 62.51±11.34; 50μg/ml: 59.37±7.98, 72.59±11.57 and 75.42±12.53 respective-ly). These results suggested that rs11558961 variant, which was related with GFAP expression, indeed increased imatinibinduced apoptosis, and reduced chemoresistance.

rs11558961 variant suppressed the migration of GBM cells
It has been proven that GFAP is a pro-tumorigenic factor in brain tumor [5][6][7]. To confirm the association  AGING 75.29±10.21, respectively). Taken together, rs11558961 C>G variant substantially reduced vimen-tin expression and suppressed the migration of GBM cells (Fig. 5).

DISCUSSION
In the present study, we investigated the genetic effects of SNPs in GFAP gene on the progression of GBM. Four candidate SNPs were genotyped using MALDI-TOF MS in 261 patients and 406 healthy controls. A significant association between rs11558961 and decreased GBM susceptibility was identified. Variant G allele of rs11558961 reduced GFAP expression via interfering the binding of miR-139 and 3'UTR, resulting in low GFAP expression level, and further suppressing the chemo-resistance and metastasis of GBM cells (Fig. 6).
rs11558961 was located at the 3'UTR of GFAP and predicted in a miR-139 binding site, with proximal transcription regulatory potential. While allele C changes to G, the secondary structure and minimum free energy of mRNA changed (Fig. 1); there was an emerging miR-139 binding site exposed. These analysis data were acquired using bioinformatical software-rSNP and SNPinfo. It confirmed the significance of this functional SNP in transcriptional regulation for GFAP gene. According to the results shown in Figs. 2-3, rs11558961 C>G change decreased the transcriptional activity, promoted miR-139 binding, therefore inhibited GFAP expression. To the best of our knowledge, the functional polymorphism-rs11558961 has not been reported before. This is the first time to report the potential function of rs11558961 in GFAP expression and GBM susceptibility.

AGING
Another interesting finding was that rs11558961 might be associated with chemoresistance, via fine tuning the GFAP expression. GFAP is important in malignant progression of brain neoplasm. It acts as an essential component of cytoskeleton and expresses almost exclusively in astrocytes [4]. Increasing evidences showed that GFAP level was elevated in high-grade brain tumors, implicating that GFAP might be involved in the aggressiveness of brain tumors [5][6][7]. Intermediate filament proteins promote cytoskeleton assembly/stabilization, and correlate with increased chemoresistance of cytotoxic agents [13]. Our study showed that rs11558961 C>G variant reduced the chemoresistance of GBM cells against imatinib. However, rs11558961 variant was proven associated with GFAP expression (Fig 2-3). Therefore, rs11558961 might decrease the chemoresistance via suppressing GFAP expression. The subject carrying C allele is more prone to high-grade GBM because the chemoresistance of their cells tend to be higher than those with G allele.
Dysfunction of GFAP has been implicated in the astrocytes abnormality, due to the defect of cytokinesis and cell signal transduction. For example, heterozygous missense mutations of GFAP gene resulted in Alexander disease, a neurodegenerative disorder that affects children typically [14]. Generous studies revealed that epigenetic activation of GFAP or high GFAP expression was correlated with aggressiveness of brain tumor [5][6][7]. These reports suggested that GFAP was very important in nervous system disease. Vimentin expression and migration also played a important role in brain tumor cell survival and development. Thus, we evaluated the relationship between rs11558961, vimentin expression and migration of GBM cells. In immunofluorescence assay, the vimentin expression level in wild type group was significantly higher than that in variant group. In concordance with this result, the migration capability of GBM cells was higher in wild type group than that in variant groups. Vimentin is a primordial component of class-III intermediate filaments. It is involved in increased cell motility and migration capability of GBM, which is a hint of malignant progression [12]. Taken together, rs11558961 C>G variant substantially reduced vimentin expression and suppressed the migration of GBM cells, which was critical in progression of GBM cells.  [15]. Moreover, changes of cellular RA balance in GBM might also be related to changes of GFAP expression and phenotypes. Therefore, it is necessary to clarify underlying mechanisms between the important intermediate filament-GFAP in GBM, cytoskeleton assembly and RA signaling responses. The further investigations should be performed.
In summary, we verified that rs11558961 variant in GFAP 3'UTR affected the miR-139 binding and GFAP expression in GBM for the first time. The G allele of AGING rs11558961 predisposed hosts to downregulated GFAP expression and reduced GBM susceptibility. rs11558961variant genotype was a significant genetic protect factor. These findings will be meaningful in precaution, diagnosis and surveillance of GBM, as well as in the advancement of targeted therapy for this brain malignancy.

Study population and postoperative follow-up
Consecutive GBM patients who received curative tumorectomy at Department of Neurosurgery in Zhenjiang first people's hospital, Changzhou first people's hospital, and the first affiliated hospital of Soochow University from Sep 2007 to Dec 2014. Healthy controls were randomly selected from people who received physical examination at Advanced Physical Examination Center. The demographics and clinical features of the GBM patients and controls involved in the study were listed in Table 2. Cases and controls were frequency-matched on gender and healthy controls were older than patients. All the patients were pathologically confirmed and signed the informed consent. Follow-up was performed via in person interview or telephone calls according to the standard epidemiologic procedure twice a year. The final date of follow-up was May 26, 2016. All the protocols conformed to the 1975 Declaration of Helsinki and were approved by the ethics committee.

Blood samples collection and GBM cells primary culture
Ethylene diamine tetraacetic acid (EDTA) anticoagulated peripheral blood samples were collected from patients before surgery and healthy controls. Genomic DNA was extracted using a RelaxGene Blood DNA System (TIANGEN biotech, Beijing, China) according to the manufacturer's instruction. The fresh surgical specimens of GBM from 11 patients were harvested and immediately immersed in ice-cold PBS containing 1% penicillin/streptomycin and 0.5% glutamine (Beyotime, Shanghai, China). Primary cell culture was performed within 60 min after surgery. As a variation of phenotype might occur at higher passages, we chose the cell cultures at the fourth passage for functional experiments.

Quantitative real-time PCR analysis of miR-139
The expression level of miR-139 in U251 cells was detected using RNA-tailing and primer-extension realtime PCR according to the instructions of All-in-One miRNA Detection Kit (Genecopoeia, Guangzhou, China). miR-139 forward primer and miRNA universal adaptor PCR primer were provided by Genecopoeia corporation (Genecopoeia, Guangzhou, China).

Luciferase reporter assay
To evaluate the binding of miR-139 to 3'UTR of GFAP, three reporter constructs carrying two copies of rs11558961-C allele, G allele or mutant sequences at the 3'UTR of luciferase gene were created. Firstly, three ~100 bp DNA sequences centered at rs11558961 (C or G allele) or mutant sequence were synthesized; then, two tandem copies of these sequences were cloned into pGL-3 Basic vector (Promega, Madison, WI, USA) using restriction enzyme sites-BamH I and Sal I. The schematic diagram for vector construction was shown in Fig. 2A

Imatinib-induced apoptosis
The GBM primary cells with rs11558961 CC (n=5), CG (n=3) or GG (n=3) genotypes (5×10 3 per well) were incubated with imatinib at the concentration of 10μg/ml or 50μg/ml for 24 hours. The cells were then fixed with 4% paraform followed by staining with 200 μl Hoechst 33258 reagent (Beyotime, Shanghai, China) in the dark for 30 min. After being washed twice with PBS, cells were immediately photographed under an inversion fluorescence microscope (Olympus IX51, Tokyo, Japan) to determine ratios of apoptotic cells. In CC, CG or GG group, cells were quantified by counting at least 3 samples, 5 independent visual fields/sample, to determine the apoptotic ratio. Data was presented as mean ± SD.

Cell migration assay
Cell migration measurements were performed using 24- well Transwell® units (8.0 μm pore, Costar Corning, NY, USA). In brief, different GBM primary cells (1×10 3 ) in 100 μl were added to each matrigel-coated insert, and 500 μl DMEM supplemented with 10% FBS was added to the lower chamber. The plates were incubated at 37 °C, 5 % CO2 for 24 h. The nonmigrated cells in the insert were wiped away and the migrated cells were stained with ponceau. Finally, the images were captured with a phasecontrast microscope (Olympus IX51, Tokyo, Japan). There were 11 groups in this experiment, rs11558961 CC (n=5), CG (n=3) or GG (n=3). In each group, the migrated cells were quantified by counting at least 3 different samples, 5 independent visual fields/sample, to determine the average invasion ratio. Each assay was performed in triplicate.

Statistical analysis
The differences of the demographic and clinical features, and frequencies of genotypes in case-control study were calculated by the Chi-square test (for categorical variables) or Student's t-test (for continuous variables). Hardy-Weinberg equilibrium (HWE) was detected by online analytical tools (https://ihg.gsf.de/ cgi-bin/hw/ hwa1.pl). The linkage disequilibrium (LD) of candidate SNPs was analyzed using HaploView v4.2 (Broad Institute, Cambridge, MA, USA). For the determination of main effect of SNPs, univariate and multivariate logistic regression models were conducted to generate odds ratio (ORs) and corresponding 95% confidence intervals (CIs) with adjustment for possible confounders, i.e. age, gender, drinking status and so on. All statistical tests were two-sided and performed using Statistical Program for Social Sciences (SPSS 16.0, Chicago, IL, USA) and R (http://www.r-project.org/). P<0.05 was considered as statistically significant.