primate-specific mir-663 functions as a tumor suppressor...

Published OnlineFirst February 12, 2014.Clin Cancer Res Yu Shi, Cong Chen, Xia Zhang, et al. glioblastomatargeting PIK3CD and predicts the prognosis of human Primate-specific miR-663 functions as a tumor suppressor by

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1

Primate specific miR-663 functions as a tumor suppressor by targeting PIK3CD 1

and predicts the prognosis of human glioblastoma 2

3

Yu Shi 1, 2, Cong Chen 1, 2, Xia Zhang 1, 2, Qing Liu 1, 2, Jin-ling Xu 1, 3, Hua-rong 4

Zhang 1, 2, Xiao-hong Yao 1, 2, Tao Jiang 4, Zhi-cheng He 1, 2, Yong Ren 1, 2, Wei Cui 1, 5

2, Chuan Xu 1, 2, Ling Liu 5, You-hong Cui 1, 2, Shi-zhu Yu 3, Yi-fang Ping 1, 2, *, 6

Xiu-wu Bian 1, 2, * 7

8

Author Affiliations 9

1 Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third 10

Military Medical University, Chongqing 400038, China; 11

2 Key Laboratory of Tumor Immunopathology of Ministry of Education of China, 12

Third Military Medical University, Chongqing 400038, China; 13

3 Department of Neuropathology, Tianjin Neurological Institute, Tianjin Medical 14

University General Hospital, Tianjin 300052, China; 15

4 Department of Neurosurgery, Tiantan Hospital, Capital Medical University, Beijing 16

100050, China; 17

5 Department of Health Statistics, College of Preventive Medicine, Third Military 18

Medical University, Chongqing 400038, China 19

20

Running title: miR-663 favors glioblastoma prognosis 21

22

Key words: miR-663, glioblastoma, prognosis, subclassification, PIK3CD. 23

24

Financial Support 25





2

This project was supported by grants from the National Basic Research Program of 1

China (973 Program, No. 2010CB529400), the National Natural Science Foundation 2

of China (No. 30930103 and 30900564) and Programs of Science and Technology 3

Commission Foundation of Tianjin Municipal (10SYSYJC28800, 12ZCDZSY17400). 4

5

*Corresponding authors: 6

Xiu-wu Bian and Yi-fang Ping, Institute of Pathology and Southwest Cancer Center, 7

Southwest Hospital, Third Military Medical University, Chongqing 400038, China. 8

Phone: +862368754431; Facsimile: +862368754431; E-mail addresses: 9

[email protected] and [email protected] 10

11

Conflicts of interest: None 12

13

Word count (excluding references): abstract, 250 words; text, 4572 words. 14

Total number of figures and tables: 5 figures and 1 table. 15

16





3

Translational relevance 1

Glioblastoma (GBM) is heterogeneous in morphology and varies in outcome. The 2

WHO classification of GBM based on histopathological characteristics is limited in 3

predicting the prognosis of GBM patients. Our present results reveal that miR-663 4

predicts better prognosis of GBM and suppresses proliferation and invasion of GBM 5

cells by directly targeting PIK3CD. This is the first report that identifies miR-663 as a 6

novel prognostic biomarker and a tumor suppressor of GBM. Thus, our study 7

provides new insights into the diagnostic and prognostic determination as well as 8

therapeutic intervention for GBM based on the functions of miR-663.9





4

Abstract 1

Purpose: To determine the prognostic significance of miR-663 in glioblastoma 2

(GBM), its effect in tumor progression and the underlying mechanism. 3

Experimental Design: Specimens from 256 cases of glioma patients, including 239 4

patients with follow-up information, were used to analyze the association between 5

miR-663 and patients’ prognosis by Kaplan-Meier and multivariate Cox regression 6

analyses. The effects of miR-663 on GBM cell proliferation and invasion were 7

examined both in vitro and in vivo. Bioinformatics prediction and signal network 8

analysis were applied to identify the putative targets of miR-663, which were further 9

verified by luciferase reporter assay, rescue experiments as well as the 10

immunohistochemistry (IHC) and Western blotting examination of downstream 11

effectors. Quantitative reverse transcriptase-PCR (qRT-PCR) and IHC were applied to 12

investigate the clinical association between miR-663 and its target in human GBM 13

specimens. 14

Results: miR-663 was inversely correlated with glioma grades but positively 15

correlated with patients’ survival. Furthermore, two distinct subgroups of GBM 16

patients with different prognoses were identified based on miR-663 expression in our 17

specimens and that from TCGA database. Overexpression of miR-663 significantly 18

suppressed the proliferation and invasion of GBM cells in vitro and in vivo. 19

Mechanistically, we discovered PIK3CD as a direct target of miR-663 and found that 20

phosphorylated AKT and three key downstream effectors of PIK3CD, i.e., CCND1, 21

MMP2 and MMP7, were down-regulated by miR-663 overexpression. Moreover, 22

PIK3CD was inversely correlated with miR-663 in GBM specimens and predicted 23

poor prognosis of GBM patients. 24

Conclusion: miR-663 is a novel prognostic biomarker and a potential therapeutic 25





5

candidate for GBM. 1

2





6

Introduction 1

Glioblastoma (GBM) is the most aggressive and lethal malignant brain tumor. Despite 2

efforts have been made to improve therapeutic strategies, the average survival of 3

GBM patients have been improved only slightly in the past decades (1). Although in 4

general, patients with GBM tend to have poor prognoses, significant intra-group 5

variations in their survival have been observed (2). Additionally, traditional 6

histological criteria for GBM diagnosis are not suitable for comprehensive assessment 7

of the status of patients, especially their survival (3). The ineffectiveness to predict 8

patients’ outcomes based on histopathological features suggests the requirement for 9

more precise criteria in GBM subclassification. Recent studies by gene expression 10

profiling and factor analyses have identified subtypes of GBM and a few biomarkers 11

associated with GBM patients’ prognoses (3-5). However, the clinicopathological 12

heterogeneities and prognostic signatures of GBM have not been fully characterized. 13

14

Since no ideal protein biomarker has been shown totally effective in predicting GBM 15

patients’ outcomes, some investigators have evaluated the prognostic significance of 16

miRNAs that have recently been recognized as important regulators of cancer 17

biological behavior (6-10). However, the expression pattern and prognostic 18

significance of miRNAs in GBM remain elusive due to the lack of large pools of 19

clinical specimens for screening. Previous studies have shown that miR-663, a 20

member of primate-specific miRNA family, is associated with many important 21

biological processes, including viral infection, inflammatory responses and 22

autoimmune diseases (11-16). However, its role in tumor progression is contradictory. 23

While it acts as an oncogene to promote the malignancy of lung cancer, 24

nasopharyngeal carcinoma and breast cancer (13, 14), miR-663 may also be a 25





7

potential tumor suppressor in gastric cancer, colorectal carcinoma, prostate cancer and 1

acute lymphoblastic leukemia (15, 16). Moreover, the prognostic significance of 2

miR-663 in cancer has not been reported. 3

4

Correlation of changes in miRNA levels with tumorigenesis and tumor progression 5

remains elusive. However, miRNA expression profiling could be useful in improving 6

the classification of tumors and predicting their behavior (17). In the present study, we 7

identified miR-663 from miRNA profile in human GBM and evaluated its expression 8

and clinical relevance. Its contribution to GBM malignancy and the underlying 9

molecular mechanisms were also investigated. Our data demonstrate that miR-663 has 10

potential values as a prognostic marker and a therapeutic target of GBM. 11

12

13





8

Materials and methods 1

Patients and tumor specimens 2

Surgical specimens were obtained from 256 patients with astrocytic gliomas 3

(Southwest Hospital, Third Military Medical University (TMMU), Chongqing, and 4

Tiantan Hospital, Capital Medical University, Beijing) with written consent. After 5

surgical removal, specimens were immediately fixed in a 4% buffered formaldehyde 6

solution. The formalin-fixed, paraffin-embedded (FFPE) samples were stored at room 7

temperature. Histological diagnoses were independently made by two 8

neuropathologists according to WHO classification of central nervous system tumors 9

(2007). Normal brain tissues adjacent to tumors were used as controls. The 10

clinicopathological features of these patients were summarized in Supplementary 11

Table S1. Two hundred and thirty-nine patients with complete information were 12

followed up after operation until February 1st, 2013, with a median follow-up time of 13

23.0 months. This study was carried out in accordance with the principles of the 14

Helsinki Declaration and approved by the Ethics Committee of TMMU and Capital 15

Medical University. 16

17

An independent cohort of 483 patient specimens from TCGA database 18

(https://tcga-data.nci.nih.gov/tcga) (18) was applied to validate the prognostic value of 19

miR-663 in GBM patients. The mircoarray data of all GBM tissue samples with Level 20

3 miRNA (Agilent 8 × 15 K Human miRNA-specific Microarray) were used and the 21

results were filtered based on miR-663 expression and clinicopathological features of 22

patients. 23

24

Cell culture 25





9

Human normal glial cell line, HEB, was generously provided by Professor Guang-mei 1

Yan (Department of Pharmacology, Sun Yat-sen University, Guangzhou) (19, 20). 2

U87-MG was obtained from American Type Culture Collection (ATCC, Manassas, 3

VA). CHG5 (21) and primary GBM cells were established from the tumor specimens 4

of glioma patients (Southwest Hospital, TMMU). HEB, U87-MG and primary GBM 5

cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 6

Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA). 7

CHG5 was grown in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) with 10% FBS. All 8

the cells were incubated at 37°C in a humidified incubator with 5% CO2/95% air. 9

10

Real-time RT-PCR 11

Total RNA from tumor cells or frozen sections were extracted using RNAiso (Takara, 12

Dalian, China). RNA from FFPE tissues was isolated with a miRNeasy FFPE Kit 13

(Qiagen, Hilden, Germany). For measurement of miR-663, Bulge-Loop™ miRNA 14

qPCR Primer Set (RiboBio, Guangzhou, China), ReverAidTM First Strand cDNA 15

Synthesis Kit (Fermentas, Burlington, Canada) and SYBR® Premix Ex Taq™ II 16

(Takara, Dalian, China) were used. For other genes, PrimeScript™ RT Master Mix 17

(Takara, Dalian, China) and SYBR® Premix Ex Taq™ II were used. Specific primers 18

for amplification were listed in Supplementary Table S2. All reactions were 19

performed on a CFX96TM Real-Time PCR Detection System (Bio-Rad, CA). The 20

results of miRNA and mRNA real-time PCR were normalized using the threshold 21

cycle (Ct) of U6 and GAPDH, respectively. 22

23

Western blotting 24

Western blotting was carried out as previously described (22). The primary antibody 25





10

used in this study were as follows: anti-PIK3CD (Abnova, Walnut, CA), 1

anti-phospho-Akt (Ser473), anti-Akt (Cell Signaling, Beverly, MA), anti-MMP-2, 2

anti-MMP-7, anti-MMP-9 (R&D Systems, Minneapolis, MN), anti-Cyclin D1 3

(Abcam, Cambridge, UK) and anti-GAPDH (Cell Signaling, Beverly, MA). 4

5

Gene cloning and lentiviral vector generation 6

Pre-miR-663 was cloned from human HEK293T cell genomic DNA using primers 7

listed in Supplementary Table S2. The expression vector containing PIK3CD coding 8

sequence (CDS) was obtained from Origene (Rockville, MD). The pre-miR-663 and 9

PIK3CD-CDS fragments were ligated into the pLOV-CMV-mCherry-EF1a-PuroR 10

vector or pLOV-CMV-eGFP-EF1a-PuroR vector (Neuron Biotech, Shanghai, China), 11

respectively. After sequence verification, the mCherry-pre-miR-663 cassette or the 12

eGFP-PIK3CD cassette, together with psPAX2 and pMD2G constructs (School of 13

Life Sciences, Lausanne, Switzerland), was transfected into 293FT packaging cells. 14

Medium was changed after overnight culture and lentiviruses in the supernatant were 15

collected and filtered through a 0.45-μm filter 48 hrs after transfection. 16

17

Lentiviral infection procedures 18

Cells were infected with concentrated mCherry-pre-miR-663 lentivirus, and/or 19

eGFP-PIK3CD lentivirus and empty control lentivirus vectors. Cells with stable 20

integration of pre-miR-663 or PIK3CD were selected and enriched by flow cytometry 21

(BD FACSAria II, San Jose, CA) to obtain mCherry or eGFP positive cells. 22

23

Transfection procedures 24

miR-663 inhibitor and Negative Control #1 (Ambion, Austin, TX) were transiently 25





11

transfected with a final concentrations of 100 nmol/L using Lipofectamine™ 2000 1

(Invitrogen, Carlsbad, CA). At 5 hr post-transfection, the medium was replaced by 2

DMEM with 10% FBS. The whole process was conducted according to 3

manufacturer’s guidelines. 4

5

Tumor cell proliferation assay 6

Tumor cells (500 cells/well for U87-MG and 800 cells/well for primary GBM cells) 7

were seeded into 96-well plates and cultured for 24, 48, 72 and 96 hrs. At the 8

indicated intervals, 20 µl of Cell Counting Kit-8 (Beyotime, Shanghai, China) were 9

added to each well and incubated for 2 hrs at 37°C. Thermo Multiskan Spectrum 10

Reader (Thermo Scientific, Watertown, MA) was used to measure the absorbance at 11

490 nm. 12

13

Tumor cell invasion assay 14

Twenty-four-well Millicell® chambers with 8.0 µm pore size (Millipore, Billerica, 15

MA) and BD Matrigel™ (BD Biosciences, Franklin Lakes, NJ) were used in tumor 16

cell invasion assay. Tumor cells were plated in the upper chamber pre-coated with a 5 17

µl mixture of BD Matrigel™ and DMEM (1:1, v/v). The lower chamber was added 18

with 500 µl of DMEM with 10% FBS. After 24 or 36 hrs of incubation, the Millicell® 19

chambers were removed from the plates and fixed in 4% paraformaldehyde, and then 20

stained with Crystal Violet Staining Solution (Beyotime, Shanghai, China). 21

Non-invasive cells on the upper surface of Millicell® chambers were wiped with a 22

cotton swab. Invaded cell number was manually counted in five randomly selected 23

fields under light microscope at 200×. 24

25





12

Luciferase plasmid construction and reporter assay 1

The pmiR-REPORT™ Luciferase miRNA Expression Reporter Vector (RiboBio, 2

Guangzhou, China) was used to construct the pmiR-3’UTR-wt vector. The primer 3

pairs for wild and mutant 3’UTR amplification were listed in Supplementary Table S2. 4

U87-MG cells seeded in 96-well plates were co-transfected with 20 ng pmiR-3’UTR 5

DNA and 100 pmol miR-663 precursor and control oligomers (Pre-miR Negative 6

Control #1) (Ambion, Austin, TX). The activities of both renilla and firefly luciferases 7

were measured using Dual-Luciferase Reporter Assay System (Promega, Madison, 8

WI) on a SpectraMAX Gemini XS Microplate Reader Fluorometer (Molecular 9

Devices, Sunnyvale, CA). The results were presented after normalization with the 10

measured values of firefly luciferase. 11

12

Tumor implantation 13

The animal experiments were approved by the Institutional Animal Care and Use 14

Committee of the Southwest Hospital, TMMU according to the Guide for the Care 15

and Use of Laboratory Animals. Each of 6-weeks-old male SCID mice (Laboratory 16

Animal Center, Southwest Hospital, TMMU) was anesthetized and intracranially 17

injected with 1 × 105 miR-663 overexpressing cells or control cells. Animals were 18

monitored daily for weight change and survival. The brains were collected until the 19

animals became moribund and then sampled for Harris hematoxylin and 20

alcoholiceosin (H&E) staining and immunohistochemistry. 21

22

Immunohistochemistry 23

The FFPE human gliomas and xenografted tumors were sectioned at 4 μm for 24

immunohistochemical staining. The whole process was conducted using Dako 25





13

REAL™ EnVision™ Detection System (DAKO, Carpinteria, CA) according to the 1

manufacturer’s instructions. Primary antibodies included anti-PIK3CD 2

(Sigma-Aldrich, St. Louis, MO), anti-Ki67, anti-vimentin, anti-Cyclin D1 (ZSGB-Bio, 3

Beijing, China), anti-IDH1 R132H, anti-GFAP (Maixin-Bio, Fujian, China), 4

anti-p-AKT (Ser473, Novus Bio, Littleton, CO), anti-MMP-2, anti-MMP-7 and 5

anti-MMP-9 (R&D Systems, Minneapolis, MN). Semi-quantitations of PIK3CD, 6

p-AKT, Cyclin D1, MMP-2, MMP-7 and MMP-9 were independently performed by 7

two neuropathologists according to the staining intensity and the percentage of 8

positive tumor cells as previously described (23). Ki67 proliferation index was 9

determined under 10 randomly selected microscopic fields at 400×. 10

11

Statistical analyses 12

All statistical analyses were performed using PASW 18.0 software. 13

Kolmogorov-Smirnov test was used to estimate the normality of distributions. 14

Student’s t-test or one-way ANOVA was conducted for normally distributed data. 15

miR-663 expression in different grade glioma specimens was assessed by 16

Bonferroni-corrected Mann-Whitney U-test. Pearson’s chi-square test was used to 17

determine the correlation between miR-663 expression and clinicopathological 18

features of patients. Patients’ survival was analyzed with Kaplan-Meier method, using 19

the log-rank test for comparison. The statistical software X-tile (24) was used to 20

determine the cutoff in the glioma cohort from our groups and TCGA database. The 21

Cox’s proportional hazard model was used for univariate and multivariate survival 22

analyses. The correlation between the levels of miR-663 and PIK3CD was measured 23

using nonparametric (Spearman's rho) correlation test and curvilinear regression 24

model. Data were presented as the mean ± SD. Statistic significance was assigned at P 25





14

< 0.05 (*) or P < 0.01 (**). All experiments were performed at least three times with 1

triplicate samples. 2

3





15

Results 1

miR-663 is associated with better prognosis in human glioblastoma 2

To identify the potential miRNAs in glioma progression, miRNA profiling was 3

conducted in primary GBM tissues and the matched adjacent normal tissues 4

(Supplementary Table S3 and S4). Among the differential expressed miRNAs, we 5

found that miR-663 was downregulated in GBM tissues (Supplementary Table S3). 6

To validate the relationship between miR-663 expression and human glioma 7

malignancy, we applied real-time PCR to quantitatively analyze endogenous miR-663 8

expression in the established human normal glia cell line (HEB), human glioma cell 9

lines (CHG5 and U87-MG) and primary human glioma cells. We found that CHG5 10

cells from low-grade human glioma expressed relatively higher level of miR-663 than 11

human GBM (grade IV) cells including U87-MG and primary cells (GBM-1, -2 and 12

-3) (Fig. 1A). We then examined miR-663 expression in the specimens from 256 13

glioma patients, and found that miR-663 expression was significantly reduced in 14

high-grade glioma as compared to normal human brain (p < 0.01) and low-grade 15

glioma (p < 0.01) (Fig. 1B and Supplementary Tables S5-S7). Furthermore, miR-663 16

expression in GBM (grade IV) was lower than that in anaplastic glioma (grade III) (p 17

< 0.01) (Fig. 1B). Kaplan-Meier analyses showed that the patients with higher level of 18

miR-663 had longer disease-free survival (DFS) (p < 0.0001) and overall survival (OS) 19

(p < 0.0001) (Fig. 1C). Significantly, we found that GBM patients could be divided 20

into two subgroups with different outcomes based on miR-663 expression, i.e., the 21

higher expression of miR-663, the better prognosis of patients (DFS: p < 0.0001; OS: 22

p < 0.0001) (Fig.1D). The prognostic value of miR-663 in GBM was further verified 23

in 483 GBM patients from TCGA database (Supplementary Fig. S1A). Univariate and 24

multivariate analyses revealed that miR-663 was an independent predictor for DFS 25





16

and OS of GBM patients (Supplementary Table S8 and Table 1). These data indicate 1

an inverse association of miR-663 expression with glioma malignancy, and reveal that 2

miR-663 may be a potential biomarker for prognosis in GBM patients. 3

4

miR-663 inhibits the proliferation and invasion of GBM cells in vitro and in vivo 5

To examine the role of miR-663 in glioma malignancy, we used GBM cells with 6

relatively different levels of miR-663 expression to respectively generate miR-663 7

overexpressing and knock-down cells (Supplementary Fig. S2A and S2B). We found 8

that overexpressed miR-663 reduced, whereas downregulated miR-663 promoted, 9

GBM cell proliferation (Fig. 2A and 2B), without any effect on cell apoptosis 10

(Supplementary Fig. S2C and S2D). Consistent with the in vitro results, xenografted 11

tumors formed by miR-663 overexpressing GBM cells showed lower proliferation 12

capacity as indicated by Ki67 labeling index (Fig. 2C). Ectopic expression of 13

miR-663 abrogated, whereas suppression of miR-663 enhanced, the migratory and 14

invasive abilities of GBM cells, as measured by cell scratch and invasion assays (Fig. 15

2D and 2E; Supplementary Fig. S2E-S2H). Histologically, we found the tumors 16

formed by miR-663 overexpressing cells might be less invasive compared with those 17

by control ones (Supplementary Fig. S2I), suggesting that miR-663 overexpression 18

might abrogate the invasive phenotype of GBM cells. Moreover, the mice bearing 19

miR-663 overexpressing xenografts survived much longer than those bearing control 20

ones (Fig. 2F). Our results reveal that miR-663 could attenuate the proliferation and 21

invasion of GBM cells in vitro and in vivo. 22

23

miR-663 directly targets PIK3CD and inhibits its signaling 24

We next performed bioinformatic analysis to identify the possible targets of miR-663, 25





17

and PIK3CD was singled out as a potential one (Supplementary Fig. S3A and S3B, 1

Supplementary Table S9). TargetScan prediction revealed that the 3’UTR of PIK3CD 2

harbored a putative miR-663 binding site (Fig. 3A), which was confirmed by 3

luciferase reporter assay (Fig. 3B), indicating PIK3CD as a direct target by miR-663. 4

Functionally, PIK3CD overexpression (Fig. 3C) partially compromised the inhibitory 5

effects of miR-663 on GBM proliferation (Fig. 3D) and invasion (Fig. 3E and 3F), 6

while suppression of PIK3CD (Supplementary Fig. S3C) had the opposite effects 7

(Supplementary Fig. S3D-S3F). Our results reveal that the inhibitory effect of 8

miR-663 on glioma is at least in part through targeting PIK3CD. 9

10

To further determine the underlying mechanisms that miR-663 suppresses GBM, we 11

focused on PIK3CD and its important downstream effectors to investigate whether 12

they were responsible for the suppressed proliferation and invasion induced by 13

miR-663. As shown in Fig. 4, PIK3CD, CCND1, MMP2 and MMP7 (Fig. 4A) 14

together with their protein abundances (Fig. 4B) were significantly reduced in the 15

miR-663 overexpressing cells as compared to control ones, possibly through the 16

reduction of PIK3CD-mediated AKT phosphorylation (Fig. 4B). Consistently, IHC 17

analysis on the specimens from the intracranially xenografted tumors indicated that 18

the abundances of PIK3CD, p-AKT, CCND1, MMP2 and MMP7 proteins were 19

reduced by miR-663 (Fig. 4C and Supplementary Fig. S4A). The results reveal that 20

targeting PIK3CD by miR-663 suppresses the activation of PI3K-AKT pathway and 21

reduces the expression of genes related to proliferation and invasion, thereby 22

inhibiting GBM progression (Supplementary Fig. S4B). 23

24

The reduced miR-663 expression correlates with the high level of PIK3CD in 25





18

human GBM 1

To investigate the correlation between miR-663 and its target PIK3CD, we detected 2

their expressions in primary human GBM. In comparison with the matched normal 3

brain tissues, miR-663 was reduced while PIK3CD mRNA (Fig. 5A) were increased 4

in the tumor tissues from 35 GBM patients, showing a significantly negative 5

correlation (r = -0.773, p < 0.01) (Fig. 5B). Consistently, IHC analysis (Fig. 5C) on 6

the specimens from 108 GBM patients revealed an inverse association between 7

miR-663 and PIK3CD (p < 0.01) (Fig. 5D). Kaplan-Meier analysis demonstrated that 8

the high level of PIK3CD expression predicted a short-term DFS (p = 0.0079) (Fig. 9

5E) and OS (p =0.0376) (Fig. 5F) in GBM patients. Univariate analysis revealed that 10

PIK3CD was a potential predictor for DFS and OS of GBM patients (Supplementary 11

Table S8), further confirming an inverse correlation between miR-663 and its target 12

PIK3CD and their functions on the malignant behaviors of human GBM. 13

14





19

Discussion 1

miR-663, which is expressed in Homo sapiens and Pan troglodytes, belongs to the 2

primate-specific miRNAs that possibly attribute to the vertebrates evolution, 3

development and carcinogenesis (24-27). The effect of miR-663 in malignant 4

progression is controversial, since it could act as a tumor promoter or suppressor in an 5

organ-specific fashion (13-16). The clinical relevance of miR-663 in glioma remains 6

unknown. In the present study, we selected miR-663 as our target molecule from 7

miRNA profiling and identified miR-663 as a tumor suppressor to inhibit the 8

proliferation and invasion of GBM, representing the first comprehensive analysis of 9

miR-663 in glioma. Mechanistically, we identified PIK3CD as a direct and functional 10

target of miR-663, which facilitated our understanding of the mechanisms underlying 11

GBM progression. Most importantly, we found that miR-663 and PIK3CD, not only 12

were correlated with each other, but also predicted the survival of GBM patients, 13

highlighting the potential values of miR-663 and PIK3CD as novel prognostic 14

biomarkers in human GBM. 15

16

Significant endeavors have been made to demonstrate the altered miRNA expressions 17

and their effects on GBM progression and patients’ survival (28). miRNA-based 18

integrated analyses have provided new perspectives for clinical validation of the 19

association between miRNA expression patterns and tumor grades or specific 20

subtypes, highlighting the potential values of miRNAs in diagnosis and 21

subclassification (29-31). Our present data indicated that miR-663 was differently 22

expressed in 17 grade I glioma, 52 grade II glioma, 62 grade III glioma and 125 GBM 23

(grade IV) specimens, suggesting miR-663 was a potential molecular biomarker in 24

distinguishing tumor grades. In GBM, a subgroup of tumors that expressed a 25





20

relatively higher level of miR-663 was correlated with better prognosis, suggesting 1

miR-663 was a specific biomarker for the prognostic-based GBM subclassification. 2

Furthermore, miRNAs stably exist in formalin-fixed and paraffin-embedded (FFPE) 3

samples and can be easily detected by real-time PCR, in situ hybridization or 4

microarrays (32, 33). Thus, miR-663 could be a novel and clinical feasible candidate 5

for miRNA-based diagnosis and subclassification for GBM. 6

7

GBM is characterized by rapid growth, relentless invasion and redundant 8

microvessels (1). We identified miR-663 as a tumor suppressor to inhibit the 9

proliferation and invasion of GBM cells thereby suppressing the growth and 10

infiltration of GBM tumors, highlighting the therapeutic potential of miR-663 in 11

GBM treatment. Notably, histopathological features of the xenografted tumors by 12

miR-663 overexpression cells were hardly distinguished from those by control ones, 13

which exhibited the pathological characteristics of GBM, including nuclear atypia, 14

cellular pleomorphism, mitotic activity, microvascular proliferation and necrosis (data 15

not shown). However, xenografted tumors with miR-663 overexpression were much 16

lower in proliferation and weaker in invasion, and animals survived longer than 17

control ones, suggesting that miR-663 could be potentially applied in the treatment of 18

GBM. 19

20

PIK3CD, which encodes PI3K subunit p110δ, is primarily expressed in the 21

hematopoietic cells and has recently been discovered to regulate the proliferation and 22

invasion of certain cancer cells (34-39). We identified PIK3CD as a direct target of 23

miR-663 by bioinformatics prediction, signal network analysis, luciferase reporter 24

assay, gain-of-function and rescue experiments. Our results suggested the oncogenic 25





21

role of PIK3CD in GBM malignant proliferative and invasive behaviors, which were 1

consistent with the previous study showing that the organ-specific PIK3CD, but not 2

other isoforms of PI3K subunits, promoted the migration and invasion of GBM cells 3

(36). We further demonstrated that miR-663-induced suppression of PIK3CD partially 4

inactivated AKT pathway, which enhanced our comprehension of the molecular 5

mechanism of GBM progression (Supplementary Fig. S4B). However, the 6

tumor-suppressive effects of miR-663 could not be fully compromised by PIK3CD 7

overexpression. Besides, the prognostic significance of PIK3CD was not as important 8

as that of miR-663, suggesting that other target genes might be involved in the 9

modulation induced by miR-663. Given the therapy-resistance feature of GBM and 10

the drug-resistance properties of some predicted targets of miR-663 (14, 40), the 11

involvement of miR-663 in chemoresistance requires further investigation. 12

13

Our study showed that miR-663 was down-regulated in GBM as compared with the 14

matched normal brain tissue, which could be illustrated by two possible mechanisms. 15

Recent studies have shown that transcription factors (TFs), such as ZEB family 16

members and p53, may directly bind to the promoter regions of miRNAs or genes that 17

harbor miRNAs (41, 42). By bioinformatic analysis, we have identified several 18

potential TFs that could bind to pre-miR-663 promoter regions (data not shown). 19

Some of them are the predicted miR-663 targets, which may form a feedback loop 20

between miR-663 and these TFs. Another potential mechanism for miR-663 21

regulation is epigenetic modification, such as histone acetylation or methylation and 22

DNA methylation (43). In fact, reduction of miR-663 by hypermethylation in its 23

promoter region has been identified in MDA-MB-231 breast cancer cells and K-562 24

leukemia cells (14, 44). Further studies are underway to examine the methylation 25





22

status of miR-663 promoter region in different grade glioma specimens and cell lines. 1

2

Several prognostic biomarkers, including IDH mutational status and ATRX 3

expression/mutation, have been established in glioma (45, 46). We investigated IDH1 4

and ATRX status in TCGA database and stratified the GBM patients into two 5

subgroups by these molecular features (Supplementary Fig. S1B and 1C). Our results 6

showed that miR-663 was a potential prognostic biomarker in IDH1WT subgroup 7

(Supplementary Fig. S1D, left) and IDH1MU subgroup (Supplementary Fig. S1D, 8

right), suggesting that the prognostic value of miR-663 might not rely on IDH1 status. 9

Besides, miR-663 could divide ATRXlow subgroup into two subsets with different 10

prognosis (Supplementary Fig. S1E, left), though its role in the ATRXhigh subgroup 11

was not significant (Supplementary Fig. S1E, right). Further perspective studies are 12

warranted. 13

14

In summary, our study revealed that miR-663 inhibited the proliferation and invasion 15

of GBM cells in vitro and in vivo by directly targeting PIK3CD, and predicted better 16

prognosis in human GBM. More importantly, miR-663 might be a novel biomarker 17

for molecular subclassification of GBM and a target for treatment of this lethal 18

disease. 19

20





23

Acknowledgements 1

We thank Prof. Peng Zhang and Prof. Marie C.M. Lin of Institute of Pathology and 2

Southwest Cancer Center, Southwest Hospital and Dr. Ji Ming Wang of Center for 3

Cancer Research, National Cancer Institute at Frederick for reviewing this 4

manuscript. 5

6

Grant Support 7

This project was supported by grants from the National Basic Research Program of 8

China (973 Program, No. 2010CB529400), the National Natural Science Foundation 9

of China (No. 30930103 and 30900564) and Programs of Science and Technology 10

Commission Foundation of Tianjin Municipal (10SYSYJC28800, 12ZCDZSY17400). 11





24

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Figure Legends 1

Fig. 1. miR-663 expression correlates with glioma grade and the prognoses of patients. 2

A, miR-663 expression in normal glial cell line (HEB), glioma cell lines (CHG5 and 3

U87-MG) and primary cells (GBM-1, -2, and -3) assessed by qRT-PCR. The relative 4

expression of miR-663 was normalized against U6 expression, which was employed 5

for the comparison between HEB and other cells. The miR-663/U6 ratio in HEB was 6

arbitrarily set to 1.0. B, Expression of miR-663 in the formalin-fixed, 7

paraffin-embedded (FFPE) glioma tissues from 256 patients assessed by qRT-PCR. 8

The transcript level of miR-663 was normalized against U6. The relative expression 9

of miR-663 in specimens was determined as compared to that in adjacent normal 10

tissues. The average expression of miR-663 in adjacent normal tissues was arbitrarily 11

set to 0 with log 10 fold change. All experiments in A and B were performed at least 12

in triplicate and the data are presented as the mean ± SD. **, P < 0.01. C and D, 13

Kaplan-Meier analysis of the correlation between miR-663 and DFS (left) or OS 14

(right) of all grade glioma patients (C) and glioblastoma (GBM) patients (D). Patients 15

were divided into two groups using X-tile analysis as mentioned in Materials and 16

methods. 17

18

Fig. 2. miR-663 inhibits the proliferation and invasion of GBM cells in vitro and in 19

vivo. A and B, Growth curve from the miR-663 up-regulated (A) or down-regulated 20

cells (B) assessed by cell proliferation assay. C, Representative images (left) and 21

proliferation index (right) in tumors indicated by Ki67 staining. Ki67 positive cells 22

were counted microscopically under 10 high powered fields per group, and the results 23

were presented as the mean number of Ki67 positive cells. Scale bar = 25 μm. D and 24

E, Quantification of invaded tumor cells analyzed by cell invasion assay in the 25





31

miR-663 up-regulated (D) or down-regulated cells (E). All experiments in A-E were 1

performed at least in triplicate and the data are presented as the mean ± SD. **, P < 2

0.01. F, Kaplan-Meier analysis of the survival of SCID mice bearing xenografted 3

tumors formed by miR-663 over-expressing cells and control cells. 4

5

Fig. 3. miR-663 directly targets PIK3CD. A, The predicted miR-663 binding sites in 6

the PIK3CD-3’-UTR region by Targetscan and the designed mutant sequence 7

(PIK3CD-3’-UTR-mutant). B, Luciferase reporter assay of U87-MG cells transfected 8

with pmiR-PIK3CD-3’-UTR, the corresponding mutant reporter, miR-663 precursor 9

oligomer or control oligomer. C, qRT-PCR analysis of PIK3CD overexpressing 10

efficiency in U87-MG cells as indicated. The transcript levels were normalized 11

against GAPDH expression. D, Growth curves from the indicated cells assessed by 12

cell proliferation assay. a, b, c and d denote statistical significance among different 13

groups. a: (miR-663 vs. miR-Ctrl), P < 0.01; b: (PIK3CD vs. miR-Ctrl), P < 0.01; c: 14

(miR-663+PIK3CD vs. miR-Ctrl), P < 0.01; d: (miR-663 vs. miR-663+PIK3CD), P < 15

0.01. E and F, Numbers (E) and representative images (F) of indicated invaded cells 16

analyzed by cell invasion assay. Scale bar = 50 μm. Experiments in B-E were 17

performed at least in triplicate and the data are presented as the mean ± SD. **, P < 18

0.01. 19

20

Fig. 4. miR-663 overexpression suppresses the activation of AKT pathway. A, 21

qRT-PCR analysis of PIK3CD, CCND1, MMP2, MMP7 and MMP9 in U87-MG with 22

miR-663 overexpressing vector or empty vector. B, Western blot analyses of PIK3CD, 23

phosphorylated AKT, AKT, CCND1, MMP2, MMP7 and MMP9 in the cells as 24

indicated. Expressions of indicated targets were normalized against GAPDH. C, 25





32

Quantification of IHC staining of PIK3CD, p-AKT, CCND1, MMP2, MMP7 and 1

MMP9 in the xenograft tumors as indicated. The expressions of targets were scored 2

by staining intensity and area as described in Materials and Methods. All experiments 3

were performed at least in triplicate and the data in A and C are presented as the mean 4

± SD. *, P < 0.05, **, P < 0.01. 5

6

Fig. 5. The relationship between miR-663 and PIK3CD in human GBM specimens. A 7

and B, the expressions of miR-663 and PIK3CD (A) and their correlation (B) in the 8

GBM specimens with matched adjacent normal tissues obtained from 35 patients. The 9

transcript levels of miR-663 and PIK3CD were respectively normalized against U6 10

and GAPDH, which were employed for the comparison between tumor and adjacent 11

brain tissue. The miR-663/U6 ratio and PIK3CD/GAPDH one in adjacent normal 12

tissues were arbitrarily set to 1.0. Experiments were performed at least in triplicate 13

and the data are presented as the mean ± SD. C, Representative IHC staining of 14

PIK3CD in GBM specimens stratified by miR-663 expression. Scale bar = 25 μm. 15

Case 7 and Case 34 in (A) represented miR-663high group and miR-663low group 16

respectively. D, The correlation of miR-663 and PIK3CD protein expression was 17

analyzed by Pearson’s chi-square test. GBM specimens were divided into two groups 18

based on miR-663 or PIK3CD expression using X-tile analysis. **, P < 0.01. E and F, 19

Kaplan-Meier analysis indicated the correlation between PIK3CD and DFS (E) or OS 20

(F) of GBM patients. The patients were divided into two groups based on the 21

conditions mentioned in (D). 22

23





Table 1. Multivariate analysis for disease-free survival and overall survival in GBM patients.

Factors Disease-free survival Overall survival

HR (95%CI) P value HR (95%CI) P value

Gender 1.463 (0.893-2.397) 0.131 1.545 (0.953-2.507) 0.078

Age 0.998 (0.981-1.016) 0.839 0.996 (0.979-1.013) 0.626

Predominant Side 1.027 (0.651-1.621) 0.909 1.271 (0.806-2.005) 0.302

Predominant Lobe 0.879 (0.707-1.092) 0.245 0.874 (0.708-1.079) 0.21

KPS Score 0.997 (0.981-1.014) 0.762 1.010 (0.992-1.027) 0.273

Extent of surgical resection 1.216 (0.757-1.953) 0.419 1.088 (0.684-1.730) 0.723

Ki67 Index 1.014 (0.998-1.031) 0.091 1.015 (0.999-1.032) 0.068

IDH1(R132H) Status 0.285 (0.103-0.794) 0.016 0.253 (0.090-0.707) 0.009

miR-663 0.833 (0.699-0.992) 0.041 0.828 (0.704-0.975) 0.024

PIK3CD 1.144 (1.028-1.271) 0.013 1.072 (0.970-1.186) 0.174

Abbreviation: HR, Hazards ratio; CI, Confidence interval; KPS, Karnofsky performance status; IDH1,

isocitrate dehydrogenase (IDH) type 1.





primate-specific mir-663 functions as a tumor suppressor...

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