Download Supplementary materials and methods. Supplementary tables 1 -6.

SUPPLEMENTAL DATA
1. Supplemental data : Lower expression levels of RAMP2-AS1 are linked to
reduced overall survival of GBM patients
1.1. In-house GBM patient samples
Glioblastoma specimens were obtained from 20 patients with GBM who underwent
surgical treatment at Navy General Hospital, China, from January 2011 to December
2013. Glioma was diagnosed according to the 2007 WHO Classification of Tumors of
the Central Nervous System. We calculated Survival curves using the Kaplan-Meier
method and compared using the log-rank test.
1.2. The Cancer Genome Atlas GBM patient samples
The influence of low expression of RAMP2-AS1 on patients’ survival was assessed
based on five year survival analysis. We performed the survival analysis using the R
software package Survival [1]. We downloaded GBM clinical follow-up data from the
Cancer Genome Atlas (TCGA) data portal on November 18, 2015. We used ‘days to
death’ column to consider event (vital status= dead) and ‘days to last follow up’
column to consider the data as censor (vital status= alive). Patients were censored
from statistical analysis if they were alive and had <5 year of clinical follow up. We
calculated Survival curves using the Kaplan-Meier method and compared using the
log-rank test.
1.3. References
[1] Therneau T, Grambsch P (2014) Modeling Survival Data: Extending the Cox Model. New York:
Springer.
2. SUPPLEMENTAL TABLES
2.1. Table S1 The primer information of selected lncRNAs and genes for
real-time RT-PCR detection
Primer name
Primer sequence(5'to3')
NOTCH3 -up
TTCTCATCCGAAACCGCTCTAC
NOTCH3- down
CTCTTCCACCATGCCCTCTACT
NKIRAS1-up
TGATTCTCCTGCCTCAGCCTC
NKIRAS1- down
CCCACAGATAACAATCCACAAACC
SRC-up
CGCTGAGGAGTGGTATTTTGG
SRC- down
CATCGGCGTGTTTGGAGTAGTA
ARHGAP32-up
GACTGAACCACAAAGAGGTAAGGCT
ARHGAP32- down
TGGGACGATTGACTGAGTGAGG
ENSG00000197291-up
GAACTCAGGCCAGATTTACAAG
ENSG00000197291- down
TTGGGTCCTACAGCAACCAT
ENSG00000226645-up
GGGCACATGGTCATCGGAA
ENSG00000226645- down
CCACTTCGCTAGCCTCTACTC
ENSG00000261684-up
GGTTGCAAAACTGTGATGCCT
ENSG00000261684- down
AACCTTCAGGCCACAAAGCA
ENSG00000234741-up
TCCCCAAGGAAGGATGAGAAT
ENSG00000234741- down
CGTTACCAGGAGCAGAACCA
ACTIN-up
ACTTAGTTGCGTTACACCCTT
ACTIN - down
GTCACCTTCACCGTTCCA
2.2. Table S2: Summary of the In-house GBM samples
Age
Diagnosis
Gender
1
Primary GBM
M
50
Surgery+TMZ+RT
113
2
Primary GBM
M
43
Surgery+TMZ+RT
102
3
Primary GBM
F
58
Surgery+TMZ+RT
127
4
Primary GBM
M
64
Surgery+TMZ+RT
87
5
Primary GBM
M
39
Surgery+TMZ+RT
93
6
Primary GBM
M
37
Surgery+TMZ+RT
133
7
Primary GBM
F
77
Surgery+TMZ+RT
120
8
Primary GBM
M
54
Surgery+TMZ+RT
118
9
Primary GBM
F
50
Surgery+TMZ+RT
189
10
Primary GBM
F
43
Surgery+TMZ+RT
164
11
Primary GBM
M
41
Surgery+TMZ+RT
131
12
Primary GBM
M
49
Surgery+TMZ+RT
123
13
Primary GBM
F
58
Surgery+TMZ+RT
101
14
Primary GBM
M
64
Surgery+TMZ+RT
339
15
Primary GBM
M
39
Surgery+TMZ+RT
480
16
Primary GBM
F
37
Surgery+TMZ+RT
341
17
Primary GBM
M
77
Surgery+TMZ+RT
531
18
Primary GBM
F
55
Surgery+TMZ+RT
489
19
Primary GBM
F
43
Surgery+TMZ+RT
396
20
Primary GBM
M
48
Surgery+TMZ+RT
441
(years)
Treatment
Survival
Number
*GBM: Glioblastoma; M:Male; F:Female; TMZ:Temozolomide; RT: Radiotherapy
time(days)
2.3. Table S3: Summary of co-expression relationship among aberrantly
expressed lncRNAs and genes in GBM
Relationship
lncRNA-gene
gene-gene
lncRNA-lncRNA
Dysregulation pattern
# pairs
# lncRNAs
# genes
down-up
11
6
9
up-down
5
4
5
down-down
118
27
81
up-up
4
3
4
down-up
4
-
7
up-down
7
-
8
down-down
474
-
68
up-up
6
-
7
down-up
2
4
-
up-down
2
4
-
down-down
18
24
-
up-up
-
-
-
2.4. Table S4: Over-represented pathways in the aberrantly expressed
lncRNA-gene co-expression network
Pathway Commons pathways
Nominal P
Adjusted P $
EphrinB-EPHB pathway
0.0004
0.0092
EPHB forward signaling
0.0001
0.0092
GP1b-IX-V activation signalling
0.0002
0.0092
Signal regulatory protein (SIRP) family interactions
0.0003
0.0092
p75(NTR)-mediated signaling
0.0007
0.0107
TNF alpha/NF-kB
0.0006
0.0107
Neurotrophic factor-mediated Trk receptor signaling
0.0016
0.0210
Reelin signaling pathway
0.0020
0.0230
Cell-Cell communication
0.0025
0.0256
ErbB2/ErbB3 signaling events
0.0034
0.0313
$
P values were adjusted using the Benjamini-Hochberg (1995) multiple testing correction method.
2.5. Table S5: Over-represented Gene Ontology biological process terms in the
aberrantly expressed lncRNA-gene co-expression network
Biological process
Nominal P
Adjusted P $
Axon extension involved in development
7.70e-06
0.0053
Regulation of axon extension involved in regeneration
2.73e-05
0.0062
Regulation of axonogenesis
0.0002
0.0227
Regulation of cellular component size
0.0003
0.0292
Regulation of cell morphogenesis involved in differentiation
0.0004
0.0303
Adult walking behavior
0.0004
0.0303
Learning
0.0010
0.0498
neuron differentiation
0.0013
0.0498
Regulation of neurogenesis
0.0011
0.0498
Neuronal ion channel clustering
0.0012
0.0498
Cellular macromolecular complex assembly
0.0019
0.0498
†
Ranking based on derived adjusted P values.
$
P values were adjusted using the Benjamini-Hochberg (1995) multiple testing correction method.
2.6. Table S6: GBM specific pathways and GO biological process (BP) terms link
to genes selected for qRT-PCR analysis
Gene symbol
Pathway
NOTCH3
GO BP
Neuron differentiation, Regulation
of neurogenesis
ARHGAP32
p75(NTR)-mediated signaling,
Neurotrophic factor-mediated Trk receptor
signaling
NKIRAS1
TNF alpha/NF-kB
SRC
EphrinB-EPHB pathway, EPHB forward
signaling, p75(NTR)-mediated signaling,
TNF alpha/NF-kB, Neurotrophic
factor-mediated Trk receptor signaling