Supplementary Materials and Methods
1 Patients and tissue specimens.
176 paraffin-embedded specimens and 12 surgically resected fresh
glioma tissues were collected from the Affiliated Hospital of Weifang
Medical University and the Weifang People’s Second Hospital from 2008
to 2013. The detail information of the patients was listed in
Supplementary Table 1 and Supplementary Table 3 respectively. All
patients provided written informed consent and approval from the
Institutional Research Ethics Committee for the use of their tissue
specimens in this study. None of them had received chemotherapy or
radiotherapy before surgery.
2 Cell lines
Human glioma (SHG44, which belongs to WHO grade II), glioblastoma
(U87, U251, LN-229, which belong to WHO grade IV) cells and normal
human astrocytes (NHA) cells were obtained from the American Type
Culture Collection (Manassas, VA, USA). The cells were cultured in
RPMI 1640 (HyClone, SH30809.01B) supplement with 10% fetal bovine
serum (HyClone, SH30070.03), at 37°C in a humidified atmosphere of
5% CO2 and 95% air. The human HGF (hepatocyte growth factor) was
purchased from R&D Systems (Minneapolis, MN, USA). 5μM DNA
methyltransferase inhibitor 5-Aza-20-deoxyazacytidine (5-aza-dC;
Sigma, St Louis, MO, USA) was used to treat the cell lines for 72h.
3 Plasmid construction and cell transfection
Cells were plated in a 35mm dish for 24h before transfection into the
complete medium. The transfection was performed with Lipofectamine
2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's
instructions. For stable transfection, a siRNA expression plasmid
containing target sequence (5'-GCTCAAGATTGGTGACTTT-3') and a
vector containing a scrambled sequence were selected by using 600 μg/ml
neomycin. The stable transfected cells are named siBMK1/U87 and
SCR/U87 cells for subsequent studies.
The 3'UTRs of BMK1 containing predicted miR-429 target sites were
amplified by PCR from SHG44 cell genomic DNA and cloned into
pcDNA3.1 expression vector (GenePharma Company, Shanghai, China).
The SHG44 cells were transfected with pcDNA3.1-BMK1 plasmid or
pcDNA3.1 vector using Lipofectamine 2000 (Invitrogen) following the
protocol. After 24h of transfection, cells were trypsinised, diluted, and
reseeded into 10 cm culture dishes. Stable transfected cells were obtained
by using selection medium (culture medium with 700 μg/ml G418).
Single cell clones were isolated for clone expansion. Stable transfected
cell clones were named SHG44/BMK1 and SHG44/CON cells. The cells
were maintained and passaged in culture medium with G418 (400 μg/ml).
The 3'UTRs of BMK1 were amplified and then cloned into the
downstream of the luciferase gene in a modified pGL3 control vector
(Promega, Madison, WI, USA).
The miR-24 mimics, miR-124 mimics, miR-143 mimics, miR-429
mimics, miR-429 inhibitor oligonucleotides, miR-429 mutants and
corresponding control oligonucleotide mimics (NC) were synthesized by
GenePharma (Shanghai, China). Transfection and transduction were
performed as described previously. Following transduction, puromycin
(1.5μg/ml) was used as a selection antibiotic to select the infected cells
for 10 days. Stable transfected cell clones were obtained.
4 Western blot
For western blot, cells or tissues were directly lysed in 1×SDS sample
buffer . Following antibodies were used: anti-BMK1 (Santa Cruz
Biotechnology; 1:1000), β-actin (Cell Signaling; 1:1000), T-cadherin
(Santa Cruz biotechnology, 1:1000)，N-cadherin (Santa Cruz
biotechnology, 1:1000), CD44 (Santa Cruz biotechnology, 1:1000), Snail
(Santa Cruz biotechnology, 1:500), Slug (Santa Cruz biotechnology,
1:1000), Twist1 (Santa Cruz biotechnology, 1:1000), Zeb1 (Santa Cruz
biotechnology, 1:1000), Zeb2 (Santa Cruz biotechnology, 1:1000), p-
GSK3β (Cell Signaling Technology, 1:1000), GSK3β(Cell Signaling
Technology, 1:1000) antibody.
5 Wound healing/scratch assay
All cells were seeded in six-well plates and grown until full confluence.
After using a 10μl pipette tip to make a straight scratch, cells were
incubated in a minimum medium (containing 0.1% BSA) in a 37%
humidified incubator at different time points and the wound distances
were measured under a light microscope.
6 Luciferase reporter assay
Cells (3 × 104) were seeded in triplicates in 48-well plates and allowed
to settle for 1d. According to the manufacturer’s recommendation, about
one nanogram of pRL-TK renilla plasmid (Promega) plus one hundred
nanogram of pGL3-BMK1-3'UTR were transfected using the
Lipofectamine 2000 reagent (Invitrogen). According to a protocol
provided by the manufacturer, renilla signals and luciferase were
measured at 48h after transfection using the Dual Luciferase Reporter
Assay Kit (Promega).
7 Intracranial Brain Tumor Xenografts
To establish intracranial brain tumor xenograft models, 5×105 U87
cells, SCR/U87 cells and SiBMK1/U87 cells were stereotactically
implanted into four-week-old male SCID mice brains individually. Each
group contains 8 mice. After 28 days, the mice were sacrificed and the
whole brains were removed and fixed with formalin immediately. Serial
sections and H&E staining were performed to detect glioma
micrometasis.
8 Statistical analysis
A cohort of 176 glioma patients was divided into two groups based on
BMK1 expression level for clinical survival analysis: the high-BMK1
expression group (above the median value) and the low-BMK1
expression group (below the median value). SPSS 16.0 statistical
software package was used to perform all statistical analyses.
Relationship among miR-429, BMK1 expression and clinicopathologic
characteristics were analyzed using the chi-square test. Survival curve
was compared using the log-rank test and was plotted using the KaplanMeier method. Statistical significance for comparisons between groups
was determined using analysis of variance (ANOVA) or Student's paired
two-tailed t-test. P < 0.05 was considered statistically significant in all
cases.
Supplementary Table 1
Clinicopathologic characteristics of studied patients (n=176)
n (%)
Gender
Male
Female
99 (56.2)
77 (43.7)
Age(years)
＜40
81 (46.0)
40-49
37 (21.0)
50-59
27 (15.3)
60-69
25 (14.2)
70-79
6 (3.4)
WHO grading
GradeⅠ
24 (13.6)
Grade Ⅱ
53 (30.1)
Grade Ⅲ
56 (31.8)
Grade Ⅳ
43 (24.4)
Patient survival (n=176)
Alive
Deceased
50 (28.4)
126 (71.6)
Supplementary Table2
Correlation between clinicopathologic features and
expressions of BMK1 and miR-429 in glioma patients
Patient
BMK1 expression
miR-429 expression
Low or none
High P
Low or none
High
P
Male
38
61
64
35
0.689
Female
27
50
52
25
≤45
41
67
70
38
＞45
24
44
46
22
Ⅰ and Ⅱ
45
32
35
42
Ⅲ and Ⅳ
20
79
81
18
characteristic
Gender
0.651
Age (years)
0.721
0.700
WHO grade
Survival (n=176)
0.000
0.000
Alive
34
16
Deceased
31
95
0.000
16
34
100
26
0.000
Supplementary Table 3
Clinicopathologic characteristics of studied patients (n=12)
n (%)
Gender
Male
5(41.7%)
Female
7(58.3%)
Age(years)
≤45
6(50%)
＞45
6(50%)
WHO grading
GradeⅠ
1(8.3%)
Grade Ⅱ
4(33.3%)
Grade Ⅲ
4(33.3%)
Grade Ⅳ
3(25%)
Supplementary Table 4
The expressions of BMK1 and miR-429 in glioma patients
miR-429 expression
BMK1 expression
Low or none High
Low or none
15
50
High
101
10
P
0.000
Supplementary Table 5
The relationship between miR-429 expression and BMK1 expression and
clinicopathologic factors by Spearman correlation analysis
miR-429 expression
Variables
Spearman’s
correlation
BMK1 expression
P
Spearman’s correlation
P
Gender
-0.030
0.691
0.034
0.653
Age
-0.029
0.701
0.027
0.723
Who grade
-0.381
0.000
0.393
0.000
Survival(n=176)
-0.451
0.000
0.406
0.000
BMK1 expression
-0.691
0.000