Supplementary Table1: Clinicopathological characteristic of HCC patients included
Variable
Cohort 1(case numbers)
Cohort 2 (case numbers)
All cases
18#
31*
Age(years), ＞55:≤55
8:10
13:18
Gender, male/female
16:2
28:3
HBsAg, positive/negative
14:4
25:6
Tumor size(cm), ＞5:≤5
10:8
14:17
10:8
19:12
Edmondson Grade,
I+II: III
Patients of group cohort 2 were cohort 1 plus additional 14 cases.
#
19 cases were used and 1 of them lost the clinical characteristics.
*33 cases were used and 2 of them lost the clinical characteristics.
Supplementary Table 2 Primers and annealing temperature of genes analyzed by
RT-PCR
Primers
Temperature
(5′to 3′)
(℃)
Amplification
Gene
size (bp)
F: TGGATGAAAGGGCATTTGAGA
PDCD4
55
164
61
324
65
110
64
270
54
201
61
220
64
62
64
90
R: AGCCTTCCCCTCCAATGCTA
F: CCGAGTTGGTGATGGTGTGTAC
DNMT1
R: AGGTTGATGTCTGCGTGGTAGC
F: TATTGATGAGCGCACAAGAGAGC
DNMT3A
R: GGGTGTTCCAGGGTAACATTGAG
F: GACTTGGTGATTGGCGGAA
DNMT3B
R: GGCCCTGTGAGCAGCAGA
F: TTCTTCGTCTGCCGTTCC
HBx
R: TCGGTCGTTGACATTGCT
F: AAAGACCTGTACGCCAACAC
ACTB
R: GTCATACTCCTGCTTGCTGAT
Stem-loop: GTCGTATCCAGTGCAGGGTCC
GAGGTATTCGCACTGGATACGACTCAACA
miR-21
F:GCCCGCTAGCTTATCAGACTGATG
R: GTGCAGGGTCCGAGGT
F:CGCTTCGGCAGCACATATACTA
U6
R: CGCTTCACGAATTTGCGTGTCA
F: TTTAGTTTCGGTTTCGTCGTTAC
PDCD4(M)
50
135
55
136
58
194
R: GAAAAATCTCTAACCCTTCTCGC
PDCD4(U)
F: TTTAGTTTTGGTTTTGTTGTTATGA
R: CAAAAAATCTCTAACCCTTCTCACT
PDCD4(BGS)
F: GGAGTTATTTTTTTATTGAGAGGGG
R: CATCTTCAAAAAATTCCCAAAAA
Supplementary materials and methods
Bisulfite treatment and promoter methylation analysis
Genomic DNA was extracted from the cells using the phenol–chloroform method
followed by bisulfite modification. Bisulfite genome sequencing was performed as
described previously (1). Bisulfite-treated genomic DNA from L02-Vector, L02-HBx
#1 and L02-HBx #8 cells were amplified using methylated and unmethylated CDH1
primers as reported previously (2). Primers designed for PDCD4 bisulfite genome
sequencing are listed in Supplementary Table 1. Cell lines were treated with 5-Aza
(Sigma-Aldrich, St Louis, MO, USA) to determine whether demethylation could
restore PDCD4 expression. Briefly, 1×105 cells were treatment with 0, 10, 50 and 100
μM of 5-Aza for 2 days.
Wound healing assays
Cell migration was assessed by measuring the movement of cells into a scraped area
created by a 200-μl pipette tip, and the spread of the wound closure was observed after
24 h. The cells were photographed under a microscope.
1. Tao Q, Huang H, Geiman TM, Lim CY, Fu L, Qiu GH et al. Defective de novo
methylation of viral and cellular DNA sequences in ICF syndrome cells. Hum
Mol Genet 2002; 11: 2091–2102.
2. Zhang h, xiao w, liang H, Fang d, Yang S, luo Y. Demethylation in the promoter
area by the antisense of human dna mtase gene. Zhonghua Zhong liu Za Zhi 2002;
24: 444-447.
Supplementary Figure Legends
Supplementary Fig. S1. A, HBx expression levels were detected in 10 liver cell lines
by RT-PCR. H2O was used as a blank control. A human gastric adenocarcinoma cell
line (AGS) was used as negative control. β-actin was used as a loading control.
B,
The methylation status of the E-cadherin promoter was analyzed using MSP in
QSG-7701 cells transfected with HBx and control cells.
C, PDCD4 and CDH1
expression was measured by RT-PCR after treatment with 10, 50 and 100 μmol/L
5’-aza for up to 2 days in QSG-7701 cells. β-actin was used as a loading control.
D,
Mapping of the methylation status of individual CpG site in the PDCD4 promoter by
bisulfite genome sequencing in L02-Vector, L02-HBx #1 and L02-HBx #8 cells. The
regions spanning the CpG island with 13 CpG sites were analyzed. Methylation status
of the PDCD4 promoter was shown. Black color represents the methylated CpG site;
achromatic color represents the unmethylated CpG site; every circle represents
bisulfite sequencing for 6 random clones. E, In clinical cases with high miR-21
expression (cases in which miR-21 expression was higher than the average expression
level in all cases), the rates of PDCD4 down-regulation (T＜N) were 63% (5/8)
compared with 45% (5/11) of clinical cases with low miR-21 expression (cases which
miR-21 expression was lower than average expression level in all cases). Case
numbers of every sub-group were represented in the chart.
Supplementary Fig. S2. A, Flow cytometry analysis of cell apoptosis. HBx did not
significantly influence cell apoptosis when compared to control cells. The values
indicate the mean+s.d. for three separate experiments (independent Student’s t-test).
B, The effects of PDCD4 on cell migration were detected by wound healing assays
and didn’t find significant difference between 7402-PDCD4 and 7402-control cells.
Microscopic observation was recorded at 0, 24, and 36 hours after scratching the
surface of a confluent layer of cells.