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E
Methylation (%)
F
Methylation (%)
100
80
60
40
20
100
80
60
40
20
0
Sall3-1 1
2
3
4
5,6
7
Sall3-1 1
2
3
4
5,6
7
tion levels in human HCC tissues are due to promoter
CpG island hypermethylation, six human HCC cell lines
(Huh7, HepG2, SK-HEP1, SMMC7721, Bel7402 and
QGY7703) were exposed to 0, 0.1, 0.5 or 2.5 mmol 5-aza-
CdR, an inhibitor of DNMTs, for 72 h. As expected,
after treatment with 0.1, 0.5 or 2.5 mmol 5-aza-CdR, promoter
CpG island methylation levels showed dose-dependent
downregulation in all six cell lines (Figure 4). sall3
mRNA levels also showed dose-dependent upregulation
10,11,12
10,11,12
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15,16
15,16
17
17
18
19
20
21,22,23
24,25,26
27,28
CpG units (SMMC7721)
18
19
20
21,22,23
24,25,26
27,28
CpG units (QGY7703)
31
31
Sall3-2 5
6
7
9
Sall3-2 5
6
7
9
10,11
10,11
in Huh7, HepG2, SK-HEP1 and SMMC7721 cells, while
it was irregularly upregulated in Bel7402 and QGY7703
cells (Figure 5). These data indicated that sall3 promoter
CpG island hypermethylation is likely responsible for the
decrease in sall3 mRNA transcription level.
DISCUSSION
12
12
13,14,15
13,14,15
20,21,22
20,21,22
Control
0.1 mmol
0.5 mmol
2.5 mmol
Control
0.1 mmol
0.5 mmol
2.5 mmol
Figure 4 Quantitative methylation analysis on each CpG unit of sall3 CpG island in HCC cells after 5-Aza-CdR treatment or control (A-F).
Relative expression
Yang XX et al . Aberrant methylation downregulate sall3 in HCC
6
5
4
3
2
1
0
0
Hep G2 Huh7 Bel7402 SK-HEP1 SMMC7721 QGY7703
Figure 5 Relative sall3 expression in six hepatocellular carcinoma cells after 5-AZA-CdR treatment. Error bars, SD from triplicates.
50
40
30
20
10
Control
0.1 mmol
0.5 mmol
2.5 mmol
HCC is one of the most common gastrointestinal ma-
2724 June 7, 2012|Volume 18|Issue 21|