Background
Results
Validity of methods
Selection of candidate genes
Symbol | Chr | Role | Methylated in other cancers | Comment |
---|---|---|---|---|
DHRS3 | 1p36 | Short-chain dehydrogenases/reductase | Melanoma cell lines [17] | |
DUSP23 | 1q23 | Dual-specificity protein phosphatase | ||
TGFBI | 5q31 | Extracellular matrix protein | Leukemia, renal cell-, lung-, esophageal cancer [24] | |
COL1A2 | 7q22 | Type I collagen | Tumors that secrete the gene have low tumorigeneic potential [27]. | |
PRKCDBP | 11p15 | Protein kinase binding | Also called hSRBC. Putative TSG. | |
SCNN1A | 12p13 | Ion transport | Breast cancer [22] | Contributes to methylator phenotype in breast cancer [22]. |
POU2F2 | 19q13 | Transcription factor | Regulator of neuronal differentiation [34]. | |
KRT19 | 17q21 | Intermediate filament protein | Renal cell carcinoma [30] |
Verification of the array data with expression analysis using real-time RT-PCR
Bisulfite sequencing and methylation-specific PCR
Differential methylation analysis
Gene | CpG site | In CpG island | Mean DOD (SD) | Mean NED (SD) | Δbeta value |
---|---|---|---|---|---|
SCNN1A | cg18738906 | yes | 0.88 (0.21) | 0.65 (0.27) |
0.23***
|
cg26215727 | no | 0.47 (0.20) | 0.31 (0.17) |
0.16***
| |
PRKCDBP | cg05628549 | yes | 0.53 (0.28) | 0.34 (0.26) |
0.18*
|
cg16245261 | yes | 0.45 (0.34) | 0.20 (0.23) |
0.25**
| |
cg18392783 | yes | 0.50 (0.32) | 0.26 (0.24) |
0.24**
| |
cg18959478 | yes | 0.55 (0.31) | 0.32 (0.27) |
0.23**
| |
KRT19 | cg11462865 | yes | 0.65 (0.24) | 0.43 (0.29) |
0.22**
|
cg16585619 | no | 0.82 (0.09) | 0.81 (0.06) | 0.01 | |
TGFBI | cg00386408 | yes | 0.40 (0.24) | 0.32 (0.23) | 0.08 |
cg21034676 | yes | 0.28 (0.28) | 0.23 (0.24) | 0.04 | |
POU2F2 | cg21608489 | yes | 0.62 (0.22) | 0.59 (0.20) | 0.03 |
cg22054191 | yes | 0.22 (0.08) | 0.24 (0.10) | -0.02 | |
DUSP23 | cg10663017 | yes | 0.20 (0.25) | 0.11 (0.16) | 0.09 |
cg11104347 | yes | 0.17 (0.25) | 0.10 (0.15) | 0.08 | |
DHRS3 | cg01346152 | yes | 0.64 (0.30) | 0.64 (0.26) | 0.00 |
cg09229231 | yes | 0.03 (0.01) | 0.03 (0.01) | 0.00 | |
COL1A2 | cg18511007 | yes | 0.42 (0.27) | 0.35 (0.25) | 0.07 |
cg25300386 | yes | 0.37 (0.29) | 0.32 (0.27) | 0.05 |
Gene | CpG site | INRG (M/L) | 1p-dela
| MNAa
| 11q-dela
| 17q-gaina
| >18 m |
---|---|---|---|---|---|---|---|
SCNN1A | cg18738906 |
0.33***
| 0.15 |
0.21**
|
0.25***
|
0.34***
|
0.15*
|
cg26215727 |
0.15**
| 0.04 |
0.13*
|
0.17**
|
0.20***
| 0.09 | |
PRKCDBP | cg05628549 | 0.09 | -0.06 | 0.12 | 0.08 | 0.09 | -0.10 |
cg16245261 |
0.17*
| 0.00 |
0.27***
| 0.15 |
0.19*
| 0.01 | |
cg18392783 | 0.12 | -0.06 |
0.18**
| 0.10 | 0.12 | -0.02 | |
cg18959478 | 0.12 | -0.06 |
0.16*
| 0.10 | 0.11 | -0.04 | |
KRT19 | cg11462865 | 0.14 | -0.01 |
0.19*
| 0.10 | 0.12 |
0.22**
|
cg16585619 | 0.01 |
-0.05*
|
0.07***
|
0.04*
| 0.03 | 0.01 | |
TGFBI | cg00386408 | 0.05 | -0.04 | 0.11 | 0.10 | 0.07 |
0.14*
|
cg21034676 | 0.04 | -0.04 | 0.13 | 0.06 | 0.05 | 0.12 | |
POU2F2 | cg21608489 | -0.08 | -0.05 | -0.08 | -0.01 | -0.04 | -0.08 |
cg22054191 | 0.03 | -0.03 | -0.01 | 0.04 | 0.02 | 0.04 | |
DUSP23 | cg10663017 | 0.06 | -0.10 |
0.27***
|
0.21***
|
0.16**
| 0.07 |
cg11104347 | 0.05 |
-0.13*
|
0.29***
|
0.16**
|
0.13**
| 0.08 | |
DHRS3 | cg01346152 | -0.13 | -0.03 |
-0.20**
| -0.03 | -0.08 | -0.10 |
cg09229231 |
0.004*
| 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
COL1A2 | cg18511007 | 0.07 | 0.00 | 0.05 | 0.07 | -0.01 |
0.18**
|
cg25300386 | 0.02 | -0.04 | -0.02 | 0.02 | -0.09 |
0.15*
|
Expression analysis with real-time RT-PCR
Discussion
Conclusions
Methods
Cell lines and tumor material
Expression analysis
Methylation analysis
Drug treatments
RNA extraction
Expression microarray analysis
Analysis of DNA methylation
cDNA preparation and real-time RT-PCR
DNA methylation analysis with bisulfite sequencing
Methylation-specific PCR (MSP)
Data analysis
Unadjusted OR | Adjusted OR for other prognostic factor | |||||
---|---|---|---|---|---|---|
Gene | CpG site | INRG (M/L) | MNA | 11q-del | >18 m | |
SCNN1A | cg18738906 |
3.4**
| 2.2 |
3.0**
|
3.1**
|
3.1**
|
PRKCDBP | cg16245261 |
2.5**
|
2.4*
|
2.3*
|
2.4*
|
2.9**
|
KRT19 | cg11462865 |
2.5*
|
2.5*
|
2.2*
|
2.3*
|
2.1*
|