Background
Results
Validation of the splice-inducing ability of SRSF2
Cross validation with 44 k Agilent microarray
Validation of the labeling method: comparison of the 15 k custom and 44 k Agilent microarrays
Detection of the mRNA regulation
Gene regulation | Gene symbol | Control condition intensity | SRSF2 condition intensity | Regulation | Fold-change | P-value |
---|---|---|---|---|---|---|
Up-regulated |
VEGFA
| 9.25 | 9.99 | up | 1.67 | 3.38E-08 |
Down-regulated |
HER1/EGFR
| 5.01 | 4.11 | down | 1.87 | 3.65E-06 |
Slightly down-regulated |
HER3
| 2.33 | 1.78 | down | 1.47 | 3.98E-03 |
HIF1A
| 6.48 | 6.06 | down | 1.33 | 2.04E-04 | |
PIR
| 7.01 | 6.62 | down | 1.32 | 1.82E-03 | |
AKT1
| 8.04 | 7.68 | down | 1.28 | 8.15E-05 | |
VEGFB
| 7.84 | 7.48 | down | 1.28 | 2.56E-04 | |
Not regulated |
PIK3R2
| 3.94 | 4.19 | up | 1.19 | 1.62E-01 |
AKT3
| 2,00 | 1.91 | down | 1.06 | 6.18E-01 | |
HER2
| 4.89 | 4.81 | down | 1.05 | 4.01E-01 | |
AKT2
| 5.81 | 5.74 | down | 1.05 | 5.57E-01 | |
VEGFC
| 5.90 | 5.89 | down | 1.01 | 9.03E-01 | |
Not expressed |
PIK3R1
| 1.35 | 1.89 | up | 1.45 | 3.71E-02 |
VEGFD
| 0.88 | 0.98 | up | 1.07 | 8.23E-02 | |
HER4
| 0.65 | 0.69 | up | 1.03 | 4.87E-01 | |
PIK3CA
| 1.79 | 1.78 | down | 1.01 | 8.58E-01 |
Regulation events among the expressed genes
Gene symbol | Region name | Region type | Confidence | Regulation | Fold-change | P-value | RT-PCR |
---|---|---|---|---|---|---|---|
AKT2
| je14_e15_5p_region | Junction | Low | Down | 1.63 | 1.36E-02 | No |
AKT3
|
je7_e8
|
Junction
|
High
|
Down
|
5.99
|
2.07E-05
|
Yes
|
AKT3
|
e8
|
Exon
|
High
|
Down
|
4.19
|
4.10E-03
|
Yes
|
HER1/EGFR
|
je16_e19
|
Junction
|
High
|
Down
|
3.66
|
1.42E-03
|
Yes
|
HER1/EGFR
| predict_exon_1_2-10 | Exon | Medium | Up | 33.98 | 0.00E + 00 | No |
HER1/EGFR
| est_1_2 | Exon | Medium | Up | 17.35 | 1.00E-09 | No |
HER1/EGFR
| predict_exon_2_3-1 | Exon | Medium | Up | 4.97 | 5.51E-05 | No |
HER1/EGFR
| e26 | Exon | Medium | Down | 2.64 | 2.30E-03 | No |
HER1/EGFR
| ae1_donor_alter | Donor_alter | Medium | Up | 5.55 | 5.40E-03 | No |
HER1/EGFR
| je14_e15 | Junction | Low | Down | 1.87 | 1.05E-02 | No |
HER1/EGFR
| je12_e13 | Junction | Low | Down | 1.76 | 1.13E-02 | No |
HER2
| ae10_prom_alter | Prom_alter | Medium | Down | 2.73 | 2.32E-02 | No |
HER3
| je22_ae23_acceptor_alter_l | Junction | Low | Up | 1.68 | 3.33E-02 | No |
HIF1A
|
je10_e11
|
junction
|
High
|
Down
|
1.87
|
1.66E-05
|
Yes
|
HIF1A
|
e10
|
Exon
|
High
|
Down
|
1.62
|
3.65E-02
|
Yes
|
HIF1A
|
e9
|
Exon
|
High
|
Down
|
1.56
|
4.78E-02
|
Yes
|
HIF1A
| e5 | Exon | Low | Down | 2.07 | 1.23E-02 | No |
HIF1A
| je1_5p_region_ae3_acceptor_alter | Junction | Low | Down | 1.56 | 1.64E-02 | No |
HIF1A
| je14_e16 | Junction | Low | Up | 2.22 | 2.08E-02 | No |
PIK3R2
| e6 | Exon | High | Down | 1.97 | 7.61E-05 | No |
PIK3R2
| je5_e6 | Junction | High | Down | 3.95 | 3.98E-04 | No |
PIK3R2
| je7_e8 | Junction | Low | Down | 1.68 | 2.14E-03 | No |
PIK3R2
| e9 | Exon | Low | Down | 1.74 | 3.34E-03 | No |
PIK3R2
| je2_e3 | Junction | Low | Down | 1.58 | 4.36E-02 | No |
VEGFA
|
e4_term_alter
|
Term_alter
|
High
|
Up
|
10.18
|
7.43E-08
|
Yes
|
VEGFA
|
ae6_donor_alter_2
|
Donor_alter
|
High
|
Down
|
2.44
|
4.18E-07
|
Yes
|
VEGFA
|
jae6_donor_alter_2_e7
|
Junction
|
High
|
Down
|
1.93
|
4.96E-03
|
Yes
|
VEGFA
|
ae7_donor_alter
|
Donor_alter
|
High
|
Up
|
1.80
|
6.98E-04
|
Yes
|
VEGFA
| e7 | Exon | Low | Up | 1.50 | 4.00E-04 | No |
VEGFB
| je2_e3 | Junction | Low | Down | 1.62 | 6.47E-05 | No |
Validation of regulation events by real-time polymerase chain reaction
SRSF2 Condition vs. Control condition | |||||
---|---|---|---|---|---|
Gene | Calculation | Transcript | Expression | Observed transcript regulation | Expected transcript regulation |
HER1/EGFR
| Relative expression | Last exon = e17 | n/a | Not expressed | No expression |
Last exon = e18 | 0.91 | Not regulated | Over-expression | ||
Last exon > e20 | 0.42 | Under-expressed | Under-expression | ||
AKT3
| Fold-change | e7+ e8- vs. e7+ e8+ | 1.46 | Over-expression of e7+ e8- | Over-expression of exon 7 and under-expression of exon 8 |
e7- e8+ vs. e7+ e8+ | -1.19 | No regulation of e7- e8+ | |||
e7- e8- vs. e7+ e8+ | n/a | No expression of e7- e8- | |||
HIF1A
| Fold-change | e9+ e10- vs. e9+ e10+ | 1.91 | Over-expression of e9+ e10- | Under-expression of exon 10 |
e9- e10+ vs. e9+ e10+ | -1.52 | Under-expression of e9- e10+ | Under-expression of exon 10 | ||
e9- e10- vs. e9+ e10+ | 1.82 | Over-expression of e9- e10- | Under-expression of exons 9 and 10 | ||
VEGFA
| Fold-change | Last exon = e4 vs. last exon > e5 | 18.93 | Over-expression of "last exon = e4" | Over-expression of exon 4 |
VEGFA
| Fold-change | Alternative vs. constitutive donor e6 | 14.46 | Over-expression of "alternative donor e6" | Over-expression of alternative donor |
HER1/EGFR protein expression analysis
Alternative splicing events in lung cancer biopsy samples
A
| |||||||||||||
Sample
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
| |||
SRSF2 Protein Score |
270
|
270
|
240
| 180 | 160 | 140 | 120 | 100 | 60 | n/a | |||
Phospho-SRSF2 Protein Score |
270
|
270
|
270
| 100 | 100 | 90 | 60 | 100 | 40 | n/a | |||
B
| |||||||||||||
Non small cell lung carcinoma
vs
. normal lung tissue
| |||||||||||||
Gene | Calculation | Transcript |
1
|
2
|
3
| 4 | 5 | 6 | 7 | 8 | 9 | 10 | Observed Transcript Regulation |
HER1/EGFR
| Relative expression | Last exon = e17 |
n/a
|
n/a
|
n/a
| n/a | 7.17 | n/a | n/a | 16.15 | 2.52 | n/a | Not expressed or over-expressed |
Last exon = e18 |
2.69
|
n/a
|
1.86
| 2.27 | 5.13 | 6.86 | 2.23 | 15.16 | 3.01 | 66.18 | Over-expressed | ||
Last exon > e20 |
0.15
|
0.29
|
0.20
| 6.71 | 6.99 | 21.45 | 0.10 | 5.39 | 2.46 | n/a | Over-expressed or under-expressed | ||
AKT3
| Fold-change | e7+ e8- vs. e7+ e8+ |
0.84
|
1.46
|
1.16
| 1.37 | 1.08 | 1.76 | 1.29 | 1.47 | 1.47 | 2.28 | Over-expression or no regulation |
e7- e8+ vs. e7+ e8+ |
n/a
|
1.33
|
n/a
| 1.35 | n/a | n/a | n/a | 0.38 | n/a | n/a | Low expression of e7- e8+ | ||
e7- e8- vs. e7+ e8+ |
n/a
|
n/a
|
n/a
| n/a | n/a | n/a | n/a | n/a | n/a | n/a | No expression of e7- e8- | ||
HIF1A
| Fold-change | e9+ e10- vs. e9+ e10+ |
0.63
|
0.54
|
1.63
| 1.40 | 0.79 | 1.77 | 0.87 | 1.22 | 1.20 | 1.15 | Depending on biopsy samples |
e9- e10+ vs. e9+ e10+ |
1.87
|
n/a
|
n/a
| 1.06 | n/a | n/a | n/a | n/a | 1.14 | 1.77 | Depending on biopsy samples | ||
e9- e10- vs. e9+ e10+ |
0.57
|
0.73
|
1.55
| 1.49 | 0.84 | 1.88 | 0.79 | 1.99 | 0.76 | 1.01 | Depending on biopsy samples | ||
VEGFA
| Fold-change | Last exon = e4 vs. Last exon > e5 |
1.33
|
1.21
|
1.54
| 1.92 | 0.84 | 2.22 | 10.37 | 2.73 | 1.21 | 1.09 | Over-expression or no regulation |
VEGFA
| Fold-change | Alternative vs. constitutive donor e6 |
3.50
|
3.33
|
5.25
| 3.99 | n/a | n/a | 19.22 | 14.29 | n/a | 45.68 | Over-expression |
Discussion
Gene | Custom array (present study) | Affymetrix™ Exon array | ||
---|---|---|---|---|
Nb probes (exonic/junction) | Average probe length (bp) | Nb probes (exonic/junction) | Average probe length (bp) | |
HIF1A | 123 (85/38) | 42.4 | 80 (80/0) | 25.0 |
VEGFA | 90 (64/26) | 42.3 | 60 (60/0) | 25.0 |