Background
Methods
Cell cultures
Library construction and PET extraction
PET-to-genome mapping
Individual PET-to-exon designation
PET clustering and cluster-to-gene annotation
Gene level melanoma-melanocyte comparison
Pathway level melanoma-melanocyte comparison
Testing the feasibility of using PET count to measure gene expression level
Identification of most significantly altered pathways with hypergeometric distribution
TSS/PAS variants
Partial pathway analysis and manual curation
Results and discussion
Libraries
Library | #Total PETs | #uPETs | #Mappable (% over uPETs) | # PET1 | #Known gene-asso. PET1 (% over PET1) | #Pathway-asso. PET1 (% over PET1) (% over known gene-asso. PET1) |
---|---|---|---|---|---|---|
B16F1 melanoma (SMT001) | 91977 | 45465 | 34146 (75.1%) | 32267 | 29439 (91.2%) | 11687 (36.2%) (39.7%) |
Melan-a2 melanocyte (SMN001) | 68880 | 51352 | 38640 (75.3%) | 36623 | 33005 (90.1%) | 13348 (36.4%) (40.4%) |
E14 Stem Cell (MoEScom) | 248234 | 135328 | 97295 (71.9%) | 93384 | 86019 (92.1%) | 28311 (30.3%) (32.9%) |
E17.5 embryo (SME006) | 81793 | 41595 | 30883 74.3% | 28387 | 24656 (86.9%) | 9314 (32.8%) (37.8%) |
Melanin de novo biosynthesis genes are highly expressed in the cells of melanocyte origin, but not in the embryonic cells
Library Gene | B16F1 melanoma cells | Melan-a2 melanocytes | E14 Stem Cell | E17.5 embryo | Descriptions |
---|---|---|---|---|---|
PET counts (cpm) | |||||
Tyr
| 185 | 798 | 0 | 0 | Tyrosinase precursor; Albino locus protein; Belong to tyrosinase gene family. |
Dct (Tyrp2) | 1468 | 3208 | 0 | 0 | Dopachrome tautomerase precursor; Tyrosinase-related protein 2; Belong to tyrosinase gene family. |
Ddt
| 65 | 58 | 44 | 0 | D-dopachrome tautomerase. |
Tyrp1
| 1750 | 2816 | 0 | 0 | 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase precursor; Belong to tyrosinase gene family. |
Si
| 4751 | 7985 | 125 | 12 | Melanocyte protein Pmel 17 precursor (Silver locus protein). |
The most significantly upregulated pathways are diversely involved in various cellular activities
Pathway ID | Description | KEGG Category/Description* | # genes | # up-regulated genes | # down-regulated genes | p-value |
---|---|---|---|---|---|---|
up-regulated | ||||||
230
|
Purine metabolism
|
Nucleotide Metabolism
|
119
|
11
|
23
|
4.37E-05
|
440
|
Aminophosphonate metabolism
|
Metabolism of Other Amino Acids
|
12
|
0
|
6
|
0.000153
|
626
|
Nitrobenzene degradation
|
Biodegradation of Xenobiotics
|
11
|
0
|
5
|
0.00098
|
363
|
Bisphenol A degradation
|
Biodegradation of Xenobiotics
|
16
|
0
|
6
|
0.00101
|
350
|
Tyrosine metabolism
|
Amino Acid Metabolism
|
44
|
6
|
10
|
0.00196
|
450
|
Selenoamino acid metabolism
|
Metabolism of Other Amino Acids
|
20
|
0
|
6
|
0.00370
|
52
|
Galactose metabolism
|
Carbohydrate Metabolism
|
27
|
2
|
7
|
0.00429
|
4540 | Gap junction | Cell Communication | 85 | 4 | 14 | 0.00670 |
340 | Histidine metabolism | Amino Acid Metabolism | 31 | 1 | 7 | 0.00964 |
4110 | Cell cycle | Growth and Death | 98 | 2 | 15 | 0.01009 |
Down-regulated | ||||||
190
|
Oxidative phosphorylation
|
Energy Metabolism
|
128
|
46
|
8
|
2.95E-27
|
193
|
ATP synthesis
|
Energy Metabolism
|
42
|
14
|
4
|
2.68E-08
|
720 | Reductive carboxylate cycle | Energy Metabolism | 10 | 6 | 0 | 5.25E-06 |
20
|
Citrate cycle (TCA cycle)
|
Carbohydrate Metabolism
|
26
|
9
|
2
|
6.56E-06
|
620
|
Pyruvate metabolism
|
Carbohydrate Metabolism
|
35
|
8
|
5
|
0.00057
|
240 | Pyrimidine metabolism | Nucleotide Metabolism | 72 | 12 | 12 | 0.00057 |
660 | C5-branched dibasic acid metabolism | Carbohydrate Metabolism | 2 | 2 | 0 | 0.00319 |
480
|
Glutathione metabolism
|
Metabolism of Other Amino Acids/Neutralization of ROS*
|
37
|
7
|
1
|
0.00400
|
280 | Valine, leucine and isoleucine degradation | Amino Acid Metabolism | 39 | 7 | 6 | 0.00543 |
PET counts (cpm) | |||||
---|---|---|---|---|---|
Substrates/Products | Products/Substrates | Enzyme | Gene name | Melan-a2 melanocytes | B16F1 melanoma cells |
galactose | galactose 1-P | galactokinase |
Galk1
| 0 | 154 |
galactose 1-P + UDP-glucose | UDP-galactose + glucose1-P | galactose 1-P uridyl transferase |
Galt
| 45 | 33 |
UDP-galactose | UDP-glucose | UDP-galactose-4-epimerase |
Gale
| 0 | 33 |
glucose 1-P | UDP-glucose | UDP-glucose pyrophosphorylase2 |
Ugp2
| 284 | 863 |
Most of the significantly downregulated pathways reside in mitochondria and are related to energy metabolisms through the electron transport chain
Upregulation of RAS-MAPK pathway genes, and c-Myc in B16F1 melanoma cells
PET counts (cpm) | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Library |
PKA
|
PKB (Akt)
|
PKC
|
Ras
|
Raf
|
Mek1
|
Mek2
|
Erk
| ||||
H-ras
|
K-ras
|
M-ras
|
N-ras
|
R-ras
| ||||||||
Melan-a2 melanocytes | 0 | 90 | 15 | 89 | 0 | 30 | 15 | 60 | 58 | 15 | 44 | 73 |
total: 194 | ||||||||||||
B16F1 melanoma cells | 33 | 176 | 97 | 22 | 11 | 0 | 11 | 186 | 108 | 98 | 130 | 98 |
total: 230 |
Upregulation of Trp53, and cell cycle progression gene expressions
Perturbations in apoptosis pathway: elevation of caspase 8 transcription and E2f1-to-E2f4 switch
Cell adhesion and metastasis related genes were expressed as diverse isoforms in melanoma cells
PET counts (cpm) | A:B ratio | |||
---|---|---|---|---|
Protein | Gene isoforms | Melan-a2 melanocytes (A) | B16F1 melanoma cells (B) | |
Collagen |
Col2a1
| *S6-7E53T53: 15 | 0 | 30:0 |
Col1a2
| S31E52T52: 15 | 0 | ||
Integrin |
Itgb1
| S1E16T16: 44 | S1E16T16: 11 S1E10T16: 11 | 44:22 |
Itgb5
| S1E16T16: 15 *S6-7E16T16: 15 S14E16T16: 15 | 0 | 45:0 | |
Actin |
Actb
| S1E2T6: 15 S1E6T6: 399 | S4E6T6: 11 S1E2T6: 1896 | 414:1907 |
Actg1
| S3E6T6: 15 S4E6T6: 15 S1E6T6: 876 | S1E6T6: 5260 | 906:5260 | |
Cadherin |
Cdh11 (not in KEGG) | *S2-3E6-7T13: 15 *S3E6-7T13: 15 | *S2-3E6-7T13: 11 | 30:11 |
JAM |
Jam2
| S1E10T10: 15 | 0 | 15:0 |
Jam3
| S1E9T9: 15 | 0 | 15:0 | |
β-catenin |
Catnb
| S1E1T15: 30 (skip*) S1E15T15: 15 | S11E15T15: 33 | 15:33 |
α-catenin |
Catna1
| S4E18T18: 15 | S1E18T18: 22 S1E5T18: 11 S15E18T18: 11 | 15:44 |
Snail, Slug |
Snail, Slug
| 0 | 0 | 0:0 |
CD44 |
Cd44
| S1E14T14: 15 | S1E14T14: 77 S1E12T14: 33 S1E4T14: 11 | l5:121 |
OPN (osteopontin) |
Spp1
| S1E7T7: 1889 | 0 | 1889:0 |
TSS/PAS variants
Category | 1 (A) | 2 (B) | 3 (C) | 4 (AB) | 5 (AC) | 6 (BC) | 7 (ABC) | Total genes | Total transcripts |
---|---|---|---|---|---|---|---|---|---|
#Gene | 1485 | 1229 | 1603 | 258 | 437 | 374 | 220 | 5606 | |
#Melan-a2 melanocyte-specific tsc (A) | 1678 | 0 | 0 | 299 | 549 | 0 | 351 | 2877 | |
#B16F1 melanoma-specific tsc (B) | 0 | 1409 | 0 | 323 | 0 | 473 | 382 | 2587 | |
#Overlapped tsc (C) | 0 | 0 | 1681 | 0 | 485 | 423 | 317 | 2906 | |
8370 |
Transcription of solute carriers (SLCs)
SLCs | Solute(s) | #A-s tsc | A-s cpm | #A/B -c tsc | A-c cpm | B-c cpm | #B-s tsc | B-s cpm | (A-s + A-c) cpm | (B-s + B-c) cpm | B – A cpm |
---|---|---|---|---|---|---|---|---|---|---|---|
Slc25a5 | adenine nucleotide (ATP/ADP) | 1 | 15 | 1 | 842 | 2631 | 3 | 109 | 857 | 2740 | 1883 |
Slc3a2 | neutral amino acids | 2 | 44 | 2 | 1553 | 1305 | 5 | 152 | 1597 | 1457 | -140 |
Slc25a3 | phosphate | 0 | 0 | 2 | 1423 | 1174 | 1 | 43 | 1423 | 1217 | -206 |
Slc25a11 | 2-oxoglutarate/malate | 1 | 15 | 1 | 131 | 120 | 2 | 33 | 146 | 153 | 7 |
Slc20a2 | phosphate | 1 | 15 | 1 | 189 | 120 | 2 | 22 | 204 | 142 | -62 |
Slc2a1 | glucose | 1 | 15 | 1 | 15 | 120 | 0 | 0 | 30 | 120 | 90 |
Slc31a1 | copper | 1 | 15 | 1 | 58 | 98 | 0 | 0 | 73 | 98 | 25 |
Slc30a9 | zinc | 1 | 15 | 1 | 29 | 65 | 0 | 0 | 44 | 65 | 21 |
Slc12a4 | K+/Cl- | 1 | 15 | 1 | 44 | 54 | 0 | 0 | 59 | 54 | -5 |
Slc35a2 | UDP-galactose | 1 | 15 | 0 | 0 | 0 | 2 | 54 | 15 | 54 | 39 |
Slc25a17 | adenine nucleotide | 1 | 15 | 1 | 116 | 33 | 0 | 0 | 131 | 33 | -98 |
Slc25a19 | adenine nucleotide | 2 | 29 | 1 | 44 | 33 | 0 | 0 | 73 | 33 | -40 |
Slc9a3r1 | Na+/H+ exchanger | 2 | 29 | 1 | 116 | 11 | 1 | 11 | 145 | 22 | -123 |
Slc6a9 | neurotransmitter, glycine | 0 | 0 | 1 | 15 | 22 | 0 | 0 | 15 | 22 | 7 |
Slc35c2 | neutral amino acids | 0 | 0 | 1 | 58 | 11 | 0 | 0 | 58 | 11 | -47 |
Slc39a6 | zinc | 1 | 15 | 1 | 15 | 11 | 0 | 0 | 30 | 11 | -19 |
Slc6a15 | neurotransmitter, aa, osmolytes | 0 | 0 | 0 | 0 | 0 | 1 | 109 | 0 | 109 | 109 |
Slc25a13 | aa Q and E | 0 | 0 | 0 | 0 | 0 | 3 | 76 | 0 | 76 | 76 |
Slc2a8 | glucose | 0 | 0 | 0 | 0 | 0 | 1 | 54 | 0 | 54 | 54 |
Slco3a1 | organic anion | 0 | 0 | 0 | 0 | 0 | 1 | 43 | 0 | 43 | 43 |
Slc35a4 | UDP-galactose | 0 | 0 | 0 | 0 | 0 | 1 | 33 | 0 | 33 | 33 |
Slc41a3 | mg++ | 0 | 0 | 0 | 0 | 0 | 1 | 33 | 0 | 33 | 33 |
Slc12a5 | Na+/Cl- | 0 | 0 | 0 | 0 | 0 | 1 | 22 | 0 | 22 | 22 |
Slc25a10 | citrate/malate, dicarboxylate | 0 | 0 | 0 | 0 | 0 | 1 | 22 | 0 | 22 | 22 |
Slc27a4 | fatty acids | 0 | 0 | 0 | 0 | 0 | 1 | 22 | 0 | 22 | 22 |
Slc37a3 | glycerol-3-P | 0 | 0 | 0 | 0 | 0 | 1 | 22 | 0 | 22 | 22 |
Slc37a4 | glycerol-6-P | 0 | 0 | 0 | 0 | 0 | 1 | 22 | 0 | 22 | 22 |
Slc4a2 | anion | 0 | 0 | 0 | 0 | 0 | 1 | 22 | 0 | 22 | 22 |
Slc7a7 | cation amino acid | 0 | 0 | 0 | 0 | 0 | 1 | 22 | 0 | 22 | 22 |
Slc35a1 | CMP-Sialic acid | 2 | 87 | 0 | 0 | 0 | 0 | 0 | 87 | 0 | -87 |
Slc37a2 | glycerol-3-P | 4 | 73 | 0 | 0 | 0 | 0 | 0 | 73 | 0 | -73 |
Slc15a4 | peptide | 1 | 58 | 0 | 0 | 0 | 0 | 0 | 58 | 0 | -58 |
Slc25a1 | citrate | 2 | 58 | 0 | 0 | 0 | 0 | 0 | 58 | 0 | -58 |
Slc37a1 | glycerol-3-P | 1 | 44 | 0 | 0 | 0 | 0 | 0 | 44 | 0 | -44 |
Slc10a3 | Na+/bile acid | 2 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |
Slc22a18 | organic cation | 1 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |
Slc33a1 | acetyl-CoA | 2 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |
Slc39a1 | zinc | 1 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |
Slc39a11 | metal ion | 1 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |
Slc39a3 | zinc | 1 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |
Slc4a8 | anion | 1 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |
Slc6a6 | taurine | 2 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |
Slco4a1 | anion | 1 | 29 | 0 | 0 | 0 | 0 | 0 | 29 | 0 | -29 |