Background
Methods
PAH models and sample collection
Transcriptomic analysis
RNA isolation, purification, and quantification
cDNA library construction and sequencing
Data analysis
Tandem mass tags analysis
Sample preparation, protein digestion and TMT labeling
High pH reverse phase separation
Low pH nano-HPLC–MS/MS analysis
Database searching and quantitative data analysis
Bioinformatics and annotations
Reduced representation bisulfite sequencing
DNA sample preparation
DNA fragmentation and bisulfite conversion
Library construction and sequencing
Bioinformatics analysis
Integrated analysis of the multiomics results
Protein–protein interaction (PPI) networks
Real-time PCR analysis
Western blot analysis
Statistical analysis
Results
Verification of hypoxia-induced PAH models
Screening the differential characteristics in PAH
Integrated analysis of multiomics characteristics
Correlation | Methylation information | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Gene symbol | mRNA_ID | Protein full name | FC | dir |
p
| FDR | Chr | Info1 | Info2 | Info3 |
S100a9 | ENSRNOT00000015351 | Protein S100-A9 | 5.8 | Up | 1.2E−04 | 1.9E−02 | chr2 | Intron | First intron | NA |
Ttc29 | ENSRNOT00000089569 | Tetratricopeptide repeat protein 29 | 0.3 | Down | 7.9E−05 | 8.8E−03 | chr19 | Exon | Internal exon | NA |
Bicc1 | MSTRG.12240.6 | BicC family RNA-binding protein 1 | 5.2 | Up | 5.0E−04 | 4.5E−02 | chr20 | Intron | Internal intron | NA |
Tnc | ENSRNOT00000084563 | Tenascin C | 0.0 | Down | 7.1E−07 | 1.6E−04 | chr5 | Intron | Internal intron | NA |
Runx1 | MSTRG.4485.1 | Runt-related transcription factor 1 | 0.6 | Down | 5.4E−05 | 8.5E−03 | chr11 | Intron | First intron | NA |
Picalm | ENSRNOT00000092945 | Clathrin-assembly lymphoid myeloid leukemia protein | 8.7 | Up | 2.1E−13 | 1.4E−10 | chr1 | Intron | Internal intron | NA |
Cdyl2 | ENSRNOT00000068440 | Chromodomain Y-like 2 | 0.3 | Down | 1.4E−04 | 1.4E−02 | chr19 | Intron | Internal intron | NA |
Gfpt2 | ENSRNOT00000003770 | Glutamine-fructose-6-phosphate aminotransferase 2 | 0.8 | Down | 3.9E−05 | 5.1E−03 | chr10 | Intron | Internal intron | NA |
Folr2 | MSTRG.1446.1 | Folate receptor 2 | 0.2 | Down | 8.7E−05 | 1.2E−02 | chr1 | Promoter | Distal | LCP |
Slc43a2 | MSTRG.3515.3 | Solute carrier family 43 member 2 | 17.7 | Up | 2.2E−19 | 3.0E−16 | chr10 | Intron | Internal intron | NA |
Myh6 | ENSRNOT00000023302 | Myosin-6 | 2.8 | Up | 4.8E−05 | 8.6E−03 | chr15 | Intron | Internal intron | NA |
RGD1563354 | ENSRNOT00000019453 | Similar to hypothetical protein D630003M21 | 0.2 | Down | 5.6E−06 | 1.1E−03 | chr3 | Promoter | Distal | LCP |
Hist2h2ac | ENSRNOT00000051917 | Histone H2A | 0.2 | down | 9.0E−08 | 3.0E−05 | chr2 | Promoter | Intermediate | ICP |
Map4k2 | ENSRNOT00000064798 | Mitogen-activated protein kinase | 4.9 | up | 3.4E−06 | 6.8E−04 | chr1 | Intron | Internal intron | NA |
Gsta3 | ENSRNOT00000088416 | Glutathione S-transferase alpha-3 | 2.7 | up | 2.3E−04 | 9.2E−06 | chr9 | Exon | Last exon | NA |
Pdk4 | ENSRNOT00000012760 | Pyruvate dehydrogenase kinase, isoenzyme 4 | 0.2 | down | 7.9E−09 | 2.7E−06 | chr4 | Promoter | Proximal | LCP |
Med12 l | ENSRNOT00000084020 | Mediator complex subunit 12-like | 6.5 | Up | 1.3E−08 | 5.0E−06 | chr2 | Promoter | Distal | LCP |
Mapk10 | ENSRNOT00000065965 | Mitogen-activated protein kinase | 2.6 | Up | 3.6E−06 | 8.0E−04 | chr14 | Intron | First intron | NA |
Cd63 | ENSRNOT00000090381 | Ad1-antigen | 0.5 | Down | 3.6E−06 | 7.0E−04 | chr7 | Promoter | Intermediate | ICP |
Krt1 | ENSRNOT00000034450 | Keratin, type II cytoskeletal 1 | 21.8 | Up | 3.6E−04 | 3.9E−02 | chr7 | Promoter | Distal | LCP |
Correlation | mRNA information | Protein information | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Gene symbol | mRNA_ID | Protein full name | FC | dir |
p
| FDR | FC | dir |
p
| FDR |
S100a9 | ENSRNOT00000015351 | Protein S100-A9 | 2.4 | Up | 0.05 | 0.82 | 1.6 | Up | 6.6E−11 | 4.5E−10 |
Ttc29 | ENSRNOT00000089569 | Tetratricopeptide repeat protein 29 | 1.0 | Down | 0.22 | 0.84 | 1.5 | Up | 1.9E−10 | 1.2E−09 |
Bicc1 | MSTRG.12240.6 | BicC family RNA-binding protein 1 | 0.8 | Down | 0.38 | 0.84 | 1.5 | Up | 1.2E−05 | 2.8E−05 |
Tnc | ENSRNOT00000084563 | Tenascin C | 2.2 | Up | 0.36 | 0.84 | 1.5 | Up | 1.5E−12 | 1.5E−11 |
Runx1 | MSTRG.4485.1 | Runt-related transcription factor 1 | 1.5 | Up | 0.3 | 0.84 | 1.5 | Up | 3.2E−10 | 1.9E−09 |
Picalm | ENSRNOT00000092945 | Clathrin-assembly lymphoid myeloid leukemia protein | 0.6 | Down | 0.04 | 0.82 | 1.5 | Up | 1.6E−10 | 1.0E−09 |
Cdyl2 | ENSRNOT00000068440 | Chromodomain Y-like 2 | 1.1 | Up | 0.24 | 0.84 | 1.4 | Up | 9.9E−16 | 2.7E−14 |
Gfpt2 | ENSRNOT00000003770 | Glutamine-fructose-6-phosphate aminotransferase 2 | 1.5 | Up | 0.1 | 0.82 | 1.4 | Up | 1.7E−17 | 9.4E−16 |
Folr2 | MSTRG.1446.1 | Folate receptor 2 | 1.4 | Up | 0.15 | 0.84 | 1.4 | Up | 6.4E−18 | 4.3E−16 |
Slc43a2 | MSTRG.3515.3 | Solute carrier family 43 member 2 | 1.7 | Up | 0.35 | 0.84 | 1.4 | Up | 5.1E−08 | 1.9E−07 |
Myh6 | ENSRNOT00000023302 | Myosin-6 | 1.0 | Down | 0.31 | 0.84 | 0.7 | Down | 6.8E−16 | 2.0E−14 |
RGD1563354 | ENSRNOT00000019453 | Similar to hypothetical protein D630003M21 | 1.7 | Up | 0.06 | 0.82 | 0.7 | Down | 1.6E−11 | 1.2E−10 |
Hist2h2ac | ENSRNOT00000051917 | Histone H2A | 1.1 | Up | 0.51 | 0.84 | 0.7 | Down | 8.6E−07 | 2.6E−06 |
Map4k2 | ENSRNOT00000064798 | Mitogen-activated protein kinase | 1.3 | Up | 0.08 | 0.82 | 0.6 | Down | 2.4E−14 | 4.2E−13 |
Gsta3 | ENSRNOT00000088416 | Glutathione S-transferase alpha-3 | 0.8 | Down | 0.31 | 0.84 | 0.6 | Down | 2.9E−15 | 6.6E−14 |
Pdk4 | ENSRNOT00000012760 | Pyruvate dehydrogenase kinase, isoenzyme 4 | 0.3 | Down | 0.05 | 0.82 | 0.6 | Down | 5.5E−19 | 5.8E−17 |
Med12 l | ENSRNOT00000084020 | Mediator complex subunit 12-like | 1.0 | Down | 0.02 | 0.82 | 0.6 | Down | 2.2E−04 | 4.4E−04 |
Mapk10 | ENSRNOT00000065965 | Mitogen-activated protein kinase | 1.0 | Down | 0.41 | 0.84 | 0.6 | Down | 6.7E−04 | 1.2E−03 |
Cd63 | ENSRNOT00000090381 | Ad1-antigen | 1.3 | Up | 0.47 | 0.84 | 0.6 | Down | 4.8E−03 | 7.7E−03 |
Krt1 | ENSRNOT00000034450 | Keratin, type II cytoskeletal 1 | 0.7 | Down | 0.02 | 0.82 | 0.4 | Down | 4.2E−17 | 1.9E−15 |
Dysregulated biological functions in PAH
PPI network identification of hub node genes
KEGG-pathway | Gene symbol | Name | Degree | FC |
---|---|---|---|---|
ECM-receptor interaction |
Itga5
|
Integrin subunit alpha 5
|
11
|
0.79
|
Spp1 | Secreted phosphoprotein 1 | 8 | 1.32 | |
Vtn | Vitronectin | 7 | 1.28 | |
Thbs4 | Thrombospondin 4 | 6 | 1.26 | |
Col4a1
|
Collagen, type IV, alpha 1
|
6
|
1.88
| |
Lamb1 | Laminin, beta 1 | 5 | 1.31 | |
Col4a6 | Collagen, type IV, alpha 6 | 5 | 0.78 | |
Col2a1
|
Collagen, type II, alpha 1
|
5
|
0.58
| |
Fn1 | Fibronectin 1 | 4 | 1.27 | |
Tnc | Tenascin C | 3 | 1.49 | |
Col6a6 | Collagen, type VI, alpha 6 | 1 | 0.72 | |
Col6a5 | Collagen, type VI, alpha 5 | 1 | 0.69 | |
Focal adhesion |
Itga5
|
Integrin subunit alpha 5
|
12
|
0.79
|
Spp1 | Secreted phosphoprotein 1 | 10 | 1.32 | |
Vtn | Vitronectin | 9 | 1.28 | |
Cav1 | Caveolin 1 | 7 | 0.73 | |
Mapk10 | Mitogen activated protein kinase 10 | 7 | 0.63 | |
Col2a1
|
Collagen, type II, alpha 1
|
6
|
0.58
| |
Col4a1
|
Collagen, type IV, alpha 1
|
6
|
1.88
| |
Flnc | Filamin C, gamma | 6 | 0.75 | |
Fn1 | Fibronectin 1 | 6 | 1.27 | |
Lamb1 | Laminin, beta 1 | 6 | 1.31 | |
Thbs4 | Thrombospondin 4 | 6 | 1.26 | |
Tnc | Tenascin C | 6 | 1.49 | |
Col4a6 | Collagen, type IV, alpha 6 | 5 | 0.78 | |
Myl10 | Myosin, light chain 10, regulatory | 2 | 0.77 | |
Cav2 | Caveolin 2 | 1 | 0.76 | |
Col6a5 | Collagen, type VI, alpha 5 | 1 | 0.69 | |
Col6a6 | Collagen, type VI, alpha 6 | 1 | 0.72 | |
Ppp1cc | Protein phosphatase 1 catalytic subunit gamma | 1 | 0.72 | |
Drug metabolism-cytochrome P450 | Gstm2 | Glutathione S-transferase mu 2 | 9 | 0.73 |
Gstt1
|
Glutathione S-transferase theta 1
|
9
|
0.80
| |
Adh1c | Alcohol dehydrogenase 1C (class I) | 8 | 0.78 | |
Gstm3
|
Glutathione S-transferase mu 3
|
8
|
0.59
| |
Mgst2
|
Microsomal glutathione S-transferase 2
|
8
|
1.50
| |
Adh5 | Alcohol dehydrogenase 5 (class III) | 7 | 0.78 | |
Aldh3a1 | Aldehyde dehydrogenase 3 family, member A1 | 7 | 0.72 | |
Aox3 | Aldehyde oxidase 3 | 0 | 0.73 | |
Gsta4 | Glutathione S-transferase alpha 4 | 7 | 0.76 | |
Gsta3 | Glutathione S-transferase alpha 3 | 5 | 0.64 | |
Aox1 | Aldehyde oxidase 1 | 2 | 0.73 | |
Gstm5 | Glutathione S-transferase, mu 5 | 2 | 0.67 | |
Fmo5 | Flavin containing monooxygenase 5 | 0 | 1.27 | |
Metabolism of xenobiotics by cytochrome P450 | Gstm2 | Glutathione S-transferase mu 2 | 10 | 0.73 |
Gstt1 | Glutathione S-transferase theta 1 | 10 | 0.80 | |
Ephx1 | Epoxide hydrolase 1 | 9 | 0.69 | |
Gstm3
|
Glutathione S-transferase mu 3
|
9
|
0.59
| |
Mgst2
|
Microsomal glutathione S-transferase 2
|
9
|
1.50
| |
Adh1c | Alcohol dehydrogenase 1C (class I) | 8 | 0.78 | |
Aldh3a1 | Aldehyde dehydrogenase 3 family, member A1 | 8 | 0.72 | |
Gsta4 | Glutathione S-transferase alpha 4 | 8 | 0.76 | |
Adh5 | Alcohol dehydrogenase 5 (class III) | 6 | 0.78 | |
Gsta3 | Glutathione S-transferase alpha 3 | 6 | 0.64 | |
Gstm5 | Glutathione S-transferase, mu 5 | 3 | 0.67 | |
Complement and coagulation cascades | C3 | Complement C3 | 16 | 1.26 |
Fgg
|
Fibrinogen gamma chain
|
14
|
1.57
| |
Fga | Fibrinogen alpha chain | 12 | 1.60 | |
Kng1
|
Kininogen 1
|
12
|
3.01
| |
F10 | Coagulation factor X | 11 | 1.32 | |
Fgb | Fibrinogen beta chain | 10 | 1.49 | |
Kng2 | Kininogen 2 | 10 | 1.80 | |
Proc | Protein C | 10 | 0.79 | |
C9 | Complement component 9 | 9 | 1.44 | |
F3 | Coagulation factor III | 9 | 0.71 | |
C4bpa | Complement component 4 binding protein, alpha | 7 | 1.64 | |
C4bpb | Complement component 4 binding protein, beta | 7 | 1.90 | |
Thbd
|
Thrombomodulin
|
6
|
0.62
| |
C1qb | Complement C1q B chain | 5 | 1.25 | |
C5 | Complement component 5 | 4 | 1.29 | |
C5ar1 | Complement C5a receptor 1 | 4 | 1.47 | |
F13a1 | Coagulation factor XIII A1 chain | 4 | 1.27 | |
A2m | Alpha-2-macroglobulin | 3 | 1.93 | |
C6 | Complement component 6 | 3 | 1.31 | |
Kng2l1 | Kininogen 2-like 1 | 0 | 1.55 |