Background
Methods
Cell culture and transduction with Ad5-HIF-1α and Ad5-siHIF-1α
In vivo CAM assay
Histological assessment of transplantation tumors in the CAM
RNA extraction, microarray hybridization and data analysis
Transcriptase-polymerase chain reaction (RT-PCR) analysis
Gene | Primer | Tm(°C) | Length(bp) |
---|---|---|---|
Human | |||
VEGF-A | sense 5'-TGGAAGAAGCAGCCCATGAC-3' | 59 | 375 |
antisense 5'-GCACTAGAGACAAAGACGTG-3' | |||
IL-6 | sense 5'-TCAATGAGGAGACTTGCCTG-3' | 55 | 410 |
antisense 5'-GATGAGTTGTCATGTCCTGC-3' | |||
PDGFC | sense 5'-GCCTCTTCGGGCTTCTCC-3' | 56 | 395 |
antisense5'-TTACTACTCAGGTTGGATTCCGC-3' | |||
FN1 | sense 5'-CGAAATCACAGCCAGTAG-3' | 51 | 278 |
antisense 5'-ATCACATCCACACGGTAG-3' | |||
MMP28 | sense 5'-CAAGCCAGTGTGGGGTCT-3' | 56 | 252 |
antisense 5'-TAGCGGTCATCTCGGAAG-3' | |||
MMP14 | sense 5'-ATGTCTCCCGCCCCA-3' | 60 | 678 |
antisense 5'-TCAGACCTTGTCCAGCAGG-3' | |||
GLUT1 | sense 5'-CGGGCCAAGAGTGTGCTAAA-3' | 62 | 283 |
antisense 5'-TGACGATACCGGAGCCAATG-3' | |||
GLUT2 | sense 5'-CCTGAATGCCAAGGGAATCCGG-3' | 48 | 368 |
antisense 5'-GCCAGATGAGGTAATCAATCATAG-3' | |||
GAPDH | sense 5'-AGAAGGCTGGGGCTCATTTG-3' | 57 | 258 |
antisense 5'-AGGGGCCATCCACAGTCTTC-3' | |||
Chicken | |||
VEGF-A | sense 5'-GTCTACGAACGCAGCTTCTG-3' | 62 | 265 |
antisense 5'-TCACATGTCCAAGTGCGCAC-3' | |||
IL-6 | sense 5'- TTGATGGACTCCCTAAGGC-3' | 50 | 395 |
antisense 5'-GATTCGGGACTGGGTTCTC-3' | |||
PDGFC | sense 5'-TTCTCAACCTGGATTCTGC-3' | 52 | 355 |
antisense 5'-AATGGTGTCAGTTCGCTTC-3' | |||
FN1 | sense 5'-ACCAACATTGACCGCCCTAA-3' | 56 | 458 |
antisense 5'-AATCCCGACACGACAGCAGA-3' | |||
MMP28 | sense 5'-TGACATCCGCCTGACCTT-3' | 57 | 376 |
antisense 5'-GTCCTGGAAGTGAGTGAAGACC-3' | |||
MMP14 | sense 5'-CGTGTTCAAGGAGCGGTGGC-3' | 61 | 114 |
antisense 5'-TAGGCGGCGTCGATGCTGT-3' | |||
GLUT1 | sense 5'-CACTGTTGTTTCGCTCTTCG-3' | 42 | 316 |
antisense 5'-AATGTACTGGAAGCCCATGC-3' | |||
GLUT2 | sense 5'-AGTTTGGCTACACTGGAG-3' | 60 | 436 |
antisense 5'-AGGATGGTGACCTTCTCC-3' | |||
GAPDH | sense 5'-CTTTCCGTGTGCCAACCC-3' | 65 | 108 |
antisense 5'-CATCAGCAGCAGCCTTCACTAC-3' |
Western blot analysis
Statistical analysis
Results
Implantation of cells on CAM in vivo
day 8 | day 11 | day 14 | day 17 | |
---|---|---|---|---|
Vessel length (pixels) | ||||
Control (n = 10 × 4) | 2106 ± 143 | 1967 ± 113 | 1457 ± 135 | 2183 ± 156 |
NCI/H446(n = 10 × 4) | 2452 ± 117 | 2564 ± 96* | 2687 ± 103* | 2798 ± 135* |
NCI/H446/HIF-1α(n = 15 × 4) | 2742 ± 83 | 2814 ± 154 | 2910 ± 137§ | 2994 ± 124§ |
NCI/H446/siHIF-1α(n = 12 × 4) | 2331 ± 53# | 2268 ± 106# | 2236 ± 162# | 2203 ± 116# |
Vessel Branch points | ||||
Control (n = 10 × 4) | 76 ± 5 | 82 ± 9 | 73 ± 8 | 89 ± 5 |
NCI/H446(n = 10 × 4) | 92 ± 7 | 101 ± 11 | 105 ± 6* | 117 ± 7* |
NCI/H446/HIF-1α(n = 15 × 4) | 116 ± 16 | 123 ± 11§ | 128 ± 9§ | 134 ± 21§ |
NCI/H446/siHIF-1α(n = 12 × 4) | 82 ± 5# | 87 ± 6# | 92 ± 11# | 102 ± 13# |
Regulation of angiogenic gene expression by HIF-1α
UniGeneID | Gene name | Gene Symbol | Fold change (ratio ≥ 2, p < 0.05) | |
---|---|---|---|---|
A | B | |||
Hs.143250 | Tenascin C (hexabrachion) | TNC | 5.28 | -3.23 |
Hs.654458 | Interleukin 6 (interferon, beta 2) | IL6 | 5.29 | -2.27 |
Hs.73793 | Vascular endothelial growth factorA | VEGF-A | 6.76 | -3.98 |
Hs.437322 | Tumor necrosis factor, alpha-induced protein 6 | TNFAIP6 | 6.96 | -4.75 |
Hs.570855 | Platelet derived growth factor C | PDGFC | 2.26 | -3.21 |
Hs.701982 | Interleukin 1 receptor, type I | IL1R1 | 2.64 | -2.21 |
Hs.203717 | Fibronectin 1 | FN1 | 2.31 | -2.57 |
Hs.567641 | Myocardin | MYOCD | 3.03 | -2.08 |
Hs.517581 | Heme oxygenase (decycling) 1 | HMOX1 | 2.64 | -2.73 |
Hs.687274 | Matrix metallopeptidase 28 | MMP28 | 4.39 | -3.67 |
Hs.2399 | Matrix metallopeptidase 14 | MMP14 | 2.97 | -2.24 |
Hs.473721 | Glucose transporter 1 | GLUT1 | 2.98 | -2.16 |
Hs.167584 | Glucose transporter 2 | GLUT2 | 3.74 | -2.05 |
Hs.485572 | Suppressor of cytokine signaling 2 | SOCS2 | -6.06 | 3.06 |
Hs.450230 | Insulin-like growth factor binding protein 3 | IGFBP3 | -4.02 | 2.17 |
Hs.653377 | Insulin-like growth factor 1 receptor | IGF1R | -2.00 | 2.89 |
Hs. 8867 | Cysteine-rich, angiogenic inducer, 61 | CYR61 | -3.03 | 2.18 |