The authors have declared that there are no conflicts of interest.
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REFERENCES
1. Sant M, Aareleid T, Berrino F, Bielska Lasota M, Carli PM, Faivre J, et al.
EUROCARE Working Group. EUROCARE-3: survival of cancer patients diagnosed 1990–94—results and commentary. Ann Oncol. 2003;5:S61–118.
2. World Health Organization. World Health Statistics 2006. Geneva, Switzerland:
World Health Organization; 2006. Available at:
http://www.who.int/whosis/whostat2006/en/index.html.
3. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA: A Cancer Journal for Clinicians. 2011;61:69–90.
4. Carney DN. Lung cancer – time to move on from chemotherapy. N Engl J Med.
2002;346:126–128.
5. Minna JD, Roth JA, Gazdar AF. Focus on lung cancer. Cancer Cell. 2002;1:49–
52.
6. Rosell R, Monz M, Pifarre A, Ariza A, Sanchez JJ, Moreno I, et al. Molecular staging of non-small cell lung cancer according to K-ras genotypes. Clin Cancer Res. 1996;2:1083–1086.
7. Malkinson AM. Molecular comparison of human and mouse pulmonary 503
504 505 506
507 508
509 510 511
512 513
514 515
516 517 518
519
adenocarcinomas. Exp Lung Res. 1998;24:541–555.
8. Wheatley-Price P, Shepherd FA. Targeting angiogenesis in the treatment of lung cancer. J Thorac Oncol. 2008;3:1173–1184.
9. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669–676.
10.Tammela T, Enholm B, Alitalo K, Pavvonen K. The biology of vascular endothelial growth factors. J Cardio Res. 2004;65:550–563.
11.Connolly DT, Olander JV, Heuvelman D, Nelson R, Monsell R, Siegel N, et al.
Human vascular permeability factor ： Isolation from U937 cells. J Biol Chem.
1989;254:20017–20024.
12.Ferrara N. VEGF and the request for tumour angiogenesis factors. Nat Rev Cancer. 2002;2:795–803.
13.Gasparini G. The rationale and future potential of angiogenesis inhibitor in neoplasia. Drugs. 1999;58:17–38.
14.Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor.
Endocr Rev. 1997;18:4–25.
15.Murphy G., Stanton H, Cowell S, Butler G, Knauper V, Atkinson S, et al.
Mechanisms for pro matrix metalloproteinase activation. Acta Pathol Microbiol 520
521 522
523 524
525 526
527 528 529
530 531
532 533
534 535
536 537
Immun Scand. 1999;107:38–44.
16.Gupta MK, Qin RY. Mechanism and its regulation of tumor-induced angiogenesis. World J Gastroenterol. 2003;9:1144–1155.
17.Wicki A, Christofori G. The angiogenic switch in tumorigenesis. In: Tumor angiogenesis. Marme D, Fusenig N (eds.) Springer Berlin Heidelberg, New York, 2008;pp 67–88.
18.Hicks SM, Vassallo JD, Dieter MZ, Lewis CL, Whiteley LO, Fix AS, et al.
Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis. Toxicology. 2003;187:217–228.
19.Roskoski R Jr. Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol. 2007;62:179–213.
20.Kerbel RS. Tumor angiogenesis. N Engl J Med. 2008;358:2039–2049.
21.Fontanini G, Vignati S, Boldrini L, Chinè S, Silvestri V, Lucchi M, et al. Vascular endothelial growth factor is associated with neovascularization and influences progression of non-small cell lung carcinoma. Clin. Cancer Res. 1997;3:861–865.
22.Yuan A, Yu CJ, Kuo SH, Chen WJ, Lin FY, Luh KT, et al. Vascular endothelial growth factor 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small-cell lung 538
539 540
541 542 543
544 545 546
547 548
549
550 551 552
553 554 555
cancer. J Clin Oncol. 2001;19:432–441.
23.Zachary I. VEGF signalling: Integration and multi-tasking in endothelial cell biology. Biochem Soc Trans. 2003;31:1171–1177.
24.Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al.
Paclitaxel–carboplatin alone or with bevacizumab for non-small-cell lung cancer.
N Engl J Med. 2006;355:2542–2550.
25.Reck M, von Pawel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, et al.
Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil. J Clin Oncol. 2009;27:1227–1234.
26.Beckmann N, Mueggler T, Allegrini PR, Laurent D, Rudin M. From anatomy to the target: contributions of magnetic resonance imaging to preclinical pharmaceutical research. Anat Rec. 2001; 265: 85–100.
27. Kennel SJ, Davis IA, Branning J, Pan H, Kabalka GW, Paulus MJ. High
resolution computed tomography and MRI for monitoring lung tumor growth in mice undergoing radioimmunotherapy: Correlation with histology. Med. Phys.
2000; 27: 1101–1107.
556
557 558
559 560 561
562 563 564 565
566 567 568
569 570 571 572
28. Hildebrandt IJ, Su H, Weber WA. Anesthesia and other considerations for in vivo
imaging of small animals. ILAR J. 2008; 49: 17–26.
29. Hua Z, Lv Q, Ye W, Wong CK, Cai G, Gu D, et al. MiRNA-directed regulation of
VEGF and other angiogenic factors under hypoxia. PLoS One. 2006;1: e116.
30.Roccaro AM, Sacco A, Thompson B, Leleu X, Azab AK, Azab F, et al.
MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma. Blood.
2009; 113: 6669-6680.
31. Sun CY, She XM, Qin Y, Chu ZB, Chen L, Ai LS, et al. miR-15a and miR-16
affect the angiogenesis of multiple myeloma by targeting VEGF. Carcinogenesis.
2013; 34: 426-35.
32.Takeshita F, Patrawala L, Osaki M, Takahashi RU, Yamamoto Y, Kosaka N et al.
Systemic delivery of synthetic microRNA-16 inhibits the growth of metastatic prostate tumors via downregulation of multiple cell-cycle genes. Mol Ther.
2010;18:181–187.
33.Liu HC, Pai SY, Cheng WT, Chen HL, Tsai TC, Yang SH, et al. Ingestion of milk containing the Dp2 peptide, a dust mite allergen, protects mice from allergic airway inflammation and hyper-responsiveness. Allergy Asthma Clin Immunol.
2013;9:21.
573 574
575 576
577 578 579
580 581 582
583 584 585 586
587 588 589 590
34.Chen HL, Huang JY, Chu TW, Tsai TC, Hung CM, Lin CC, et al. Expression of VP1 protein in the milk of transgenic mice: a potential oral vaccine protects against enterovirus 71 infection. Vaccine. 2008;26:2882–2889.
35.Yen CC, Lim CY, Chong KY, Tsai TC, Shen CJ, Lim MF, et al. Lactoferrin as a natural regimen for selective decontamination of the digestive tract: recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonates from pathogenic challenge in the gastrointestinal tract. J Infect Dis.
2009;199:590–598.
36.Yen CC, Lai YW, Chen HL, Lai CW, Lin CY, Chen W, et al. Aerosolized human extracellular superoxide dismutase prevents hyperoxia-induced lung injury. PLoS
One. 2011;6:e26870.
37.Lai CW, Chen HL, Lin KY, Liu FC, Chong KY, Cheng WT, et al. FTSJ2, a heat shock-inducible mitochondrial protein, suppresses cell invasion and migration.
PLoS One. 2014;9:e90818.
38.Tung YT, Chen HL, Lee CY, Chou YC, Lee PY, Tsai HC, et al. Active component of danshen (Salvia miltiorrhiza Bunge), tanshinone I, attenuates lung tumorigenesis via inhibitions of VEGF, Cyclin A, and Cyclin B expressions. Evid Based Complement Alternat Med. 2013;2013:319247.
591 592 593
594 595 596 597 598
599 600 601
602 603 604
605 606 607 608
39.Huang JT, Tseng CP, Liao MH, Lu SC, Yeh WZ, Sakamoto N, et al. Hepatitis C virus replication is modulated by the interaction of nonstructural protein NS5B and fatty acid synthase. J Virol. 2013;87:4994-5004.
40.Richardson M., Singh G. Observations on the use of the avian chorioallantoic membrane (CAM) model in investigations into angiogenesis. Curr Drug Targets-Cardiovasc Hematol Disord. 2003;3:155–185.
41.Wen ST, Chen W, Chen HL, Lai CW, Yen CC, Lee KH, et al. Amniotic fluid stem cells from EGFP transgenic mice attenuate hyperoxia-induced acute lung injury. PLoS One. 2013;8:e75383.
42.Chen HL, Yen CC, Wang SM, Tsai TC, Lai ZL, Sun JY, et al. Aerosolized bovine lactoferrin reduces lung injury and fibrosis in mice exposed to hyperoxia.
Biometals. 2014;27:1057–1068.
43.Cheng PH, Li CL, Chang YF, Tsai SJ, Lai YY, Chan AW, et al. miR-196a ameliorates phenotypes of Huntington disease in cell, transgenic mouse, and induced pluripotent stem cell models. Am J Hum Genet. 2013;93:306-312.
44.Fulzele SV, Chatterjee A, Shaik MS, Jackson T, Singh M. Inhalation delivery and
anti-tumor activity of celecoxib in human orthotopic non-small cell lung cancer
xenograft model. Pharm Res. 2006;23(9):2094-2106.
609 610 611
612 613 614
615 616 617
618 619 620
621 622 623
624 625 626
Table 1. Oligonucleotide primers used for transgene detection, RT-PCR and real-time PCR analyses
Primer set Oligonucleotide sequence Size (bp) Tm (C)
VEGF 94 (+) 5’-AAGGAGGAGGGCAGAATCATC-3’ 243 63
VEGF 315 (-) 5’-GAGGTTTGATCCGCATAATCTG-3’
cyclin B1 (+) 5’-CCAGAGGCGGATGAGAACA-3’ 101 57.8
cyclin B1 (-) 5’-ATGGAGGGTGGGTTGGAAAT-3’
cdc2 (+) 5’-CACCTGTGGCTGTCAGAGAA-3’ 153 57
cdc2 (-) 5’-GGGTAGAGTAAACTCCGTGA-3’
egfr (+) 5’-GCCATCTGGGTACGTTCAAT-3’ 128 58.5
egfr (-) 5’-GCAAGAGGGCAGAATCTGAG-3’
mmp9 (+) 5’-TGAATCAGCTGGCTTTTGTG-3’ 225 53
mmp9 (-) 5’-GTGGATAGCTCGGTGGTGTT-3’
brca1 (+) 5’-TTCAAGTGCCAGTGTCAAGG-3’ 160 56
brca1 (-) 5’-AGGGAGCAAAAGGGAAGAAA-3’
myc (+) 5’-CTGTCCATTCAAGCAGACGA-3’ 113 57.8
myc (-) 5’-TCCAGCTCCTCCTCGAGTTA-3’
nrp1 (+) 5’-AGCTTCAATGAGCGTCACCT-3’ 184 50
nrp1 (-) 5’-CCACCACAGGGTAAGGAGAA-3’
kdr (+) 5’-GGACCTCAGACTGCAAGGAG-3’ 202 57
kdr (-) 5’-TCTTGGAGGACAGAGCCACT-3’
β-actin (+) 5’-ACACCCGCCACCAGTTCGC-3’ 164 65
β-actin (-) 5’-ACCATTCCCACCATCACAC-3’
collagen α1 (+) 5′-CCAAGGGTAACAGTGGTGAA-3′ 149 57
collagen α1 (-) 5′-CCTCGTTTTCCTTCTTCTCCG-3′
α-SMA (+) 5′-GGCTCTGGGCTCTGTAAGG-3′ 124 57
α-SMA (-) 5′-CTCTTGCTCTGGGCTTCATC-3
TGF-β1 (+) 5′-GGTTCATGTCATGGATGGTGC-3′ 170 59
TGF-β1 (-) 5′-TGACGTCACTGGAGTTGTACGG-3′
TIMP1 (+) 5′-GCATCTCTGGCATCTGGCATC-3′ 214 61
TIMP1 (-) 5′-GCGGTTCTGGGACTTGTGGGC-3′
627 628 629
630
FIGURE LEGENDS
Figure 1: Schematic map of the hVEGF-A165 transgene construct and detection of its integration into the transgenic mouse genome. A. hVEGF-A165 overexpression construct. B. Verification of germline transmission and the transgenic patterns in the genomic DNA of the transgenic mice by Southern blot analysis. The Southern blot data showed that the foreign gene was found in similar patterns within the genomes of the transgenic founder mice and their offspring. C. The pulmonary Vegf-A165 mRNA expression level in the offspring of transgenic mice was determined by semi-quantitative RT-PCR. The data showed that the Vegf-A165 mRNA was expressed in the Tg 2 and Tg 3 offspring, but it was not expressed in the Tg 1 offspring. β-actin was used as an internal control. D. The Vegf-A165 mRNA expression in specific tissues was determined using semi-quantitative RT-PCR. RT-PCR showed that Vegf-A165 mRNA was expressed specifically in the lung tissue of transgenic mice. β-actin was used as an internal control.
Figure 2: The exterior and histopathologic staining and immunohistochemical (IHC) staining of lung tissues from wild-type and transgenic mice. A. Exterior and histopathologic section images of the lung tissues in the mice: pulmonary adenomas.
(A) and (B) present the exterior of the lungs from wild-type and transgenic mice, respectively. The arrows in (B) indicate adenomas. (C) The bronchia and alveoli of lung tissue from a wild-type mouse are shown. (D) The bronchia, alveoli and adenomas of lung tissue from a transgenic mouse are shown. (E) and (F) show the 631
2.5X magnification images of (C) and (D), respectively. (“b” indicates the bronchia).
B. Histopathologic section images of the lung tissues from the mice: fibrosis. (A) The bronchia and alveoli of the lung tissue in a wild-type mouse are shown. (B) The fibrosis (arrow) in the bronchia of the transgenic mice was due to bleeding. (C) and (D) are the 6X magnification images of (A) and (B), respectively (“b” indicates the bronchia). C. Histopathologic section images of lung tissues from the mice:
inflammation. (A) The normal alveoli in a wild-type mouse are shown. (B) Inflammation of the alveoli in a transgenic mouse is shown. (C) and (D) are the 6X magnification images of (A) and (B), respectively. D. Histopathologic section images of lung tissues from the mice: abnormal bronchial epithelium. (A) The normal bronchia in a wild-type mouse are shown. (B) The proliferation of the cells (arrow) on the bronchial epithelium of a transgenic mouse is shown. (C) The flattened bronchial epithelium (arrow) of a transgenic mouse is shown. (D) The cyst (arrow) on the bronchial epithelium of a transgenic mouse is shown. (“b” indicates the bronchia, “a”
indicates the alveoli). E. Detection of hVEGF-A165 expression in the lung tissue of mice by IHC staining is shown. F. Detection of the bronchia mCCSP in the lung tissue of mice using fluorescence microscopy is shown.
Figure 3: The exterior and histopathologic sections and neovascular and angiogenic activity of the lung tissues in the wild-type mice and in the three tumorigenesis levels of transgenic mice (Tg-level-1, Tg-level-2 and Tg-level-3). A.
Exterior and histopathologic section images of the lung tissues of the mice. B. Vegf-A165 protein expression in the lung tissues of mice was determined by western blot analysis; mean ± SD (n = 3). C. The neovasculature of the lung tissues of the mice was analyzed using an Angiogenesis Image Analyzer. D. The angiogenic activity of 654
the lung tissues of the mice was analyzed using chick chorioallantoic membrane (CAM).
Figure 4: The cDNA microarray data. A. Representative images of the lung tissues of wild-type and transgenic mice (Tg-level-3/wild type) after using two-color fluorescent probe hybridization. B. Clustar display of a mouse cDNA microarray of the lung tissues of wild-type and transgenic mice (Tg-level-3/wild type). The Cy5/Cy3 ratios of 580 cDNAs that deviated by more than 2-fold are shown. C. The ingenuity pathway analysis (IPA) of the mouse cDNA microarray data obtained from wild-type and Tg-level-3 mice.
Figure 5: The expression levels of mRNA and protein in the lung tissues of wild-type mice and of three different tumorigenesis levels of transgenic mice (Tg-level-1, Tg-level-2 and Tg-level-3). A. The quantitative mRNA expression levels of the hVegf-A165, cyclin b1 (cell cycle), cdc2 (cell cycle), egfr (cell proliferation), mmp9 (cell migration), brca1 (oncogenes), myc (oncogenes), nrp1 (co-receptor), and vegfr2-kdr (receptor) genes in the lung tissues of the mice were determined using qRT-PCR.
Means ± SDs (n = 3). *p < 0.05; **p < 0.01 vs. wild-type mice. B. The protein expression levels of hVEGF-A165, CYCLIN B1, CDC2, p-CDC2, BRCA-1, MYC, and GADPH in the lung tissues of the mice were determined by western blot analysis.
GADPH was used as an internal control.
Figure 6: The expression levels of CD105, mRNA, and protein in the lung tissues of wild-type mice and three different tumorigenesis levels of transgenic mice (Tg-679
level-1, Tg-level-2, and Tg-level-3). A. The CD105 level in the serum of the mice was determined using ELISA kit. **p < 0.01 vs. wild-type mice. B. The quantitative mRNA expression levels of the fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1) in the lung tissues of the mice were determined using qRT-PCR. Means ±
SD (n = 3). *p < 0.05; **p < 0.01 vs. wild-type mice. C. The inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lung tissues of the mice were determined by ELISA kit. *p < 0.05; **p < 0.01 vs. wild-type mice.
Figure 7: The MRI images of tumors (arrows) in the lung tissues of wild type mice and in two different tumorigenesis levels of transgenic mice (Tg-level-2 and Tg-level-3). Top panel: wild-type mouse. Middle panel: lower hVEGF-A165 -expressing transgenic mouse (Tg-level-2). Lower panel: higher hVEGF-A165 -expressing transgenic mouse (Tg-level-3). Red arrows indicate the solitary nodules of lung tumor.
Figure 8: MicroRNA-16 reduces pulmonary tumorigenesis in transgenic mice.
A. Histopathologic sections of lung tissues from Tg-level-3 mice are shown. B. The immunohistochemical (IHC) staining of the lung tissues of Tg-level-3 mice is shown.
C. The protein expression levels of hVEGF-A165 and GADPH in the lung tissues of Tg-level-3 mice were determined by western blot analysis. GADPH was used as an internal control. D. VEGF concentrations in the serum of Tg-level-3 mice were determined using ELISA. **p < 0.01 vs. Tg-level-3/Mock mice. E. The protein expression levels of cleavage caspase 3, cleavage caspase 8, cleavage caspase 9, cleavage PARP, and BCL2 in the lung tissues of Tg-level-3 mice were determined by 703
western blot analyses. β-actin was used as an internal control. *p < 0.05; **p < 0.01 vs. Tg-level-3/Mock mice.
Figure 9: MicroRNA-16 reduces pulmonary tumorigenesis in human orthotopic non-small cell lung cancer xenograft model. A. Exteriors, histopathologic sections and immunohistochemical (IHC) staining of lung tissue are shown. B. VEGF concentrations in the serum of the mice were determined using ELISA. ††p < 0.01 vs.
Healthy lungs/Saline mice. **p < 0.01 vs. Tumor-bearing lungs/Saline mice. C. The CD105 level in the serum of the mice was determined using ELISA kit. ††p < 0.01 vs.
Healthy lungs/Saline mice. *p < 0.05 vs. Tumor-bearing lungs/Saline mice.
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