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Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16.

Tung YT, Huang PW, Chou YC, Lai CW, Wang HP, Ho HC, Yen CC, Tu CY, Tsai TC, Yeh DC, Wang JL, Chong KY, Chen CM - Oncotarget (2015)

Bottom Line: Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses.Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues.We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan.

ABSTRACT
Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A165 (hVEGF-A165) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A165 in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A165 mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A165 overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A165 in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A165-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A165 transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.

No MeSH data available.


Related in: MedlinePlus

MicroRNA-16 reduces pulmonary tumorigenesis in transgenic miceA. 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 western blot analyses. β-actin was used as an internal control. *p < 0.05; **p < 0.01 vs. Tg-level-3/Mock mice.
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Figure 8: MicroRNA-16 reduces pulmonary tumorigenesis in transgenic miceA. 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 western blot analyses. β-actin was used as an internal control. *p < 0.05; **p < 0.01 vs. Tg-level-3/Mock mice.

Mentions: The generated mouse model can be used to investigate the role of VEGF in pulmonary adenocarcinoma. Therefore, we further investigated the inhibitory effect of microRNA-16 (miR-16) on lung tumors (Figure 8) because recent reports have linked the expression of specific microRNAs with tumorigenesis and metastasis. After three intranasal administrations of 20 μg miR-16 or mock miR per mouse once a week, we found that miR-16, which lowered the expression of VEGF in both lung tissues (Figure 8B and 8C) and circulation blood (Figure 8D) (p < 0.01), affected the formation of lung tumors in > 12-month-old lung-specific hVEGF-A165 overexpressing transgenic mice (Figure 8A). Thus, miR-16 can inhibit lung cancer growth by suppressing VEGF expression. To investigate the molecular mechanisms underlying miR-16-induced apoptosis, apoptosis-related proteins were examined via western blot analyses. The results of the western blot analyses revealed that the cleaved forms of caspase 3, 8, 9, and poly (ADP-ribose) polymerase (PARP) were activated after miR-16 treatment in the hVEGF-A165 overexpressing transgenic mice (Figure 8E). Based on these results, miR-16 may induce apoptosis via both the intrinsic and extrinsic pathways.


Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16.

Tung YT, Huang PW, Chou YC, Lai CW, Wang HP, Ho HC, Yen CC, Tu CY, Tsai TC, Yeh DC, Wang JL, Chong KY, Chen CM - Oncotarget (2015)

MicroRNA-16 reduces pulmonary tumorigenesis in transgenic miceA. 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 western blot analyses. β-actin was used as an internal control. *p < 0.05; **p < 0.01 vs. Tg-level-3/Mock mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496351&req=5

Figure 8: MicroRNA-16 reduces pulmonary tumorigenesis in transgenic miceA. 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 western blot analyses. β-actin was used as an internal control. *p < 0.05; **p < 0.01 vs. Tg-level-3/Mock mice.
Mentions: The generated mouse model can be used to investigate the role of VEGF in pulmonary adenocarcinoma. Therefore, we further investigated the inhibitory effect of microRNA-16 (miR-16) on lung tumors (Figure 8) because recent reports have linked the expression of specific microRNAs with tumorigenesis and metastasis. After three intranasal administrations of 20 μg miR-16 or mock miR per mouse once a week, we found that miR-16, which lowered the expression of VEGF in both lung tissues (Figure 8B and 8C) and circulation blood (Figure 8D) (p < 0.01), affected the formation of lung tumors in > 12-month-old lung-specific hVEGF-A165 overexpressing transgenic mice (Figure 8A). Thus, miR-16 can inhibit lung cancer growth by suppressing VEGF expression. To investigate the molecular mechanisms underlying miR-16-induced apoptosis, apoptosis-related proteins were examined via western blot analyses. The results of the western blot analyses revealed that the cleaved forms of caspase 3, 8, 9, and poly (ADP-ribose) polymerase (PARP) were activated after miR-16 treatment in the hVEGF-A165 overexpressing transgenic mice (Figure 8E). Based on these results, miR-16 may induce apoptosis via both the intrinsic and extrinsic pathways.

Bottom Line: Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses.Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues.We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan.

ABSTRACT
Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A165 (hVEGF-A165) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A165 in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A165 mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A165 overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A165 in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A165-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A165 transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.

No MeSH data available.


Related in: MedlinePlus