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Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo.

Xu C, Gui Q, Chen W, Wu L, Sun W, Zhang N, Xu Q, Wang J, Fu X - J. Exp. Clin. Cancer Res. (2011)

Bottom Line: TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels.The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner.Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of General Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.

ABSTRACT

Background: The human coagulation trigger tissue factor (TF) is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi) technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo.

Methods: The specific small interfering RNA (siRNA) designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated.

Results: TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma.

Conclusions: Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

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Related in: MedlinePlus

Knockdown of TF with TF-siRNA attenuated the migration ability of lung adenocarcinoma cells in vitro. Representative images of the wound healing assay were shown (×40).
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Figure 7: Knockdown of TF with TF-siRNA attenuated the migration ability of lung adenocarcinoma cells in vitro. Representative images of the wound healing assay were shown (×40).

Mentions: Tumor cell migration and invasion are two critical steps in cancer metastatic process [23]. To verify the effect of TF-siRNA on the migration ability, A549 cells were tested by wound healing assay and the mobility assay. Figure 7 and Figure 8 show that the cells in 50 nM and 100 nM SiTF groups demonstrated an attenuated capacity of impaired migration, when compared to control and mock groups. Moreover, untreated and transfected cells were seeded on transwell chambers with uncoated filters. After incubation for 24 h, the motility potential of transfected cells at 50 nM and 100 nM TF-siRNA was significantly suppressed (Figure 9 and Figure 10). In addition, the invasion assay using Matrigel-coated Transwell chambers showed that 50 nM and 100 nM TF-siRNA transfected cells that passed through the Matrigel-coated membranes were much more than parental cells and the cells transfected with scrambled siRNA, and it indicated that the invasive capacity was markedly decreased (Figure 11 and Figure 12). These results suggested that TF-siRNA attenuated the metastatic potential of lung adenocarcinoma cells in vitro.


Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo.

Xu C, Gui Q, Chen W, Wu L, Sun W, Zhang N, Xu Q, Wang J, Fu X - J. Exp. Clin. Cancer Res. (2011)

Knockdown of TF with TF-siRNA attenuated the migration ability of lung adenocarcinoma cells in vitro. Representative images of the wound healing assay were shown (×40).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3118969&req=5

Figure 7: Knockdown of TF with TF-siRNA attenuated the migration ability of lung adenocarcinoma cells in vitro. Representative images of the wound healing assay were shown (×40).
Mentions: Tumor cell migration and invasion are two critical steps in cancer metastatic process [23]. To verify the effect of TF-siRNA on the migration ability, A549 cells were tested by wound healing assay and the mobility assay. Figure 7 and Figure 8 show that the cells in 50 nM and 100 nM SiTF groups demonstrated an attenuated capacity of impaired migration, when compared to control and mock groups. Moreover, untreated and transfected cells were seeded on transwell chambers with uncoated filters. After incubation for 24 h, the motility potential of transfected cells at 50 nM and 100 nM TF-siRNA was significantly suppressed (Figure 9 and Figure 10). In addition, the invasion assay using Matrigel-coated Transwell chambers showed that 50 nM and 100 nM TF-siRNA transfected cells that passed through the Matrigel-coated membranes were much more than parental cells and the cells transfected with scrambled siRNA, and it indicated that the invasive capacity was markedly decreased (Figure 11 and Figure 12). These results suggested that TF-siRNA attenuated the metastatic potential of lung adenocarcinoma cells in vitro.

Bottom Line: TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels.The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner.Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of General Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.

ABSTRACT

Background: The human coagulation trigger tissue factor (TF) is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi) technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo.

Methods: The specific small interfering RNA (siRNA) designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated.

Results: TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma.

Conclusions: Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

Show MeSH
Related in: MedlinePlus