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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

Western blot analysis of PI3K/Akt by silencing TF by siRNA in lung adenocacinoma cells in vitro. Representative images were shown and bar represented that the protein relative expression levels of PI3K in 100 nM SiTF group and phosphorylated Akt (P-AKT) in 25 nM, 50 nM and 100 nM SiTF groups were decreased. *P < 0.05, **P < 0.01 versus mock.
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Figure 15: Western blot analysis of PI3K/Akt by silencing TF by siRNA in lung adenocacinoma cells in vitro. Representative images were shown and bar represented that the protein relative expression levels of PI3K in 100 nM SiTF group and phosphorylated Akt (P-AKT) in 25 nM, 50 nM and 100 nM SiTF groups were decreased. *P < 0.05, **P < 0.01 versus mock.

Mentions: The protein from transfected cells was extracted to examine the effects of TF-siRNA on some important cytokines and signaling molecules. After 48 h of transfection, the protein relative expression levels of phosphorylated Erk1/2 and PI3K in 100 nM SiTF group and phosphorylated Akt in 25 nM, 50 nM and 100 nM SiTF groups were decreased, while that in control and mock groups had no differences (Figure 14 and Figure 15). Furthermore, compared to control and mock groups, transfection with high concentrations of 50 nM and 100 nM TF-siRNA suppressed the MMP-9/-2 expression (Figure 16), and the protein expression of VEGF of 100 nM SiTF group was decreased (Figure 17). These data demonstrated that knockdown of TF by siRNA may inhibit Erk1/2 MAPK, PI3K/Akt signaling pathway, MMP-9/-2 and VEGF, which all play an important role in tumor progress.


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)

Western blot analysis of PI3K/Akt by silencing TF by siRNA in lung adenocacinoma cells in vitro. Representative images were shown and bar represented that the protein relative expression levels of PI3K in 100 nM SiTF group and phosphorylated Akt (P-AKT) in 25 nM, 50 nM and 100 nM SiTF groups were decreased. *P < 0.05, **P < 0.01 versus mock.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 15: Western blot analysis of PI3K/Akt by silencing TF by siRNA in lung adenocacinoma cells in vitro. Representative images were shown and bar represented that the protein relative expression levels of PI3K in 100 nM SiTF group and phosphorylated Akt (P-AKT) in 25 nM, 50 nM and 100 nM SiTF groups were decreased. *P < 0.05, **P < 0.01 versus mock.
Mentions: The protein from transfected cells was extracted to examine the effects of TF-siRNA on some important cytokines and signaling molecules. After 48 h of transfection, the protein relative expression levels of phosphorylated Erk1/2 and PI3K in 100 nM SiTF group and phosphorylated Akt in 25 nM, 50 nM and 100 nM SiTF groups were decreased, while that in control and mock groups had no differences (Figure 14 and Figure 15). Furthermore, compared to control and mock groups, transfection with high concentrations of 50 nM and 100 nM TF-siRNA suppressed the MMP-9/-2 expression (Figure 16), and the protein expression of VEGF of 100 nM SiTF group was decreased (Figure 17). These data demonstrated that knockdown of TF by siRNA may inhibit Erk1/2 MAPK, PI3K/Akt signaling pathway, MMP-9/-2 and VEGF, which all play an important role in tumor progress.

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