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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

Efficient delivery of siRNA into lung adenocarcinoma cells. (A): Detection of transfection efficiency by flow cytometry. Transfection efficiency was maintained at over 85% for 6 h post-transfection. (B): Detection of transfection efficiency by fluorescence microscopy. High efficiency of transfection with fluorescent siRNA (green) in A549 cells were easily identified for 48 h post-transfection (×100).
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Figure 1: Efficient delivery of siRNA into lung adenocarcinoma cells. (A): Detection of transfection efficiency by flow cytometry. Transfection efficiency was maintained at over 85% for 6 h post-transfection. (B): Detection of transfection efficiency by fluorescence microscopy. High efficiency of transfection with fluorescent siRNA (green) in A549 cells were easily identified for 48 h post-transfection (×100).

Mentions: To make sure the transfection efficiency of siRNA in A549 cells, uptake of fluorescently labeled scrambled siRNAs (25 nM, 50 nM and 100 nM) was detected by flow cytometry and fluorescence microscopy after 6 h and 48 h post-transfection. It showed a high-efficiency transfection that more than 85% cells displayed green fluorescence with 100 nM fluorescent siRNA (Figure 1). When cells were treated with TF-targeting siRNA (25 nM, 50 nM and 100 nM SiTF) and the scramble siRNA (Mock, 100 nM) for 48 h, the mRNA and protein expressions of TF were examined by RT- PCR and Western blot. As shown in Figure 2 and Figure 3, the Mock did not affect the expression levels of TF, but in 25 nM, 50 nM and 100 nM SiTF groups, compared with mock, the TF expression decreased at both protein and mRNA levels. Specially, 100 nM SiTF indicated a 80-85% reduction of TF expression in A549 cells. These results demonstrated that the TF-targeting siRNA was efficient to knock down the expression of TF in A549 cells.


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)

Efficient delivery of siRNA into lung adenocarcinoma cells. (A): Detection of transfection efficiency by flow cytometry. Transfection efficiency was maintained at over 85% for 6 h post-transfection. (B): Detection of transfection efficiency by fluorescence microscopy. High efficiency of transfection with fluorescent siRNA (green) in A549 cells were easily identified for 48 h post-transfection (×100).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Efficient delivery of siRNA into lung adenocarcinoma cells. (A): Detection of transfection efficiency by flow cytometry. Transfection efficiency was maintained at over 85% for 6 h post-transfection. (B): Detection of transfection efficiency by fluorescence microscopy. High efficiency of transfection with fluorescent siRNA (green) in A549 cells were easily identified for 48 h post-transfection (×100).
Mentions: To make sure the transfection efficiency of siRNA in A549 cells, uptake of fluorescently labeled scrambled siRNAs (25 nM, 50 nM and 100 nM) was detected by flow cytometry and fluorescence microscopy after 6 h and 48 h post-transfection. It showed a high-efficiency transfection that more than 85% cells displayed green fluorescence with 100 nM fluorescent siRNA (Figure 1). When cells were treated with TF-targeting siRNA (25 nM, 50 nM and 100 nM SiTF) and the scramble siRNA (Mock, 100 nM) for 48 h, the mRNA and protein expressions of TF were examined by RT- PCR and Western blot. As shown in Figure 2 and Figure 3, the Mock did not affect the expression levels of TF, but in 25 nM, 50 nM and 100 nM SiTF groups, compared with mock, the TF expression decreased at both protein and mRNA levels. Specially, 100 nM SiTF indicated a 80-85% reduction of TF expression in A549 cells. These results demonstrated that the TF-targeting siRNA was efficient to knock down the expression of TF in A549 cells.

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