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Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma.

Hamamoto J, Soejima K, Naoki K, Yasuda H, Hayashi Y, Yoda S, Nakayama S, Satomi R, Terai H, Ikemura S, Sato T, Arai D, Ishioka K, Ohgino K, Betsuyaku T - Cancer Sci. (2014)

Bottom Line: Interestingly, we found that TMPRSS4 expression was associated with tissue factor pathway inhibitor 2 (TFPI-2) expression in these clinical samples.Knockdown of TMPRSS4 reduced the proliferation rate in several lung cancer cell lines.We found a novel molecular mechanism that TFPI-2 negatively regulates cell growth by inhibiting transcription of TMPRSS4.

View Article: PubMed Central - PubMed

Affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, Tokyo, Japan.

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TFPI-2 downregulates the expression of TMRPSS4 in lung cancer cells. (a) NCI-H358, NCI-H520, NCI-H1975, A549, and NCI-H2228 cells were transfected with GFP plasmid alone (lane 1) or with GFP-TFPI-2 (lane 2) for 48 h, and protein levels of TFPI-2 were detected with α-GFP antibody. (b) mRNA expression levels of TMPRSS4/GAPDH in the same experiments as (a) were detected with RT-PCR. Cells treated with GFP plasmid were calculated as 1 in (b). (c) Five cell lines were co-transfected with a GFP or GFP-TFPI-2 expression plasmid and a Luc or TMPRSS4-Luc reporter plasmid for 24 h together with an internal control RL-TK plasmid, and the effect on the reporter activity of TMPRSS4 by TFPI-2 was evaluated by the luciferase assay. (d, e) NCI-H358, NCI-H520, and NCI-H1975 cells were transfected with negative control or TFPI-2 siRNAs for 48 h, and mRNA expression levels of TFPI-2/GAPDH (d) and TMPRSS4/GAPDH (e) were evaluated by RT-PCR. Each value from cells treated with negative siRNA was calculated as 1. *P < 0.05 compared to GFP alone or to negative control siRNA. All experiments were carried out three times independently. Representative data are shown for Western blotting (a).
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fig03: TFPI-2 downregulates the expression of TMRPSS4 in lung cancer cells. (a) NCI-H358, NCI-H520, NCI-H1975, A549, and NCI-H2228 cells were transfected with GFP plasmid alone (lane 1) or with GFP-TFPI-2 (lane 2) for 48 h, and protein levels of TFPI-2 were detected with α-GFP antibody. (b) mRNA expression levels of TMPRSS4/GAPDH in the same experiments as (a) were detected with RT-PCR. Cells treated with GFP plasmid were calculated as 1 in (b). (c) Five cell lines were co-transfected with a GFP or GFP-TFPI-2 expression plasmid and a Luc or TMPRSS4-Luc reporter plasmid for 24 h together with an internal control RL-TK plasmid, and the effect on the reporter activity of TMPRSS4 by TFPI-2 was evaluated by the luciferase assay. (d, e) NCI-H358, NCI-H520, and NCI-H1975 cells were transfected with negative control or TFPI-2 siRNAs for 48 h, and mRNA expression levels of TFPI-2/GAPDH (d) and TMPRSS4/GAPDH (e) were evaluated by RT-PCR. Each value from cells treated with negative siRNA was calculated as 1. *P < 0.05 compared to GFP alone or to negative control siRNA. All experiments were carried out three times independently. Representative data are shown for Western blotting (a).

Mentions: Because TMPRSS4 is a serine protease and TFPI-2 encodes a broad-spectrum serine protease inhibitor, we speculated that there is some relationship between TMPRSS4 and TFPI-2. Although we first hypothesized that TFPI-2 inhibits the protease activity of TMPRSS4, surprisingly, we found that the mRNA expression level of TMPRSS4 was reduced when TFPI-2 was overexpressed by plasmid transfection in NCI-H358, NCI-H520, and NCI-H1975 cells (Fig.3a,b). We checked whether the reporter activity of endogenous TMPRSS4 is inhibited by the overexpression of TFPI-2. The reporter activity of the TMPRSS4 promoter region was partially inhibited by TFPI-2 in these three cell lines (Fig.3c). Conversely, the knockdown of TFPI-2 by siRNA induced an increase in TMPRSS4 mRNA expression (Fig.3d,e).


Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma.

Hamamoto J, Soejima K, Naoki K, Yasuda H, Hayashi Y, Yoda S, Nakayama S, Satomi R, Terai H, Ikemura S, Sato T, Arai D, Ishioka K, Ohgino K, Betsuyaku T - Cancer Sci. (2014)

TFPI-2 downregulates the expression of TMRPSS4 in lung cancer cells. (a) NCI-H358, NCI-H520, NCI-H1975, A549, and NCI-H2228 cells were transfected with GFP plasmid alone (lane 1) or with GFP-TFPI-2 (lane 2) for 48 h, and protein levels of TFPI-2 were detected with α-GFP antibody. (b) mRNA expression levels of TMPRSS4/GAPDH in the same experiments as (a) were detected with RT-PCR. Cells treated with GFP plasmid were calculated as 1 in (b). (c) Five cell lines were co-transfected with a GFP or GFP-TFPI-2 expression plasmid and a Luc or TMPRSS4-Luc reporter plasmid for 24 h together with an internal control RL-TK plasmid, and the effect on the reporter activity of TMPRSS4 by TFPI-2 was evaluated by the luciferase assay. (d, e) NCI-H358, NCI-H520, and NCI-H1975 cells were transfected with negative control or TFPI-2 siRNAs for 48 h, and mRNA expression levels of TFPI-2/GAPDH (d) and TMPRSS4/GAPDH (e) were evaluated by RT-PCR. Each value from cells treated with negative siRNA was calculated as 1. *P < 0.05 compared to GFP alone or to negative control siRNA. All experiments were carried out three times independently. Representative data are shown for Western blotting (a).
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fig03: TFPI-2 downregulates the expression of TMRPSS4 in lung cancer cells. (a) NCI-H358, NCI-H520, NCI-H1975, A549, and NCI-H2228 cells were transfected with GFP plasmid alone (lane 1) or with GFP-TFPI-2 (lane 2) for 48 h, and protein levels of TFPI-2 were detected with α-GFP antibody. (b) mRNA expression levels of TMPRSS4/GAPDH in the same experiments as (a) were detected with RT-PCR. Cells treated with GFP plasmid were calculated as 1 in (b). (c) Five cell lines were co-transfected with a GFP or GFP-TFPI-2 expression plasmid and a Luc or TMPRSS4-Luc reporter plasmid for 24 h together with an internal control RL-TK plasmid, and the effect on the reporter activity of TMPRSS4 by TFPI-2 was evaluated by the luciferase assay. (d, e) NCI-H358, NCI-H520, and NCI-H1975 cells were transfected with negative control or TFPI-2 siRNAs for 48 h, and mRNA expression levels of TFPI-2/GAPDH (d) and TMPRSS4/GAPDH (e) were evaluated by RT-PCR. Each value from cells treated with negative siRNA was calculated as 1. *P < 0.05 compared to GFP alone or to negative control siRNA. All experiments were carried out three times independently. Representative data are shown for Western blotting (a).
Mentions: Because TMPRSS4 is a serine protease and TFPI-2 encodes a broad-spectrum serine protease inhibitor, we speculated that there is some relationship between TMPRSS4 and TFPI-2. Although we first hypothesized that TFPI-2 inhibits the protease activity of TMPRSS4, surprisingly, we found that the mRNA expression level of TMPRSS4 was reduced when TFPI-2 was overexpressed by plasmid transfection in NCI-H358, NCI-H520, and NCI-H1975 cells (Fig.3a,b). We checked whether the reporter activity of endogenous TMPRSS4 is inhibited by the overexpression of TFPI-2. The reporter activity of the TMPRSS4 promoter region was partially inhibited by TFPI-2 in these three cell lines (Fig.3c). Conversely, the knockdown of TFPI-2 by siRNA induced an increase in TMPRSS4 mRNA expression (Fig.3d,e).

Bottom Line: Interestingly, we found that TMPRSS4 expression was associated with tissue factor pathway inhibitor 2 (TFPI-2) expression in these clinical samples.Knockdown of TMPRSS4 reduced the proliferation rate in several lung cancer cell lines.We found a novel molecular mechanism that TFPI-2 negatively regulates cell growth by inhibiting transcription of TMPRSS4.

View Article: PubMed Central - PubMed

Affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus