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NRD1, which encodes nardilysin protein, promotes esophageal cancer cell invasion through induction of MMP2 and MMP3 expression.

Uraoka N, Oue N, Sakamoto N, Sentani K, Oo HZ, Naito Y, Noguchi T, Yasui W - Cancer Sci. (2013)

Bottom Line: Furthermore, nardilysin expression was significantly associated with poorer prognosis (P = 0.0258).The invasiveness of NRD1-knockdown TE1 and TE5 esophageal cancer cell lines was less than that of the negative control siRNA-transfected cell lines.Expression of MMP2 and MMP3 mRNA was significantly lower in NRD1-knockdown TE5 cells than in negative control siRNA-transfected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.

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Functional analysis of nardilysin in esophageal cancer cell lines. (a) Western blot analysis of nardilysin in six esophageal cancer cell lines. (b) Western blot analysis of nardilysin in cell lysates from TE1 and TE5 cells transfected with NRD1 siRNA (siRNA1–3) and negative control siRNA. (c) Effect of NRD1 knockdown on cell invasion of TE1. TE1 cells transfected with NRD1 siRNA1 and negative control siRNA were incubated in Boyden chambers. After 1 and 2 days, invading cells were counted. Bars and error bars indicate mean and SE of three different experiments. (d) Wound healing assay in NRD1 knockdown TE1 cells and negative control siRNA-transfected TE1 cells. TE1 cells transfected with NRD1 siRNA1 or negative control siRNA were wounded. (e) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of MMP2 and MMP3 mRNA in TE1 and TE5 cells transfected with NRD1 siRNA1 or negative control siRNA. Bars and error bars represent mean and standard error (SE) of three different experiments. N.S., not significant. *P = 0.0244; **P = 0.0200; ***P < 0.0200.
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fig03: Functional analysis of nardilysin in esophageal cancer cell lines. (a) Western blot analysis of nardilysin in six esophageal cancer cell lines. (b) Western blot analysis of nardilysin in cell lysates from TE1 and TE5 cells transfected with NRD1 siRNA (siRNA1–3) and negative control siRNA. (c) Effect of NRD1 knockdown on cell invasion of TE1. TE1 cells transfected with NRD1 siRNA1 and negative control siRNA were incubated in Boyden chambers. After 1 and 2 days, invading cells were counted. Bars and error bars indicate mean and SE of three different experiments. (d) Wound healing assay in NRD1 knockdown TE1 cells and negative control siRNA-transfected TE1 cells. TE1 cells transfected with NRD1 siRNA1 or negative control siRNA were wounded. (e) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of MMP2 and MMP3 mRNA in TE1 and TE5 cells transfected with NRD1 siRNA1 or negative control siRNA. Bars and error bars represent mean and standard error (SE) of three different experiments. N.S., not significant. *P = 0.0244; **P = 0.0200; ***P < 0.0200.

Mentions: We showed that high levels of NRD1 mRNA expression were correlated with T classification, N classification and tumor stage in ESCC tissues. Furthermore, nardilysin protein expression was correlated with patients’ prognosis. Therefore, we studied the biological significance of NRD1 using esophageal cancer cell lines. Western blot analysis showed that all six esophageal cancer cell lines expressed nardilysin at various levels (Fig.3a). NRD1 mRNA expression and nardilysin protein expression were well correlated. The highest nardilysin expression was detected in TE1 cells, and the other five remaining cell lines had moderate or low nardilysin expression. Next, we examined the transition of nardilysin expression by Western blot analysis of protein extracts of TE1 and TE5 cell lines transfected with NRD1 specific siRNAs because the highest nardilysin expression was detected in TE1 cells, and moderate nardilysin expression was detected in TE5 cells. Three different siRNAs (siRNA1, 2, and 3) were transfected into TE1 and TE5 (Fig.3b). The expression of nardilysin protein in TE1 was most suppressed by treatment with siRNA1. Similar results were observed in TE5 cells. Thus, to knockdown the endogenous NRD1, we used siRNA1 in the following experiments.


NRD1, which encodes nardilysin protein, promotes esophageal cancer cell invasion through induction of MMP2 and MMP3 expression.

Uraoka N, Oue N, Sakamoto N, Sentani K, Oo HZ, Naito Y, Noguchi T, Yasui W - Cancer Sci. (2013)

Functional analysis of nardilysin in esophageal cancer cell lines. (a) Western blot analysis of nardilysin in six esophageal cancer cell lines. (b) Western blot analysis of nardilysin in cell lysates from TE1 and TE5 cells transfected with NRD1 siRNA (siRNA1–3) and negative control siRNA. (c) Effect of NRD1 knockdown on cell invasion of TE1. TE1 cells transfected with NRD1 siRNA1 and negative control siRNA were incubated in Boyden chambers. After 1 and 2 days, invading cells were counted. Bars and error bars indicate mean and SE of three different experiments. (d) Wound healing assay in NRD1 knockdown TE1 cells and negative control siRNA-transfected TE1 cells. TE1 cells transfected with NRD1 siRNA1 or negative control siRNA were wounded. (e) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of MMP2 and MMP3 mRNA in TE1 and TE5 cells transfected with NRD1 siRNA1 or negative control siRNA. Bars and error bars represent mean and standard error (SE) of three different experiments. N.S., not significant. *P = 0.0244; **P = 0.0200; ***P < 0.0200.
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fig03: Functional analysis of nardilysin in esophageal cancer cell lines. (a) Western blot analysis of nardilysin in six esophageal cancer cell lines. (b) Western blot analysis of nardilysin in cell lysates from TE1 and TE5 cells transfected with NRD1 siRNA (siRNA1–3) and negative control siRNA. (c) Effect of NRD1 knockdown on cell invasion of TE1. TE1 cells transfected with NRD1 siRNA1 and negative control siRNA were incubated in Boyden chambers. After 1 and 2 days, invading cells were counted. Bars and error bars indicate mean and SE of three different experiments. (d) Wound healing assay in NRD1 knockdown TE1 cells and negative control siRNA-transfected TE1 cells. TE1 cells transfected with NRD1 siRNA1 or negative control siRNA were wounded. (e) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of MMP2 and MMP3 mRNA in TE1 and TE5 cells transfected with NRD1 siRNA1 or negative control siRNA. Bars and error bars represent mean and standard error (SE) of three different experiments. N.S., not significant. *P = 0.0244; **P = 0.0200; ***P < 0.0200.
Mentions: We showed that high levels of NRD1 mRNA expression were correlated with T classification, N classification and tumor stage in ESCC tissues. Furthermore, nardilysin protein expression was correlated with patients’ prognosis. Therefore, we studied the biological significance of NRD1 using esophageal cancer cell lines. Western blot analysis showed that all six esophageal cancer cell lines expressed nardilysin at various levels (Fig.3a). NRD1 mRNA expression and nardilysin protein expression were well correlated. The highest nardilysin expression was detected in TE1 cells, and the other five remaining cell lines had moderate or low nardilysin expression. Next, we examined the transition of nardilysin expression by Western blot analysis of protein extracts of TE1 and TE5 cell lines transfected with NRD1 specific siRNAs because the highest nardilysin expression was detected in TE1 cells, and moderate nardilysin expression was detected in TE5 cells. Three different siRNAs (siRNA1, 2, and 3) were transfected into TE1 and TE5 (Fig.3b). The expression of nardilysin protein in TE1 was most suppressed by treatment with siRNA1. Similar results were observed in TE5 cells. Thus, to knockdown the endogenous NRD1, we used siRNA1 in the following experiments.

Bottom Line: Furthermore, nardilysin expression was significantly associated with poorer prognosis (P = 0.0258).The invasiveness of NRD1-knockdown TE1 and TE5 esophageal cancer cell lines was less than that of the negative control siRNA-transfected cell lines.Expression of MMP2 and MMP3 mRNA was significantly lower in NRD1-knockdown TE5 cells than in negative control siRNA-transfected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.

Show MeSH
Related in: MedlinePlus