Anat Cell Biol.  2012 Mar;45(1):17-25. 10.5115/acb.2012.45.1.17.

Expression and roles of NUPR1 in cholangiocarcinoma cells

• Kim KS
• Jin DI
• Yoon S
• Baek SY
• Kim BS
• Oh SO

• KMID: 1447449
• DOI: http://doi.org/10.5115/acb.2012.45.1.17

Abstract

Nuclear protein-1 (NUPR1) is a small nuclear protein that is responsive to various stress stimuli. Although NUPR1 has been associated with cancer development, its expression and roles in cholangiocarcinoma have not yet been described. In the present study, we found that NUPR1 was over-expressed in human cholangiocarcinoma tissues, using immunohistochemistry. The role of NUPR1 in cholangiocarcinoma was examined by its specific siRNA. NUPR1 siRNA decreased proliferation, migration and invasion of human cholangiocarcinoma cell lines (HuCCT1 and SNU1196 cells). From these results, we conclude that NUPR1 is over-expressed in cholangiocarcinoma and regulates the proliferation and motility of cancer cells.

Keyword

NUPR1; Cholangiocarcinoma; Migration; Invasion; Proliferation

MeSH Terms

Cell Line
Cholangiocarcinoma
Humans
Immunohistochemistry
Nuclear Proteins
RNA, Small Interfering
Nuclear Proteins
RNA, Small Interfering

Figure

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  Fig. 1 Nuclear protein-1 (NUPR1) was over-expressed in cholangiocarcinoma. Immunohistochemistry was carried out using cholangiocarcinoma patient tissues (n=10) to examine the expression of NUPR1. Representative stainings from normal (A) or cancerous areas (B-D) are presented. Hematoxyin and eosin staining was used for counterstaining. Scale bars=50 µm (A-D).

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  Fig. 2 Nuclear protein-1 (NUPR1) siRNA specifically down-regulated NUPR1 expression in HuCCT1 cholangiocarcinoma cells. The mRNA level of NUPR1 was reduced by siRNA (A). The protein level of NUPR1 was also reduced by siRNA (B). Protein and RNA were extracted two days after transfection with indicated concentrations of the siRNA. Three independent experiments were repeated. β-actin and α-tubulin were used for normalization of RNA and protein loading respectively. SCR, scrambled.

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  Fig. 3 Effects of knock-down of nuclear protein-1 (NUPR1) on proliferation of HuCCT1 cholangiocarcinoma cells. Cell proliferation was measured three and seven days after the siRNA transfection using WST-1 assay. In the presence of 10% (A) or 1% fetal bovine serum (FBS) (B), proliferation was examined. Data show the means±SDs of three independent experiments performed in quintuplicate (*P<0.05). SCR, scrambled.

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  Fig. 4 Nuclear protein-1 (NUPR1) affected epidermal growth factor (EGF)- or serum-induced migration of HuCCT1 cholangiocarcinoma cells. To examine effects on migration, Boyden chamber assays were performed. Two days after siRNA transfection, 5×104 cells were seeded into Boyden chambers. Addition of 100 nM EGF or 10% fetal bovine serum (FBS) into lower but not upper Boyden chambers induced migration of the cells. To rule out effects of proliferation, mitomycin C (5 µg/ml) was added. Four hours later, cells were fixed, stained with Diff-quik solution and pictured. The representative staining of migrated cells is presented (A). Scale bar=50 µm. (B) Migrated cells were counted and the data are presented as a graph. Data show the means±SDs of three independent experiments performed in triplicate (*P<0.05). SCR, scrambled.

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  Fig. 5 Nuclear protein-1 (NUPR1) affected epidermal growth factor (EGF)- or serum-induced migration of HuCCT1 cholangiocarcinoma cells. To examine the effects on migration, wound healing assays were performed. To rule out effects of proliferation, mitomycin C (5 µg/ml) was added. A day after scratch with a yellow tip, migration was documented. Three independent experiments were carried out. FBS, fetal bovine serum; SCR, scrambled.

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  Fig. 6 Nuclear protein-1 (NUPR1) affected epidermal growth factor (EGF)- or serum-induced invasion of HuCCT1 cholangiocarcinoma cells. To examine the effects on invasion, matrigel invasion assays were performed. Two days after siRNA transfection, 5×104 cells were seeded into matrigel-coated transwells. To rule out effects of proliferation, mitomycin C (5 µg/ml) was added. Eighteen hours later, the cells were fixed, stained with Diff-quik solution, and pictured. The representative staining of invaded cells is presented (A). Scale bar=50 µm. (B) Invaded cells were counted and the data are presented as a graph. Data show means±SDs of three independent experiments performed in triplicate (*P<0.01). FBS, fetal bovine serum; SCR, scrambled.

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