Dickkopf3 过表达抑制体外胰腺癌细胞生长。

Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro.

机构信息

Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100730, China.

出版信息

World J Gastroenterol. 2011 Sep 7;17(33):3810-7. doi: 10.3748/wjg.v17.i33.3810.

DOI:10.3748/wjg.v17.i33.3810

PMID:21987623

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3181442/

Abstract

AIM

To elucidate the role of dickkopf3 (Dkk3) in human pancreatic cancer cell growth.

METHODS

Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blotting and immunofluorescence. Methylation of the Dkk3 promoter sequence was examined by methylation-specific polymerase chain reaction (MSP) and Dkk3 mRNA expression was determined by real-time RT-PCR after 5-aza-2'-deoxycytidine (5-aza-dC) treatment. The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells. The expression of β-catenin, phosphorylated extracellular signal-regulated protein kinases (pERK) and extracellular signal-regulated protein kinases (ERK) was also examined by real-time RT-PCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells.

RESULTS

The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested. The Dkk3 promoter sequence was methylated in the MIA PaCa-2 and AsPC-1 cell lines, which showed reduced Dkk3 expression. These two cell lines, which initially had a methylated Dkk3 promoter, showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent, 5-aza-dC, treatment (P < 0.05 or P < 0.01). When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa-2 cells, the ability of cells to proliferate decreased (P < 0.01), and the expression of β-catenin and pERK was downregulated (P < 0.01). Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmid-transfected cells.

CONCLUSION

Our findings, for the first time, implicate Dkk3 as a tumor suppressor in human pancreatic cancer, through the downregulation of β-catenin expression via the ERK-mediated pathway.

摘要

目的

阐明 dickkopf3（Dkk3）在人胰腺癌细胞生长中的作用。

方法

通过实时逆转录聚合酶链反应（real-time RT-PCR）、Western blot 和免疫荧光检测人胰腺癌细胞系中 Dkk3 mRNA 和蛋白的表达。通过甲基化特异性聚合酶链反应（MSP）检测 Dkk3 启动子序列的甲基化，并用 5-氮杂-2′-脱氧胞苷（5-aza-dC）处理后通过 real-time RT-PCR 测定 Dkk3 mRNA 的表达。通过将 Dkk3 表达质粒转染到人胰腺癌细胞中，用 CellTiter 96® AQueous One Solution Cell Proliferation Assay（MTS）研究 Dkk3 对癌细胞增殖和体外吉西他滨敏感性的影响。在人胰腺癌细胞中上调 Dkk3 表达后，通过 real-time RT-PCR 和 Western blot 检测β-catenin、磷酸化细胞外信号调节激酶（pERK）和细胞外信号调节激酶（ERK）的表达。

结果

结果表明，在所测试的所有胰腺癌细胞系中，Dkk3 mRNA 和蛋白的表达水平均较低。Dkk3 启动子序列在 MIA PaCa-2 和 AsPC-1 细胞系中被甲基化，导致 Dkk3 表达减少。这两个细胞系最初具有甲基化的 Dkk3 启动子，表现出依赖于 DNA 去甲基化剂 5-aza-dC 处理剂量和时间的增加的 Dkk3 mRNA 表达（P<0.05 或 P<0.01）。当用 Dkk3 表达质粒转染 MIA PaCa-2 细胞上调 Dkk3 表达时，细胞增殖能力下降（P<0.01），β-catenin 和 pERK 的表达下调（P<0.01）。转染 Dkk3 表达质粒的细胞对吉西他滨的敏感性增强。