Zuo Yun, Lv Yan, Qian Xiaolan, Wang Shaokai, Chen Zhipen, Jiang Qin, Cao Cong, Song Yu
Department of Oncology, The First People Hospital of Zhangjiagang City, Soochow University, Suzhou, China.
Center of Translational Medicine, The First People Hospital of Zhangjiagang City, Jiangsu Key Laboratory of Neuropsychiatric Diseases, Soochow University, Suzhou, China.
Cell Physiol Biochem. 2018;45(5):1840-1850. doi: 10.1159/000487875. Epub 2018 Feb 28.
BACKGROUND/AIMS: Human hedgehog-interacting protein (HHIP) is a negative regulator of the hedgehog (HH) signaling pathway. It is deregulated in gastric cancer. The underlying molecular mechanism of HHIP-induced inhibition of HH signaling remains to be determined.
A lentiviral HHIP expression vector ("LV-HHIP") was established to exogenously over-express HHIP in gastric cancer cells. HHIP protein and mRNA were tested by Western blotting assay and quantitative real-time PCR assay, respectively. Cell survival was tested by the Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was examined by the BrdU ELISA assay and [H3] Thymidine DNA incorporation assay. Cell invasion and migration were tested by the phagokinetic track assay and the "Transwell" assay. The bisulfite-sequencing PCR was applied to test HHIP promoter methylation.
In the established (AGS cell line) and primary human gastric cancer cells, LV-HHIP transfection increased HHIP expression and inhibited cancer cell survival and proliferation as well as cell migration and invasion. Furthermore, LV-HHIP significantly attenuated promoter methylation of the endogenous HHIP gene in AGS cells, causing it upregulation. Inhibition of methylation by 5-aza-dc similarly induced HHIP expression in gastric cancer cells, which inhibited cancer cell proliferation and migration.
Our results suggest that inhibition of HHIP promoter methylation can efficiently inhibit human gastric cancer cell proliferation and migration.
背景/目的：人刺猬因子相互作用蛋白（HHIP）是刺猬因子（HH）信号通路的负调节因子。它在胃癌中表达失调。HHIP诱导HH信号抑制的潜在分子机制仍有待确定。
构建慢病毒HHIP表达载体（“LV-HHIP”）以在胃癌细胞中外源过表达HHIP。分别通过蛋白质免疫印迹法和定量实时PCR法检测HHIP蛋白和mRNA。通过细胞计数试剂盒-8（CCK-8）法检测细胞活力。通过BrdU ELISA法和[H3]胸苷DNA掺入法检测细胞增殖。通过吞噬动力学轨迹法和“Transwell”法检测细胞侵袭和迁移。应用亚硫酸氢盐测序PCR检测HHIP启动子甲基化。
在已建立的（AGS细胞系）和原发性人胃癌细胞中，LV-HHIP转染增加了HHIP表达，并抑制了癌细胞的存活、增殖以及细胞迁移和侵袭。此外，LV-HHIP显著减弱了AGS细胞中内源性HHIP基因的启动子甲基化，导致其上调。5-氮杂-2'-脱氧胞苷抑制甲基化同样诱导了胃癌细胞中的HHIP表达，抑制了癌细胞的增殖和迁移。
我们的结果表明，抑制HHIP启动子甲基化可有效抑制人胃癌细胞的增殖和迁移。
