Inhibition of HHIP Promoter Methylation Suppresses Human Gastric Cancer Cell Proliferation and Migration
Inhibition of HHIP Promoter Methylation Suppresses Human Gastric Cancer Cell Proliferation and Migration
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抑制 HHIP 启动子甲基化可抑制人胃癌细胞增殖和迁移。
DOI：
10.1159/000487875
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发表时间：
2018-01-01
影响因子：
--
通讯作者：
Song, Yu
中科院分区：
文献类型：
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作者：
Zuo, Yun;Lv, Yan;Song, Yu
背景/目的：人刺猬相互作用蛋白（HHIP）是刺猬（HH）信号通路的负调节因子。在胃癌中是失调的。HHIP诱导的HH信号传导抑制的潜在分子机制仍有待确定。方法：构建HHIP慢病毒表达载体（“LV-HHIP”），在胃癌细胞中外源性过表达HHIP。Western blotting检测HHIP蛋白表达，实时荧光定量PCR检测HHIP mRNA表达。通过细胞计数试剂盒-8（CCK-8）测定来测试细胞存活。用BrdU ELISA法和[H3] TdR掺入法检测细胞增殖情况。通过吞噬动力学示踪试验和“Transwell”试验检测细胞侵袭和迁移。亚硫酸氢盐测序PCR检测HHIP基因启动子甲基化。结果如下：在建立的（AGS细胞系）和原代人胃癌细胞中，LV-HHIP转染增加HHIP表达，抑制癌细胞存活和增殖以及细胞迁移和侵袭。此外，LV-HHIP显着减弱AGS细胞中内源性HHIP基因的启动子甲基化，导致其上调。5-aza-dc抑制甲基化类似地诱导胃癌细胞中HHIP表达，其抑制癌细胞增殖和迁移。结论：我们的结果表明，抑制HHIP启动子甲基化可以有效地抑制人胃癌细胞的增殖和迁移。
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. Methods: 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. Results: 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. Conclusions: Our results suggest that inhibition of HHIP promoter methylation can efficiently inhibit human gastric cancer cell proliferation and migration.