Yun Zuo1, Yan Lv2, Xiaolan Qian1, Shaokai Wang1, Zhipen Chen1, Qin Jiang3, Cong Cao4,2,5, Yu Song1. 1. Department of Oncology, The First People Hospital of Zhangjiagang City, Soochow University, Suzhou, China. 2. Center of Translational Medicine, The First People Hospital of Zhangjiagang City, Jiangsu Key Laboratory of Neuropsychiatric Diseases, Soochow University, Suzhou, China. 3. The Affiliated Eye Hospital of Nanjing Medical University, Nanjing, China. 4. North District, The Municipal Hospital of Suzhou, Suzhou, China. 5. Jiangsu Key Laboratory of Neuropsychiatric Diseases and Institute of Neuroscience, Soochow University, Suzhou, China.
Abstract
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.
BACKGROUND/AIMS: Humanhedgehog-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 humangastric 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 humangastric cancer cell proliferation and migration.