MT1G inhibits the growth and epithelial-mesenchymal transition of gastric cancer cells by regulating the PI3K/AKT signaling pathway

Gastric carcinoma (GC) is a malignant tumor that has high mortality and morbidity worldwide. Although many efforts have been focused on the development and progression of GC, the underlying functional regulatory mechanism of GC needs more clarification. Metallothionein 1G (MT1G) is a member of the metallothionein family (MTs), and hypermethylation of MT1G occurred in a variety of cancers, including gastric cancer. However, the functional mechanism of MT1G in GC remains unclear. Here, we demonstrated that MT1G was down-regulated in GC tissues and cells. Overexpression of MT1G inhibited cell proliferation, foci formation and cell invasion, while knockdown of MT1G increased cell proliferation, foci formation and cell invasion. In addition, MT1G overexpression inhibited cell cycle progression and MT1G deficiency exerted opposite phenotype. p-AKT was negatively regulated by MT1G. In summary, our study reveals that MT1G exerts crucial role in regulating of cell proliferation and migration of gastric cancer, providing new insights for MT1G-related pathogenesis and a basis for developing new strategies for treatment of GC.

Keywords:
MT1G; GC; PI3K/AKT signaling pathway; cell growth; EMT

Authorship

Guofeng Xu

First Affiliated Hospital of Gannan Medical University, Department of Gastroenterology, Ganzhou City, Jiangxi Province, China.First Affiliated Hospital of Gannan Medical UniversityChinaGanzhou City, Jiangxi Province, ChinaFirst Affiliated Hospital of Gannan Medical University, Department of Gastroenterology, Ganzhou City, Jiangxi Province, China.

Linfeng Fan

First Affiliated Hospital of Gannan Medical University, Department of Gastrointestinal Surgery, Ganzhou City, Jiangxi Province, China.First Affiliated Hospital of Gannan Medical UniversityChinaGanzhou City, Jiangxi Province, ChinaFirst Affiliated Hospital of Gannan Medical University, Department of Gastrointestinal Surgery, Ganzhou City, Jiangxi Province, China.

Shufeng Zhao

Canhui OuYang

http://orcid.org/0000-0002-7451-195X

Send correspondence
Canhui OuYang. First Affiliated Hospital of Gannan Medical University, Department of Gastroenterology, No. 128 Jinling Road, Zhanggong District, Ganzhou City, Jiangxi Province, China. Email: oych@gmu.edu.cn.

Conflict of Interest

The authors declare that there is no conflict of interest that could be perceived as prejudicial to the impartiality of the reported research.

Authors Contributions

XGF and LF designed the study, supervised the data collection; FLF designed the study, supervised the data collection; ZHSHF analyzed the data, interpreted the data; OYCH prepare the manuscript for publication and reviewed the draft of the manuscript. All authors read and approved the final version.

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Figures

Figures (6)

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Figure 1 -
MT1G is low expression in gastric cancer tissues and cells. (A) The expression of MT1G in GC tissue and para-carcinoma normal tissue specimens was determined from TCGA analysis. (B&C) The expression of MT1G in GC cells was measured by RT-qPCR (B) and western blotting (C). Error bars represent data from three independent experiments (mean±SD). *P<0.05, **P<0.01.

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Figure 2 -
MT1G facilitates gastric cells growth. (A) NCI-N87 and HGC-27 cells were transfected with indicated plasmids, and the expression of MT1G was determined by western blotting. (B) The cell proliferation in MT1G overexpression or knockdown NCI-N87 and HGC-27 cells was tested by the CCK-8 assay. Error bars represent data from three independent experiments (mean±SD). **P<0.01, #P<0.05, ## P<0.01.

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Figure 3 -
MT1G inhibits cell cycle progression of GC cells. (A and B) Clonogenic assay was performed to measure the capacity of foci formation in knockdown or overexpression of MT1G NCI-N87 and HGC-27 cells. (C) The effects of MT1G overexpression, knockdown on cell-cycle progression in NCI-N87 and HGC-27 cells were determined by propidium iodide staining and flow cytometry analysis. Error bars represent data from three independent experiments (mean±SD). *P<0.05, **P<0.01, #P<0.05, ## P<0.01.

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Figure 4 -
MT1G negatively regulates cell migration and invasion. (A and B) Transwell assay and transwell matrigel assay were used to measure cell migration and invasion in MT1G overexpression or knockdown NCI-N87 and HGC-27 cells. (C) Western blotting analysis EMT related proteins expression in knockdown or overexpression of CKS2 NCI-H2170 cells. MT1G overexpression or knockdown was shown in NCI-N87 and HGC-27 cells. Error bars represent data from three independent experiments (mean±SD). **P<0.01, ## P<0.01.

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Figure 5 -
MT1G deficiency induces PI3K/AKT signaling in gastric cancer cell. (A-B) Gastric cancer cells (NCI-N87 and HGC-27) were divided into four groups based on its different treatment with plasmids transfection, including MT1G overexpression plasmids and related control plasmids, MT1G knockdown plasmids-shMT1G and matched negative control plasmids-shNC. The expression of AKT, p-AKT, PI3K, p-PI3K in MT1G overexpression or knockdown NCI-N87 (A) and HGC-27 (B) cells was measured by western blotting. Error bars represent data from three independent experiments (mean±SD). **P<0.01. ##P<0.01.

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Figure 6 -
MT1G deficiency induces PI3K/AKT signaling in gastric normal cell. Gastric normal cells (GES-1) were divided into four groups based on its different treatment with plasmids transfection, including MT1G overexpression plasmids and related control plasmids, MT1G knockdown plasmids-shMT1G and matched negative control plasmids-shNC. The expression of AKT, p-AKT, PI3K, p-PI3K in MT1G overexpression or knockdown GES-1 cells was measured by western blotting. Error bars represent data from three independent experiments (mean±SD). ＊＊P<0.01. ##P<0.01.

Figure 1 - MT1G is low expression in gastric cancer tissues and cells. (A) The expression of MT1G in GC tissue and para-carcinoma normal tissue specimens was determined from TCGA analysis. (B&C) The expression of MT1G in GC cells was measured by RT-qPCR (B) and western blotting (C). Error bars represent data from three independent experiments (mean±SD). *P<0.05, **P<0.01.

Figure 2 - MT1G facilitates gastric cells growth. (A) NCI-N87 and HGC-27 cells were transfected with indicated plasmids, and the expression of MT1G was determined by western blotting. (B) The cell proliferation in MT1G overexpression or knockdown NCI-N87 and HGC-27 cells was tested by the CCK-8 assay. Error bars represent data from three independent experiments (mean±SD). **P<0.01, #P<0.05, ## P<0.01.

Figure 3 - MT1G inhibits cell cycle progression of GC cells. (A and B) Clonogenic assay was performed to measure the capacity of foci formation in knockdown or overexpression of MT1G NCI-N87 and HGC-27 cells. (C) The effects of MT1G overexpression, knockdown on cell-cycle progression in NCI-N87 and HGC-27 cells were determined by propidium iodide staining and flow cytometry analysis. Error bars represent data from three independent experiments (mean±SD). *P<0.05, **P<0.01, #P<0.05, ## P<0.01.

Figure 4 - MT1G negatively regulates cell migration and invasion. (A and B) Transwell assay and transwell matrigel assay were used to measure cell migration and invasion in MT1G overexpression or knockdown NCI-N87 and HGC-27 cells. (C) Western blotting analysis EMT related proteins expression in knockdown or overexpression of CKS2 NCI-H2170 cells. MT1G overexpression or knockdown was shown in NCI-N87 and HGC-27 cells. Error bars represent data from three independent experiments (mean±SD). **P<0.01, ## P<0.01.

Figure 5 - MT1G deficiency induces PI3K/AKT signaling in gastric cancer cell. (A-B) Gastric cancer cells (NCI-N87 and HGC-27) were divided into four groups based on its different treatment with plasmids transfection, including MT1G overexpression plasmids and related control plasmids, MT1G knockdown plasmids-shMT1G and matched negative control plasmids-shNC. The expression of AKT, p-AKT, PI3K, p-PI3K in MT1G overexpression or knockdown NCI-N87 (A) and HGC-27 (B) cells was measured by western blotting. Error bars represent data from three independent experiments (mean±SD). **P<0.01. ##P<0.01.

Figure 6 - MT1G deficiency induces PI3K/AKT signaling in gastric normal cell. Gastric normal cells (GES-1) were divided into four groups based on its different treatment with plasmids transfection, including MT1G overexpression plasmids and related control plasmids, MT1G knockdown plasmids-shMT1G and matched negative control plasmids-shNC. The expression of AKT, p-AKT, PI3K, p-PI3K in MT1G overexpression or knockdown GES-1 cells was measured by western blotting. Error bars represent data from three independent experiments (mean±SD). ＊＊P<0.01. ##P<0.01.

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MT1G inhibits the growth and epithelial-mesenchymal transition of gastric cancer cells by regulating the PI3K/AKT signaling pathway

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[1] Send correspondence
Canhui OuYang. First Affiliated Hospital of Gannan Medical University, Department of Gastroenterology, No. 128 Jinling Road, Zhanggong District, Ganzhou City, Jiangxi Province, China. Email: oych@gmu.edu.cn.