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. 2017 May 9;8(19):31288-31296.
doi: 10.18632/oncotarget.16126.

AMPKα Phosphatase Ppm1E Upregulation in Human Gastric Cancer Is Required for Cell Proliferation

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Free PMC article

AMPKα Phosphatase Ppm1E Upregulation in Human Gastric Cancer Is Required for Cell Proliferation

Min-Bin Chen et al. Oncotarget. .
Free PMC article

Abstract

Activation of AMP-activated protein kinase (AMPK) is a valuable anti-cancer strategy. In the current study, we tested expression and potential function of Ca2+/calmodulin-dependent protein kinase phosphatase (Ppm1E), an AMPKα phosphatase, in human gastric cancers. Ppm1E expression was elevated in human gastric cancer tissues (vs. normal tissues), which was correlated with AMPK (p-AMPKα, Thr-172) dephosphorylation and mTOR complex 1 (mTORC1) activation. Ppm1E upregulation, AMPK inhibition and mTORC1 activation were also observed in human gastric cancer cell lines (AGS, HGC-27, and SNU601). Intriguingly, Ppm1E knockdown by shRNA induced AMPK activation, mTORC1 inactivation, and proliferation inhibition in AGS cells. On the other hand, forced over-expression of Ppm1E induced further AMPK inhibition and mTORC1 activation to enhance AGS cell proliferation. Remarkably, microRNA-135b-5p ("miR-135b-5p"), an anti-Ppm1E microRNA, was downregulated in both human gastric cancer tissues and cells. Reversely, miR-135b-5p exogenous expression caused Ppm1E depletion, AMPK activation, and AGC cell proliferation inhibition. Together, Ppm1E upregulation in human gastric cancer is important for cell proliferation, possible via regulating AMPK-mTOR signaling.

Keywords: AMPKα; Ppm1E; gastric cancer; mTOR; miR-135b-5p.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicts of interests.

Figures

Figure 1
Figure 1. Ppm1E upregulation in human gastric cancer tissues
The fresh human gastric cancer tissues (“Tumor”, n = 12) and the surrounding normal gastric tissues (“Normal”) were lysed; Expressions of Ppm1E mRNA (A, qRT-PCR assay) and listed proteins (B, Western blotting assay, Data were quantified) were tested. *p < 0.05 vs. “Normal”.
Figure 2
Figure 2. Ppm1E upregulation in human gastric cancer cells
Human gastric cancer cell lines (AGS, HGC-27, and SNU601) and the gastric mucosal epithelial cell line GES-1 were subjected to qRT-PCR assay (A) and Western blotting assay (B) to test listed genes. Western blot data were quantified. *p < 0.05 vs. GES-1 cells. Experiments in this figure were repeated three times, and similar results were obtained.
Figure 3
Figure 3. Ppm1E silence induces AMPK activation and inhibits gastric cancer cell survival and proliferation
Expressions of Ppm1E mRNA (A) and listed proteins (B) in AGS cells with Ppm1E shRNA (“1#” or “2#”) or scramble control shRNA (“shRNA-C”) were shown. Above cells were also subjected to MTT assay (C), colony formation assay (D), BrdU ELISA assay (E) and DNA [H3] thymidine DNA incorporation assay (F) to test cell survival and proliferation. For these assays, exact same number of viable cells with listed shRNA was initially plated. *p < 0.05 vs. “shRNA-C” cells. Experiments in this figure were repeated four times, and similar results were obtained.
Figure 4
Figure 4. Exogenous Ppm1E over-expression promotes gastric cancer cell survival and proliferation
Expressions of Ppm1E mRNA (A) and listed proteins (B, Data were quantified in the Lower panel) in stable AGS cell lines with exogenous Ppm1E (“Line1” and “Line2”, Flag-tagged) or empty vector (“Vector”) were shown. Above cells were also subjected to MTT assay (C) and BrdU ELISA assay (D) to test cell survival and proliferation, respectively. For these assays, exact same number of viable cells with listed shRNA was initially plated. *p < 0.05 vs. “Vector” cells. Experiments in this figure were repeated three times, and similar results were obtained.
Figure 5
Figure 5. Exogenous expression of miR-135b-5p leads to Ppm1E depletion, AMPK activation, and proliferation inhibition in AGC cells
The fresh human gastric cancer tissues (“Tumor”, n = 12) and the surrounding normal gastric tissues (“Normal”, n = 12), as well as gastric cancer cells (AGS, HGC-27, and SNU601) or GES-1 epithelial cells were subjected to qRT-PCR assay of microRNA-135b-5p (“miR-135b-5p”) expression (A and B). AGC cells, expressing miR-135b-vector or the miR-control vector (“miR-Ctrl”) were subjected to qRT-PCR assay testing expression of miR-135b-5p (C) and Ppm1E mRNA (D) Listed proteins were also tested by Western blotting assay, and blot data were quantiifed (E) Cells were also subjected to MTT assay (F) and BrdU ELISA assay (G). For these assays, exact same number of viable cells with listed vector was initially plated. *p < 0.05 vs. “Normal” tissues (A) or GES-1 cells (B). *p < 0.05 vs. “miR-Ctrl” cells (C–G). Experiments in this figure were repeated three times, and similar results were obtained.

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