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PTPRD demethylation regulates the proliferation, migration, and chemoresistance of gastric cancer cells through the PI3K/Akt/mTOR pathway
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Abstract:
[Abstract] Objective: To investigate the effect of protein tyrosine phosphatase D (PTPRD) demethylation on the proliferation, migration, and chemoresistance of gastric cancer (GC) cells through the phosphatidyl inositol 3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. Methods: The gastric cancer MKN-74 and MKN-45 cells, as well as human gastric mucosal epithelial GES-1 cells, GES-1 were cultured in vitro and PTPRD expression was detected. MKN-45 cells and their drug-resistant variant MKN-45/ 5-FU cells were routinely cultured and transfected with various vectors: PTPRD empty vector (NC group, NC/5-FU group), PTPRD overexpressing adenovirus (PTPRD group, PTPRD/5-FU group), shRNA empty vector (sh-NC group, sh-NC/5-FU group), shRNA PTPRD lentivirus (sh-PTPRD group, sh-PTPRD/5-FU group), and PTPRD overexpressing adenovirus + 10 μmol/L 740Y-P treatment (PTPRD + 740Y-P group, PTPRD + 740Y-P/5-FU group). MTT assay and wound healing assay were used to assess cell proliferation and migration. Cell autophagy levels were assessed using autophagy assay, and the expression of epithelial-mesenchymal transition (EMT) and PI3K/Akt/mTOR pathway related proteins was detected using western blot (WB). MKN-45 cells were treated with 0, 2.5, 5, 10, 20 and 40 μmol/L 5-aza solutions, and the PTPRD mRNA expression and cell proliferation in MKN-45 cells were detected using qPCR and MTT assays. Results: PTPRD mRNA and protein were significantly downregulated in gastric cancer cells (P < 0.05). Compared with the MKN-45 group, the numbers of autophagosomes and autophagosomes, as well as the protein expression of PTPRD, E-cadherin, and BAX significantly increased in the PTPRD group (all P < 0.05), while cell proliferation, migration rate, and protein expression of p-PI3K, vimentin, p-Akt, and p-mTOR decreased significantly (all P < 0.05); However, in the sh-PTPRD group, cell proliferation activity, migration rate, and protein expression of p-PI3K, vimentin, p-Akt, and p-mTOR increased notably, while the quantity of autophagosomes, autophagosomes, and protein expression of PTPRD, E-cadherin, and BAX decreased (all P < 0.05). Compared with the PTPRD group, the PTPRD + 740Y-P group showed an increase in cell proliferation activity, migration rate, protein expression of p-PI3K, vimentin, p-Akt, and p-mTOR (all P < 0.05), and a decrease in number of autophagosomes, autophagosomes, and protein expression of PTPRD, E-cadherin, and BAX (all P < 0.05). With the increase of 5-aza concentration, the mRNA expression of PTPRD in MKN-45 cells increased (P < 0.05), while the cell proliferation activity decreased (P < 0.05). Compared with the MKN-45/5-FU group, the cell migration rate and proliferation activity decreased in PTPRD/5-FU group, while the sh-PTPRD/5-FU group showed an increase in cell migration rate and proliferation activity (all P < 0.05). Compared with the PTPRD/5-FU group, the PTPRD + 740Y-P/5-FU group showed an increase in cell migration rate and proliferation activity (all P < 0.05). Conclusion: PTPRD is downregulated in GC cells, and its demethylation may inhibit proliferation and migration of GC cells and enhance chemosensitivity by suppressing the PI3K/Akt/mTOR pathway.
Keywords:
[Key words] protein tyrosine phosphatase D (PTPRD) ; gastric cancer (GC) ; proliferation ; migration ; PI3K/Akt/mTOR pathway ; chemotherapy resistance ; methylation
