Mechanism Of MiR-365 In Metformin-mediated Apoptosis Of Gastric Cancer Cells

Objective: Metformin has been mainly used as a guanidine drug for the treatment of type 2 diabetes for more than thirty years.In recent years,the phenomenon that metformin can regulate cancer has been proposed.However,the molecular mechanism of metformin’s involvement in cancer cell regulation is not very clear,and it has a direct correlation with dysregulated microRNAs.Tumor cells are different from healthy cells in energy metabolism,and AMPK is not only related to energy conversion,but also important for tumor cell apoptosis.In short,we established the role of metformin and miR-365 / PTEN / AMPK signaling pathways in the apoptosis of gastric cancer cells,providing new ideas for the diagnosis and treatment of gastric cancer.Methods: Human gastric cancer cells AGS,MGC-803,and SGC-7901 in logarithmic growth phase were inoculated into culture plates,metformin at different concentrations(1,1.5,3,4.5,and 6 mM)were used for 24,48,72 hours,respectively,the same amount of sterile water culture medium was used as a control group.CCK-8and EdU kits were used to detect the proliferation of gastric cancer cells and calculate the IC50 value of metformin on gastric cancer cells 5mmol / L;5mmol / L metformin was used to intervene three gastric cancer cells at 24,48 and 72 hours,Promotes apoptosis protein(Caspase-3,BAX,and cytochrome C)and anti-apoptotic protein(BCL-2)were detected by Western Blot and calculate the ratio of BAX /BCL-2,flow cytometry was used to detect cell apoptosis;Metformin was used to detect the expression of miR-365 in cancer cells at different time and concentrations,and the effects of miR-365 mimics and inhibitors on apoptosis were measured,optimal concentration time of 5mmol / L for 48 hours was used for subsequent experiments;Biological methods to construct wild-type and mutant expression plasmids of PTEN and luciferase reporter gene plasmids pmirGLO-PTEN-WT and pmirGLO-PTEN-MUT,to detect the binding of miR-365 to PTEN;Effects of dosing and / or transfection of PTEN on AMPK phosphorylation expression and localization,flow cytometry was used to detect apoptosis,Western Blot was used to detect the expression of apoptosis-related proteins;Established mouse tumor models to detection of tumor changes after metformin and / or miR-365 in mice,tumor tissues with different treatments were taken for H & E staining,immunohistochemistry,and TUNEL staining,Western Blot was used to detect the expression of Caspase-3,BAX,and BCL-2.Results: Metformin inhibits the proliferation of gastric cancer cells and promotes apoptosis,while up-regulating the expression of miR-365.MiR-365 targets PTEN to inhibit its translation,limiting the enrichment of PTEN.PTEN can inhibit the phosphorylation expression of AMPK.Conversely,knocking down PTEN can activate the phosphorylation expression of AMPK,and metformin can directly activate the phosphorylation of AMPK,so that it forms a signal pathway loop,and induces the bi-directional apoptosis.Conclusion: These results indicate that metformin,miR-365,PTEN and AMPK forms a signal pathway,thereby regulating the expression of Phosphorylation of AMPK to promote the apoptosis of gastric cancer cells.These findings shed light on new pathways of apoptosis in gastric cancer and provide possible potential targets for the treatment of gastric cancer.