microRNA-218 promotes sensitivity of human pancreatic cancer cells to gemcitabine through HMGB1 and the PI3K/Akt pathway

Objective: The purpose of the present study was to investigate the mechanisms and signaling pathways by which microRNA-218 (miR-218) regulates the chemosensitivity of pancreatic cancer cells to gemcitabine (GEM), to explore the mechanisms underlying the resistance of pancreatic cancer cells to GEM and to provide novel approaches and strategies for treatment of pancreatic cancer. Methods: MiR-218 was overexpressed in GEM-resistant PANC-1 cells through transfection of the cells with a miR-218 mimic. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the changes in miR-218 expression in PANC-1 cells. The Cell Counting Kit-8 (CCK-8) was used to examine the effect of miR-218 on the viability of GEM-induced PANC-1 pancreatic cancer cells. An enzyme-linked immunosorbent assay (ELISA) was conducted to investigate effect of miR-218 and GEM on the secretion of high mobility group box l (HMGB1) protein by PANC-1 cells. Western blot analysis was performed to analyze the effect of miR-218 on the expression of HMGB1 and beclin 1 in PANC-1 cells. In addition, beclin 1 expression was knocked down in PANC-1 pancreatic cancer cells using RNA interference (RNAi) technology. After knockdown of beclin 1, the CCK-8 assay was performed to examine the viability of GEM-induced PANC-1 cells. Finally, the effects of miR-218 overexpression on the expression of AKT and phospho-AKT (p-AKT) in GEM-induced PANC-1 cells as well as on cell viability were examined using the phosphatidylinositol-3-kinase (PI3K) pathway inhibitor, wortmannin, with western blot analysis and the CCK-8 assay. Results: The qRT-PCR results showed that compared with the control group, miR-218 expression was significantly increased in PANC-1 cells at 48 h after transfection with the miR-218 mimic (P<0.01). The CCK-8 assay results showed that the viability of PANC-1 cells was markedly reduced after transfection with the miR-218 mimic and treatment with 5 mu M GEM (miR-218 mimic+GEM group) compared with the mimic ctrl+GEM group and the normal control group (P<0.01). The ELISA results showed that GEM induced the secretion of HMGB1 by PANC-1 cells (P<0.01), whereas miR-218 and quercetin inhibited the secretion of HMGB1 by GEM-induced PANC-1 cells (P<0.01). The western blot analysis showed that miR-218 inhibited the expression of HMGB1 and beclin 1 in PANC-1 cells (P<0.01). Transfection of PANC-1 cells with beclin 1 shRNA effectively reduced the expression of beclin 1 in PANC-1 cells (P<0.01). Knockdown of beclin 1 expression in PANC-1 cells enhanced the sensitivity of PANC-1 cells to GEM (P<0.01), whereas overexpression of HMGB1 effectively reversed the beclin 1 knockdown-induced susceptibility to GEM (P<0.01). The results of the western blot analysis and CCK-8 assay showed that compared with the control group, wortmannin significantly inhibited the expression of p-AKT in PANC-1 cells (P<0.01). In addition, wortmannin promoted the effect of miR-218 on the sensitivity of GEM-induced PANC-1 cells (P<0.05). Conclusion: Overexpression of miR-218 promotes the sensitivity of PANC-1 cells to GEM. The effect of miR-218 is achieved mainly through inhibiting the secretion of HMGB1 by PANC-1 cells and the PI3K/Akt pathway.