ABSTRACT
Objective To investigate the effects of DRP1 gene silencing on the chemosensitivity of human gastric cancer MKN-45 cells to oxaliplatin.Methods A lentiviral vector containing short hairpin RNA (shRNA) targeting DRP1 was constructed and cotransfected to MKN-45 cells.Levels of DRP1mRNA was detected by quantitative RT-PCR,With a final concentration of 0.5,1.0,2.0,4.0,8.0,16.0 and 32.0 μg/ml of oxaliplatin processing Lv-shRNA-DRPI group (experimental group),negative control group (NC group),and blank control group (CON group) respectively.Cellular proliferation was determined by MTT assay and calculate half maximal inhibitory concentration (IC5o).Celluar apoptosis was analyzed by flowcytometry.Using Western blot to detect LC3-Ⅱ and p62 expression.Results DRP1mRNA levels in experimental group (0.087 ± 0.025) was significantly inhibited as compared with NC group (1.040 ± 0.020) and CON group (1.027 ± 0.021) (P ＜ 0.001).Cellular survival rate decreased in a oxaliplatin dose-dependent manner (all P ＜ 0.001).IC50 value in experimental group (14.1 ± 0.4) μg/ml dropped compared with NC group (20.0 ± 1.1) μg/ml and CON group (20.0 ± 1.8) pg/ml (both P ＜ 0.01).Celluar apoptosis rates in experimental group (9.4％ ± 1.1％) elevated in contrast with NC group (6.4％ ± 0.8％) and CON group (6.5％ ± 1.0％) (both P ＜ 0.05),expression of LC3 in experimental group(0.36 ± 0.03) dropped compared with NC group (0.62 ± 0.04) and CON group (0.59 ± 0.07) (both P ＜ 0.05),expression of P62 in experimental group (0.58 ±-0.04) increased in contrast with NC group (0.39±0.04) and CON group (0.36 ±0.04) (both P＜0.05).Conclusions DRP1-mediated overactive mitophagy plays a vital role in increasing the sensitivity of gastric cancer cell MKN-45 to oxaliplatin.
ABSTRACT
Objective To investigate the effects of DRP1 gene silencing on the chemosensitivity of human gastric cancer MKN-45 cells to oxaliplatin.Methods A lentiviral vector containing short hairpin RNA (shRNA) targeting DRP1 was constructed and cotransfected to MKN-45 cells.Levels of DRP1mRNA was detected by quantitative RT-PCR,With a final concentration of 0.5,1.0,2.0,4.0,8.0,16.0 and 32.0 μg/ml of oxaliplatin processing Lv-shRNA-DRPI group (experimental group),negative control group (NC group),and blank control group (CON group) respectively.Cellular proliferation was determined by MTT assay and calculate half maximal inhibitory concentration (IC5o).Celluar apoptosis was analyzed by flowcytometry.Using Western blot to detect LC3-Ⅱ and p62 expression.Results DRP1mRNA levels in experimental group (0.087 ± 0.025) was significantly inhibited as compared with NC group (1.040 ± 0.020) and CON group (1.027 ± 0.021) (P ＜ 0.001).Cellular survival rate decreased in a oxaliplatin dose-dependent manner (all P ＜ 0.001).IC50 value in experimental group (14.1 ± 0.4) μg/ml dropped compared with NC group (20.0 ± 1.1) μg/ml and CON group (20.0 ± 1.8) pg/ml (both P ＜ 0.01).Celluar apoptosis rates in experimental group (9.4％ ± 1.1％) elevated in contrast with NC group (6.4％ ± 0.8％) and CON group (6.5％ ± 1.0％) (both P ＜ 0.05),expression of LC3 in experimental group(0.36 ± 0.03) dropped compared with NC group (0.62 ± 0.04) and CON group (0.59 ± 0.07) (both P ＜ 0.05),expression of P62 in experimental group (0.58 ±-0.04) increased in contrast with NC group (0.39±0.04) and CON group (0.36 ±0.04) (both P＜0.05).Conclusions DRP1-mediated overactive mitophagy plays a vital role in increasing the sensitivity of gastric cancer cell MKN-45 to oxaliplatin.