干扰Twist基因对胰腺癌PANC1细胞糖酵解的影响及机制研究

Effects of interfering Twist gene on glycolysis in pancreatic cancer PANC1 cells and its mechanism

目的观察干扰Twist基因表达对胰腺癌PANC1细胞糖酵解的影响,探讨其可能机制。方法设计并合成靶向Twist的siRNA(siRNA-Twist),采用脂质体法转染胰腺癌PANC1细胞,以非特异性siRNA(siRNA-NC)作为阴性对照,以未转染细胞作为空白对照。采用RT-PCR和蛋白质印迹法检测各组细胞TwistmRNA和蛋白表达以及蛋白激酶B(Akt)、磷酸化Akt(p-Akt)蛋白表达;MTT法检测细胞增殖能力;检测糖酵解限速酶(丙酮酸激酶、己糖激酶)活性及培养上清中乳酸含量。结果对照组、siRNA-NC组和siRNA-Twist组TwistmRNA表达量分别为1.00±0.09、1.01±0.08、0.36±0.02,蛋白表达量分别为0.41±0.05、0.42±0.04、0.12±0.06,siRNA-Twist组mRNA和蛋白表达量均显著低于对照组和siRNA-NC组,差异有统计学意义(P值均<0.05);细胞存活率分别为(100.02±9.36)%、(100.01±10.25)%、(59.32±7.26)%,siRNA-Twist组显著低于对照组和siRNA-NC组,差异有统计学意义(P值均<0.05);丙酮酸激酶活性分别为(0.73±0.05)、(0.72±0.08)、(0.43±0.05)U/mg,己糖激酶活性为(4.98±0.48)、(4.96±0.52)、(2.54±0.21)U/mg,乳酸含量分别为(20.36±2.61)、(20.64±3.05)、(9.48±1.09)mmol/L;p-Akt/Akt比值分别为0.65±0.04、0.63±0.07、0.25±0.04,siRNA-Twist组均较对照组和siRNA-NC组显著降低,差异有统计学意义(P值均<0.05)。结论干扰Twist基因表达能够抑制胰腺癌细胞增殖和糖酵解途径,其作用机制可能与Akt信号通路有关。

ObjectiveTo observe the effects of interfering Twist expression on glycolysis in pancreatic cancer PANC1 cells and explore the potential mechanism.MethodsTwist targeting siRNA (siTWIST) was designed and synthesized, and pancreatic cancer PANC1 cells were transfected with siTWIST using liposome, with nonspecific siRNA (siNC) transfected cells as negative control group and untransfected cells as blank control group. RT-PCR and Western blot were used to detect the expression levels of Twist mRNA and protein, and Akt and p-Akt protein expression in different groups;cell proliferation was detected by MTT;and glycolysis key enzyme (pyruvate kinase and hexokinase) activity and the content of lactic acid in the supernatant of culture fluid were detected.ResultsTwist mRNA level in control group, siRNA group and siTwist group expression was 1.00±0.09, 1.01±0.08 and 0.36±0.02;Twist protein level was 0.41±0.05, 0.42±0.04 and 0.12±0.06;the mRNA and protein expression in siTWIST group was obviously lower than those in control group and the difference was statistically significant (P<0.05). The cell survival rate was (100.02±9.36)%, (100.01±10.25)% and (59.32±7.26)%, which in siTWIST group was obviously lower than that in control group;pyruvate kinase activity was (0.73±0.05)U/mg, (0.72±0.08) U/mg and (0.43±0.05)U/mg;the activities of hexokinase were (4.98±0.48)U/mg, (4.96±0.52)U/mg and (2.54±0.21)U/mg;the content of lactic acid was (20.36±2.61)mmol/L, (20.64±3.05)mmol/L and (9.48±1.09)mmol/L;the ratio of p-Akt/Akt was 0.65±0.04, 0.63±0.07, and 0.25±0.04, which in siTWIST group was obviously lower than that in control group.ConclusionsInterference with Twist expression could inhibit the proliferation and glycolysis pathway in pancreatic cancer cells, and the mechanism may be related to the Akt signaling pathway.