Erastin和BIBR1532联合应用对胃癌细胞增殖的影响

Effects of the combination of Erastin and BIBR1532 on the proliferation of gastric cancer cells

目的:探究Erastin和BIBR1532(端粒酶抑制剂)联合应用对胃癌细胞增殖的影响。方法:使用TIMER2.0、GEPIA2.0数据库比较胃癌组织和相应的正常胃黏膜组织中谷胱甘肽过氧化物酶(GPX4)基因的表达水平,并通过GEPIA2.0预测GPX4是否影响胃癌患者的生存和预后。利用STRING数据库构建GPX4蛋白相互作用网络。10、20μmol/L Erastin干扰GPX4表达,通过Western印迹及TRAP(端粒酶活性测定)实验检测TERT蛋白表达量以及端粒酶活性的变化。应用CCK8增殖实验、克隆形成实验检测细胞活力。结果:TIMER2.0和GEPIA2.0数据库检索结果显示胃癌组织中的GPX4表达水平明显高于正常组织,高水平的GPX4与胃癌患者不良预后相关(P<0.05)。STRING蛋白相互作用网络分析显示,GPX4蛋白相互作用网络包括TERT、SLC7A11、PINX1、HSP90AA1、DKC1、SLC3A2、SMARCA4、PIF1、CTNNB1、WRAP53、SMG6、RUVBL1。10、20μmol/L Erastin干扰GPX4蛋白表达后,TRAP实验结果显示细胞端粒酶活性明显降低(t=34.29、14.28,均P<0.001),Western印迹结果显示TERT蛋白表达量明显降低(t=3.599、8.144,均P<0.05)。CCK8和克隆形成实验结果显示细胞生长增殖能力降低(t=8.662、27.88,均P<0.001)。10μmol/L Erastin与75μmol/L BIBR1532联合使用导致胃癌细胞端粒酶活性和TERT蛋白表达量进一步降低(t=9.931、4.918,均P<0.01),导致细胞增殖能力降低更明显(t=4.157、25.46,均P<0.05)。结论:GPX4在胃癌组织中高表达,Erastin和BIBR1532联合使用在干扰GPX4表达的同时,细胞内端粒酶活性和TERT蛋白表达量进一步降低,抑制胃癌细胞增殖。

Objective:To explore the effects of the combination of Erastin and BIBR1532(telomerase inhibitor)on the proliferation of gastric cancer cells.Methods:Relevant data from TIMER2.0 and GEPIA2.0 databases regarding glutathione peroxidase(GPX4)gene expression level in gastric cancer samples and corresponding normal gastric mucosal samples were analyzed.GEPIA 2.0 was used to predict whether GPX4 affects the survival and prognosis of gastric cancer(GC)patients.The GPX4 protein interaction network was constructed using STRING database.GPX4 expression was interfered with 10,20μmol/L Erastin,the changes of TERT protein expression and telomerase activity were detected by Western blotting and TRAP(telomerase activity assay)experiments.Cell viability was detected through CCK8 proliferation and clone formation experiments.Results:The results of TIMER2.0 and GEPIA2.0 databases showed that GPX4 expression level in GC tissues was significantly higher than that in normal tissues,and high GPX4 level was associated with poor prognosis in GC patients(P<0.05).The analysis of STRING protein interaction network showed that GPX4 protein interaction network included TERT,SLC7A11,PINX1,HSP90AA1,DKC1,SLC3A2,SMARCA4,PIF1,CTNNB1,WRAP53,SMG6 and RUVBL1.The results of TRAP experiment showed that telomerase activity significantly decreased after GPX4 expression was interfered with 10,20μmol/L Erastin(t=34.29,14.28,both P<0.001).Western blotting results showed that TERT protein expression level was significantly decreased(t=3.599,8.144,both P<0.05).The results of CCK8 and clone formation experiments showed a decrease in cell growth and proliferation(t=8.662,27.88,both P<0.001).The combined treatment of 10μmol/L Erastin and 75μmol/L BIBR1532 reduced the telomerase activity and the expression of TERT protein in GC cells(t=9.931,4.918,both P<0.01),resulting in a more significant decrease in cell proliferation(t=4.157,25.46,both P<0.05).Conclusion:GPX4 is highly expressed in GC tissues.The combination of Erastin and BIBR1532 further reduces intracellular telomerase activity and TERT protein expression after interfering with GPX4 expression,ultimately inhibiting the proliferation of GC cells.