甲硫氨酸饥饿通过抑制程序性死亡配体1诱导胃癌细胞凋亡

Methionine starvation induces apoptosis of gastric cancer cells by inhibiting programmed death ligand-1

目的研究甲硫氨酸饥饿诱导胃癌细胞凋亡的分子机制。方法利用癌症基因组图谱(TCGA)数据库分析胃癌组织中程序性死亡配体1(PD-L1)的表达及其与临床病理特征的关系。用普通培养液和甲硫氨酸饥饿培养液分别培养胃癌AGS细胞及抑制PD-L1表达的siRNA(siPD-L1)胃癌细胞,根据处理方法分为对照组、甲硫氨酸饥饿处理组、siPD-L1处理组、甲硫氨酸饥饿联合siPD-L1处理组,采用CCK-8检测胃癌细胞活力,吖啶橙/溴化乙锭双染检测胃癌细胞凋亡数目,流式细胞术检测胃癌细胞凋亡率,蛋白质印迹法检测胃癌细胞PD-L1、Bcl-2、B淋巴细胞瘤2相关X蛋白(Bax)、Caspase 3蛋白表达。starBase数据库分析PD-L1与Bcl-2抗凋亡蛋白家族(BCL2A1、MCL1、BCL2、BCL2L1)之间的联系。结果PD-L1在胃癌组织中高表达(P<0.01),且PD-L1的表达与胃癌G分级有关(P<0.01)。甲硫氨酸饥饿和siPD-L1处理均能够降低胃癌细胞存活率(P<0.01),促进凋亡(P<0.01),抑制PD-L1和Bcl-2表达(P<0.05),上调促凋亡蛋白Bax、Caspase 3表达(P<0.01);甲硫氨酸饥饿联合siPD-L1处理上述作用更强(P<0.05)。PD-L1与Bcl-2抗凋亡蛋白家族表达水平之间呈正相关(P<0.01),说明PD-L1是一个关键抗凋亡基因。结论甲硫氨酸饥饿通过抑制PD-L1的表达下调抗凋亡蛋白Bcl-2并上调促凋亡蛋白Bax和Caspase 3表达,从而诱导胃癌细胞凋亡。

Objective To explore the molecular mechanism of methionine starvation induced apoptosis in gastric cancer cells.Methods The expression of programmed death ligand-1(PD-L1)in gastric cancer tissues was analyzed by the cancer genome atlas(TCGA)database.The gastric cancer AGS cells and the gastric cancer cells treated with siRNA that inhibit the expression of PD-L1(siPD-L1)were cultured with ordinary medium and methionine-starved medium.According to the treatment method,they were divided into control group,methionine starvation treatment group,siPD-L1 treatment group,and methionine starvation combined siPD-L1 treatment group.The cell viability of gastric cancer was detected by cell counting kit 8,the apoptosis of gastric cancer cells was detected by acridine orange/ethidium bromide double staining,and the apoptosis rate of gastric cancer cells was detected by flow cytometry.The expression levels of PD-L1,B-cell lymphoma-2(Bcl-2),B-cell lymphoma-2-related X protein(Bax)and Caspase 3 were detected by Western blotting analysis.Finally,the relationship between PD-L1 and Bcl-2 anti-apoptotic protein family(Bcl-2 related protein A1[BCL2 A1],myeloid cell leakemia 1[MCL1],BCL2,and Bcl-2 like 1[BCL2 L1])was analyzed by starBase database.Results PD-L1 was highly expressed in gastric cancer tissues(P<0.01),and the expression of PD-L1 was correlated with G grade of gastric cancer(P<0.01).Methionine starvation and siPD-L1 significantly decreased the survival rate of gastric cancer cells(P<0.01),promoted apoptosis(P<0.01),inhibited the expression of PD-L1 and Bcl-2(P<0.05),and up-regulated the expression of proapoptotic proteins Bax and Caspase 3(P<0.01).The effect of methionine starvation combined with siPD-L1 was even stronger(P<0.05).There was a positive correlation between PD-L1 and Bcl-2 anti-apoptotic protein family(P<0.01),indicating that PD-L1 is a key anti-apoptotic gene.Conclusion Methionine starvation can induce gastric cancer cell apoptosis by inhibiting the expression of PD-L1,downregulating the expression of anti-apoptotic protein Bcl-2 and up-regulating the expression of Bax and Caspase 3.