阻滞腺苷A2A受体抑制胶质瘤细胞干性特征并诱导保护性自噬

目的探究阻滞腺苷A2A受体（A2AR）对胶质瘤细胞干性特征与保护性自噬的影响。方法将人胶质瘤U87细胞分为四组：对照组、shNC组、shA2AR组、SCH58261组，分别将shNC、shA2AR转染至shNC组与shA2AR组细胞中，SCH58261组使用A2AR拮抗剂SCH58261处理细胞，收集处理后的4组U87细胞，应用实时荧光定量聚合酶链式反应（qRT-PCR）检测细胞中A2AR mRNA表达水平，蛋白质免疫印记（Western Blot）检测细胞中A2AR 蛋白表达水平，流式细胞术检测细胞周期分布，细胞肿瘤球形成实验检测细胞干性，LC3自噬双标腺病毒实验检测自噬溶酶体与自噬体形成，流式细胞术检测细胞凋亡，Western Blot检测细胞中干细胞标记物巢蛋白（Nestin）、SRY相关高迁移率族盒蛋白2（SOX2）、八聚体结合转录因子4（OCT4）以及自噬相关分子微管相关蛋白3（LC3）II/LC3I、Beclin1的蛋白表达水平。 结果与对照组比较，shA2AR组和SCH58261组U87细胞中A2AR mRNA相对表达量与蛋白相对表达量降低，差异具有统计学意义（P<0.05）；G1期细胞比例增加，差异具有统计学意义（P<0.05）；S期细胞比例减少，差异具有统计学意义（P<0.05）；肿瘤球形成数目减少，差异具有统计学意义（P<0.05）；细胞中红色斑点代表的自噬溶酶体与黄色斑点代表的自噬体均明显增多，细胞凋亡率增加，差异具有统计学意义（P<0.05）；细胞中Nestin、SOX2、OCT4蛋白相对表达量减少，差异具有统计学意义（P<0.05）；同时，LC3II/LC3I蛋白比值升高，差异具有统计学意义（P<0.05）；Beclin1蛋白相对表达量也增加，差异具有统计学意义（P<0.05）。结论阻滞A2AR能够使人胶质瘤U87细胞发生G1期阻滞，抑制肿瘤细胞干性特征，并促进保护性自噬，诱导细胞发生凋亡。

Objective To investigate the effects of blocking adenosine A2A receptor(A2AR) on the stemness characteristics and protective autophagy of glioma cells. Methods Human glioma U87 cells were divided into four groups, control group, shNC group, shA2AR group and SCH58261 group, shNC and shA2AR cells were transfected into shNC group and shA2AR group, respectively. SCH58261 group was treated with A2AR antagonist SCH58261. Four groups of U87 cells were collected after treatment, real-time quantitative fluorescent polymerase chain reaction(qRT-PCR) was used to detect A2AR mRNA expression level in cells. Western blot was used to detect A2AR protein expression level in cells. Flow cytometry was used to detect cell cycle distribution. Cell tumor pellet formation assay was used to detect cell dryness. LC3 autophagy double-label adenovirus assay was used to detect the formation of autophagosome and autophagosome. Flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression levels of stem cell marker Nestin(Nestin), SRY-associated high mobility group box protein 2(SOX2), octamer binding transcription factor 4(OCT4) and autophagy related molecules microtubule associated protein 3(LC3) II/LC3I and Beclin1. Results Compared with the control group, the mRNA relative expression level and protein relative expression level of A2AR in U87 cells of shA2AR group and SCH58261 group were decreased. The difference were statistically significant(P<0.05). The proportion of cells in G1 phase was increased. The difference was statistically significant(P<0.05). The proportion of cells in S phase was decreased. The difference was statistically significant(P<0.05). The number of tumor spheres formed was decreased. The difference was statistically significant(P<0.05). The number of autophagosomes represented by red spots and autophagosomes represented by yellow spots in cells significantly increased, and the apoptosis rate increased, with a statistically significant difference(P<0.05). The relative expression levels of Nestin, SOX2, and OCT4 proteins in cells decreased with statistical significance(P<0.05). Meanwhile, the ratio of LC3II/LC3I protein increased, and the difference was statistically significant(P<0.05). The relative expression level of Beclin1 protein also increased, and the difference was statistically significant(P<0.05). Conclusion Blocking A2AR can induce G1 phase arrest of human glioma U87 cells, inhibit the stemness characteristics of tumor cells, promote protective autophagy, and induce cell apoptosis.