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.
维普
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