Prostaglandin D2 regulation of autophagy affects stemness of gastric cancer stem cells.

Objective: To explore the effect and mechanism of prostaglandins D2 (PGD2) on the stemness of gastric cancer stem cells (GCSCs). Methods: 7901-GCSCs were enriched by serum-free culture method; then the positivity rate of CD44, a stemness marker, was detected by flow cytometry in serum-free cultured 7901-GCSCs; the sphere-forming ability was detected by the sphere-forming assay after stimulation with different concentrations of PGD2 (2.5, 5, 10) μg/mL, and the expression of stemness-related indicators (OCT4, CD44) and autophagyrelated proteins (LC3, Beclin-1) after PGD2 stimulation was detected by the western blot assay in different concentrations. The expression of stemness-related indexes (OCT4, CD44) and autophagy-related proteins (LC3, Beclin-1) were detected by Western blot assay after stimulation with different concentrations of PGD2. The expression of autophagy-related proteins after stimulation with different concentrations of CQ (2.5, 5, 10) μM was detected by Western blot experiment. The protein expression of autophagy-related proteins (LC3, Beclin-1) and stemness-related indexes (OCT4, CD44) was detected by Western blot experiment after PGD2 as well as PGD2+CQ treatment. Results: Flow cytometry results showed that the expression of CD44 positivity was increased in serum-free cultured 7901-GCSCs compared with gastric cancer cells SGC-7901 (P<0.05), which fulfilled the needs of subsequent experiments. The results of stem cell spheroid formation assay showed that the spheroid formation ability of 7901-GCSCs in the PGD2 group was significantly weakened compared with that of the DMSO group (P<0.05).Western blot results showed that the protein expression of stemness-related indexes (OCT4, CD44) was down-regulated in the 7901-GCSCs in the PGD2 group compared with that of the DMSO group (P<0.05), and the expression of autophagy-related proteins (LC3, Beclin-1) expression increased (P<0.05).Compared with the DMSO group, the expression of autophagy-related proteins (LC3, Beclin-1) was decreased in the CQ group (P<0.05).Western blot results also showed that the expression of cellular autophagy-related proteins and stemness-related indexes in the PGD2+CQ group was not significantly changed compared with that of the DMSO group (ns: the difference was not significant),suggesting that the CQ could block the effect of PGD2 on the expression of stemness markers in 7901-GCSCs. 7901-GCSCs stemness inhibition. Conclusion: PGD2 may affect the stemness of 7901-GCSCs by regulating autophagy. [ABSTRACT FROM AUTHOR]